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Apparatus for Photo-micrography. 
 
PRACTICAL METHODS 
 
 MICROSCOPY 
 
 CHARLES H. CLARK, A.M. 
 
 Principal of Sanborn Seminary 
 
 BOSTON, U.S.A. 
 
 D. C. HEATH & CO., PUBLISHERS 
 1894 
 
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 P07 
 
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 / S' i 
 
 Copyright, 1893, 
 
 By CHARLES H. CLARK. 
 
 Nortonoti TPrrsg : 
 
 J. S. Cushing & Co. — Berwick & Smith. 
 Boston, Mass., U.S.A. 
 
 THE GETTY CENT art 
 
 LIBRARY 
 
PREFACE. 
 
 There are many excellent books on the microscope, and 
 many on special subjects requiring the use of the microscope 
 as a working tool for investigation. In the most of these 
 books, however, the descriptions of methods are so inter¬ 
 woven with other matters that the inexperienced student is 
 confused and, in many cases, is unable to separate the essential 
 from the non-essential. Too much is assumed to be known, 
 or is left to be filled in by an instructor. None of these books 
 gives to the private worker, in simple and concise language, 
 detailed directions for the many processes that he must learn 
 in order to make practical use of the microscope. 
 
 A large amount of apparently unproductive work must be 
 done before the microscope can be advantageously used as a 
 working tool. It is of the highest importance that this work 
 be done at as early a stage as possible, and without waste of 
 time. This book is professedly for beginners and private 
 workers, and is designed to afford the means of acquiring this 
 necessary training. The methods here described are practical 
 methods which have stood the test of general use. They are 
 the methods employed by scientific men the world over. It 
 would not be possible, in most cases, to give credit to the 
 originators of the processes, as the processes have been the 
 result of slow growth, and have been so modified by different 
 workers that they have become common property. Credit 
 can, therefore, be given only in this general way to the books 
 and workers that have furnished the material which has 
 been drawn upon. 
 
 iii 
 
IV 
 
 PKEFACE. 
 
 These pages are the outgrowth of the author’s experience 
 in the use of the microscope in the various branches of scien¬ 
 tific study pursued in the secondary schools. They are the 
 result of many hours of pleasant work, and of many hours of 
 laborious reading in the search of what, in many cases, could 
 not be found in the books consulted. 
 
 Only so much of the mechanical construction of the 
 microscope and of the theory of light is given as seems 
 absolutely essential to an intelligent understanding of the 
 instrument; other books treat exhaustively of these subjects. 
 The theory of polarized light has been somewhat fully consid¬ 
 ered, in part because of its important applications and great 
 interest, and in part because this subject is so briefly and so 
 vaguely treated in most books on the microscope. The sub¬ 
 ject is deep, and it is not easy to treat it in a popular way. 
 An attempt has been made, while adhering to scientific accu¬ 
 racy, to explain in simple language the principal phenomena 
 of polarized light so far as they have practical application in 
 the use of the microscope. 
 
 The author wishes to express his sincere thanks to the 
 following gentlemen who have carefully read the proof and 
 offered many valuable suggestions: Professor Leslie A. Lee. 
 Bowdoin College; Dr. S. G. Shanks, Albany, N.Y.; Principal 
 Clarence E. Kelley, High School, Haverhill, Mass.; Dr. S. G. 
 Bonney, Denver, Col.; Mr. George L. Chandler, High School, 
 Newton, Mass.; Rev. J. D. King, Ph.D., Cottage City, Mass .; 
 Dr. W. H. Sylvester, Natick, Mass. 
 
 Acknowledgment is also made to the Bausch and Lomb 
 Optical Co., Rochester, N. Y.; Queen & Co., Philadelphia; 
 and Williams, Brown & Earle, Philadelphia, for the use of 
 electrotypes illustrating microscopes and accessory apparatus. 
 
 C. H. C. 
 
 Kingston, N.H., December, 1893. 
 
CONTENTS. 
 
 CHAPTER I. 
 
 Light and the Microscope .. 
 
 The Undulatory Theory of Light — Refraction of Light — 
 Explanation of Refraction — Dispersion of Light — How Dis¬ 
 persion is overcome in the Case of a Prism — Index of Refrac¬ 
 tion — Absolute Indices of Refraction — Use of Indices of Re¬ 
 fraction — Total Reflection — The Double-Convex Lens — 
 Spherical Aberration — Dispersion in Lenses — How overcome 
 — Achromatic Lenses — The Simple Microscope — Different 
 Manners of Mounting — The Compound Microscope — Simplest 
 Form of — Principle of the Objective — Of the Eye-Piece — 
 The Microscope Stand —Its Various Parts — Selection of a 
 Microscope — Care of the Microscope — Cleaning Lenses — 
 Dust on Lenses —The Glass Slip —The Cover-Glass— Impor¬ 
 tance of — Effect of Thickness of Cover-Glass — How remedied 
 — Cover-Glass Gauge. 
 
 CHAPTER II. 
 
 Some Accessories for Advanced Work. 
 
 Immersion Objectives — Why they are used — How used — 
 Dry, Water-Immersion, and Homogeneous-Immersion Objec¬ 
 tives compared — Adjustable Objectives — Manipulation of — 
 Angular and Numerical Aperture — Sub-stage Condenser — 
 The Camera Lucida — How used — Micrometers and Microm¬ 
 etry — How to measure the Magnification of a Camera Lucida 
 Drawing — The Turn-Table — The Section-Cutter — How used 
 — The Microtome — How used — The Water-Bath and Oven 
 — Their Use. 
 
 v 
 
VI 
 
 CONTENTS. 
 
 CHAPTER III. 
 
 PAGE 
 
 Polarized Light and the Polarizer. 41 
 
 Difference of Elasticity of the Ether in Crystalline and Non- 
 Crystalline Substances — Resolution of the Ether Waves into 
 Two Sets of Vibrations in Crystalline Substances — Two Rays 
 the Result — Each of the Rays caused by Vibrations in a Single 
 Direction — The Directions at Right Angles to Each Other — 
 
 Both Rays refracted —Different Degrees of Refraction — Large 
 Number of Double-refracting Substances —Iceland Spar —The 
 Nicol Prism — Polarizer and Analyzer of the Microscope — 
 
 How arranged — Ray of Light traced from the Mirror through 
 the Nicols — Colors in Thin Plates — Explanation of — Com¬ 
 plementary Colors — Explanation of — The Dark Cross on Cer¬ 
 tain Crystals and Starches — Uses of Polarized Light — Polar¬ 
 izing Substances — The Selenite — Single-refracting and Double- 
 refracting Substances. 
 
 CHAPTER IV. 
 
 Instructive Practice in Manipulation.54 
 
 Importance of this Practice — Suitable Objects with which 
 to work — How to focus correctly — Illumination — How to 
 use the Mirror — The Draw-Tube — Examination of Opaque 
 Objects — Air Bubbles — Oil Globules — Eibres and Hairs — 
 Currents and the Brownian Movement — Practice with Immer¬ 
 sion Objectives—Use of Collar Adjustment — Effects of Cen¬ 
 tral and Oblique Illumination. 
 
 CHAPTER V. 
 
 Methods op Studying Fresh Objects.63 
 
 The Microscope only a Working Tool — Neither Possible 
 nor Desirable to mount permanently All Objects — The Pur¬ 
 pose of this Chapter — Equipment needed — Importance of 
 Neatness—'Vegetable Specimens — Water the Usual Medium 
 for their Examination — No Preparation Necessary in Some 
 Cases — Microscopic Water-Plants — Yeast —Pollen — Spores 
 — Fungi — Maceration and Teasing — Vegetable Sections — 
 
CONTENTS. 
 
 Methods of Studying — Starch — Cellulose — Lignified Cellular 
 Tissue — Protoplasm — Chromatophores — Plant Crystals — 
 Animal Specimens — Animalcules — Water Sediment Stiiped 
 
 Muscular Fibre — Unstriped Muscular Fibre — Sections of Ani¬ 
 mal Tissues — Blood — The Circulation of Blood Cartilage 
 Bone — Epithelium. 
 
 CHAPTER VI. 
 
 Mounting Objects for the Microscope. 
 
 Mounting in Canada Balsam—Glycerine Jelly — Thin 
 Fluids — Dry Mounting — Mounting in Cells with Fluid Media 
 
 _Finishing Slides — Ornamentation — Methods of applying 
 
 Cements —Labelling —Cleaning Glass Slips and Cover-Glasses. 
 
 CHAPTER VII. 
 
 Practice Mounting. 
 
 Necessary Equipment — Insect Scales, Starch, and Similar 
 Light Objects — Insects — Vegetable Sections — Double Stain¬ 
 ing of — Animal Sections — Staining of — Fixing on Glass Slip 
 — Diatoms. 
 
 CHAPTER VIII. 
 
 Botanical Sections. 
 
 Classification of Specimens — Preparation of Hard Speci¬ 
 mens — Transverse, Tangential, and Radial Sections Cutting 
 with the Microtome — Soft Specimens — Preservation and 
 Preparation of — Imbedding in Paraffin — Cutting Imbedded 
 Specimens — Imbedding in Celloidin — Imbedding in Soap — 
 Staining — Order of Procedure with Vegetable Specimens to 
 be cut in Paraffin — Order of Procedure with Vegetable 
 Specimens to be cut in Celloidin. 
 
 CHAPTER IX. 
 
 Sections of Animal Tissues . .....•• 
 
 Hardening — Staining — Imbedding in Paraffin — Imbed¬ 
 ding in Celloidin — Cutting Sections — Mounting — Fixing on 
 
CONTENTS. 
 
 viii 
 
 Glass Slip — Order of Procedure with Animal Tissues when the 
 Object is to be stained in toto and cut in Paraffin — Order of 
 Procedure with Animal Tissues to be cut in Paraffin and 
 stained after they are cut — Order of Procedure with Animal 
 Tissues to be stained in toto and then cut in Celloidin — Order 
 of Procedure with Animal Tissues to be cut in Celloidin, the 
 Sections to be stained after cutting and fixed in Position on the 
 Glass Slip. 
 
 CHAPTER X. 
 
 Chemical Crystals.125 
 
 Simple Processes—Different Forms obtained by Different 
 Treatment — Various Solvents — Methods for Temporary Ex¬ 
 amination— Crystals in the “Mother Liquor” — Preparation 
 for Permanent Mounting — Mounting Media — Methods of 
 Mounting — Dry Mounts — Examination of Crystals — Polar¬ 
 ized Light. 
 
 CHAPTER XI. 
 
 Rock Sections by Simple Means. 131 
 
 Importance of the Microscopical Study of Rocks — Its Inter¬ 
 est — Apparatus — Method of preparing Thin Sections — 
 Mounting — Fossil Woods — Rocks of Crystalline Structure — 
 Studying Sections of Crystals — Measuring Angles of Crystals 
 — Cleavage — Impurity of Crystals — Crystals in Crystals — 
 Liquid Carbonic Acid in Crystals — Applications of Polarized 
 Light — Singly-refractive and Doubly-refractive Crystals — 
 Amorphous Substances — Isometric Crystals, how distinguished 
 — Crystals of the Tetragonal and Hexagonal Systems — Crys¬ 
 tals of the Orthorhombic, Monoclinic, and Triclinic Systems — 
 Rolling Sand. 
 
 CHAPTER XII. 
 
 The Study of Bacteria . ... 143 
 
 To what Extent the Microscope is used in the Study of Bac¬ 
 teria — Minuteness of the Organisms — What Bacteria are — 
 
CONTENTS. 
 
 IX 
 
 Where found — Their Effects — Manner of Reproduction — 
 Some General Divisions of Bacteria — Colors of — Movements 
 of — Movements not to he confused with the Brownian Move¬ 
 ment — Harmless and Harmful Forms — Methods of studying 
 without Cultures — Bacteria of the Fur of the Teeth — Putre¬ 
 faction Bacteria — Hay Bacillus — Pure Culture of — Spiro- 
 chceta plicatalis —Movements of — Beggiatoa alba — Detection 
 of Sulphur in—Zooglsea Stage—Swarming Stage — Resting 
 Spores — Cover-Glass Preparations — Staining of — Double 
 Staining — What Staining Fluids to use, and how to prepare 
 them — Bacillus tuberculosis — Description of — Demonstra¬ 
 tion of—Ehrlich’s Method — The Ziehl-Neelsen Carbol-Fuchsin 
 Method — The Typhoid Bacillus — Description of — Does not 
 contain Spores — Examination for — The Cholera Bacillus — 
 Description of — Examination for. 
 
 CHAPTER XIII. 
 
 The Study of Bacteria : Culture Methods .... 157 
 
 Why Culture Methods are Necessary — Methods outlined — 
 Sterilization — How it is accomplished — Importance of 
 Thoroughness and Carefulness — The Sterilizers — Expensive 
 Forms not Necessary — Simple Forms described — The Hot-Air 
 Sterilizer — The Steam Sterilizer — Food Materials — Gelatine 
 Medium — Agar-agar Medium — Test-Tube Cultures — Cultures 
 at Elevated Temperatures — Plate Cultures — Method of iso¬ 
 lating Bacteria — Cultures without Oxygen — Milk Cultures — 
 Bouillon Cultures—Potato Cultures — Hanging-Drop Cultures 
 — Practical Application of the Methods. 
 
 CHAPTER XIV. 
 
 Photo-Micrography. 
 
 Practical Uses — Simple Apparatus — Arrangement of Appa¬ 
 ratus— Focussing — Dry Plates — Exposure — Measurement of 
 Magnification — Development — Fixing the Negative — Printing 
 — Toning — Fixing the Prints — Mounting — Omega Prints — 
 Bromide Prints — Lantern Slides. 
 
X 
 
 CONTENTS. 
 
 APPENDIX. 
 
 Useful Formulae .. 
 
 Staining Fluids — Fixatives for holding Sections in Position 
 on the Glass Slip — Cements — Hardening Fluids — Macerating 
 Fluids — Mounting Media — Solutions for cleaning Glass Slips 
 and Cover-Glasses — Alcohol of Percentage Strengths — Solu¬ 
 tions used in Photo-micrography — Metric-English Equivalents. 
 
 PAGE 
 
 201 
 
 Index 
 
 213 
 
LIST OF ILLUSTRATIONS. 
 
 PAGE 
 
 Apparatus for Photo-micrography. 
 
 Frontispiece 
 
 1. 
 
 Path of a Ray of Light through a Prism . 
 
 
 3 
 
 2. 
 
 Explanation of the Action of a Prism 
 
 
 3 
 
 3. 
 
 Compound Prism to overcome Dispersion 
 
 
 5 
 
 4. 
 
 Figure illustrating the Index of Refraction 
 
 
 6 
 
 5. 
 
 Figure illustrating Total Reflection .... 
 
 
 8 
 
 6. 
 
 Principle of the Double-Convex Lens 
 
 
 9 
 
 7. 
 
 The Action of the Double-Convex Lens; also 
 
 Spherical 
 
 
 
 Aberration. 
 
 
 9 
 
 8. 
 
 The Achromatic Lens. 
 
 
 11 
 
 9. 
 
 The Principle of the Simple Microscope . 
 
 
 11 
 
 10. 
 
 Watchmaker’s Glass. 
 
 
 12 
 
 11. 
 
 Pocket Lens. 
 
 
 12 
 
 12. 
 
 Dissecting Microscope. 
 
 
 12 
 
 13. 
 
 Principle of the Compound Microscope ; the Simplest Form . 
 
 13 
 
 14. 
 
 Principle of the Compound Microscope ; the Ordinary Form . 
 
 13 
 
 15. 
 
 The Huyghenian Eye-Piece ..... 
 
 
 14 
 
 16. 
 
 Beck’s Improved Continental Microscope . 
 
 
 17 
 
 17. 
 
 The Action of the Cover-Glass. 
 
 
 20 
 
 18. 
 
 The Cover-Glass Gauge ...... 
 
 
 23 
 
 19. 
 
 Figure showing Difference of Action of Dry, Water Immer- 
 
 
 
 sion, and Homogeneous Immersion Objectives . 
 
 
 25 
 
 XI 
 
Xll LIST OF ILLUSTRATIONS. 
 
 PAGE 
 
 20. Construction of Sub-stage Condenser, N. A. 1.20 ... 30 
 
 21. Construction of Sub-stage Condenser, N. A. 1.42 ... 30 
 
 22. The Camera Lucida..31 
 
 23. Principle of the Abbe Camera Lucida . . . . .31 
 
 24. Improved Form of the Abbe Camera Lucida . . .32 
 
 25. The Turn-Table.36 
 
 26. The Section-Cutter. 37 
 
 27. The Microtome . 38 
 
 28. The Water-Bath and Oven.39 
 
 29. Crystal of Iceland Spar showing Double Refraction ... 42 
 
 30. Principle of the Nicol Prism..43 
 
 31. The Polarizer.44 
 
 32. The Analyzer.44 
 
 33. The Bausch and Lomb Continental Microscope ... 62 
 
 34. Leitz’s Stand III.104 
 
 35. Queen & Co.’s Acme Continental Microscope . . . .116 
 
 36. The Bausch and Lomb Petrographical Microscope . . . 130 
 
 37. Leitz’s Stand II..142 
 
 38. The Bausch and Lomb Large Continental Microscope . 156 
 
 39. Dry-Air Sterilizer.. . 159 
 
 40. Steam Sterilizer.160 
 
 PHOTOGRAPHIC REPRODUCTIONS OF PHOTO¬ 
 MICROGRAPHS BY THE AUTHOR. 
 
 Plate I. Peduncle of Yellow Water-Lily, Nuphar advena . . 185 
 
 Part of transverse section, showing stellate cells or internal 
 hair’s, fibro-vascular bundles, large intercellular spaces, and the 
 circular sections of characteristic uncompressed cylindrical 
 cells. 
 
LIST OF ILLUSTRATIONS. 
 
 xiii 
 
 PAGE 
 
 Plate II. Scotch Pine, Pinus sylvestris ...... 185 
 
 Part of transverse section of the stem from the bark to the 
 pith, showing the loose cortical parenchyma, resin ducts, xylem, 
 or woody tissue, with incipient medullary rays, and pith paren¬ 
 chyma. 
 
 Plate III. Common Milkweed, Asclepias cornuti .... 187 
 
 Part of transverse section of the stem from the cuticle to the 
 pith, showing cork cells of the bark, cortical parenchyma, sieve 
 tubes in which are situated the principal milk ducts, the cam¬ 
 bium layer, bundles of vascular tissues, and crystals in the pith 
 parenchyma. 
 
 Plate IV. Northern Pitch Pine, Pinus rigida .... 187 
 
 Part of transverse section of the stem, showing cork cells of 
 the bark, large resin ducts in the cortical parenchyma and 
 smaller ones in the wood, with the glandular cells around them, 
 which secrete the pitch, the cambium layer, the wood with in¬ 
 cipient medullary rays, and the pith parenchyma. 
 
 Plate Y. Common Juniper, Juniperus communis . . . .187 
 
 Part of transverse section of the stem, showing annual layers, 
 large cells of early growth, and small cells of later growth. 
 
 Plate VI. Southern Yellow Pine, Pinus australis .... 189 
 
 Part of radial section, showing one whole and parts of two 
 other annual layers, tracheides, and medullary rays. 
 
 Plate VII. Hogweed, Ambrosia artimisicefolia .... 189 
 
 Part of transverse section of the stem, showing fibro-vascular 
 
 bundles and parenchyma. 
 
 Plate VIII. Foetal Thigh Bone. Transverse section . . . 191 
 
 Plate IX. Human Liver.193 
 
 Plate X. Tongue of Cat.193 
 
 Longitudinal vertical section of the upper portion near the 
 tip, showing the papillae. 
 
XIV 
 
 LIST OF ILLUSTRATIONS. 
 
 PAGE 
 
 Plate XL Fossil Wood. Silicified Conifer from Yellowstone Park 195 
 
 Transverse section of the stem, showing annual layers of 
 the wood ; the wood tissue appears as trachei'des cut across. 
 
 Plate XII. Fossil Wood. Silicified Conifer from Yellowstone 
 
 Park 195 
 
 Radial section of the stem, showing annual layers, medullary 
 rays, and trachei'des with bordered pits. 
 
 Plate XIII. Burdock, Lappa major .197 
 
 Part of transverse section of the petiole, showing fibro-vascu- 
 lar bundles in different stages of development, parenchyma, 
 and hairs. 
 
 Plate XIV. Cinnamon Fern, Osmunda cinnamommia . . . 197 
 
 Part of transverse section of stipe, showing the fibro-vascu¬ 
 lar bundle, which is made up mainly of scalariform vessels and 
 sieve tubes. 
 
 Plate XV. Male Fern . . ■.199 
 
 Part of a leaf, showing fructification. 
 
 Plate XVI. Triticum junceum .199 
 
 Transverse section of the leaf, showing hairs. 
 
 Plate XVII. Petiole of Yellow Water-Lily, Nuphar advena . . 199 
 
 Part of transverse section, showing structure similar to that 
 in Plate I. 
 
PRACTICAL METHODS IN MICROSCOPY. 
 
 CHAPTER I. 
 
 LIGHT AND THE MICROSCOPE. 
 
 The Undulatory Theory of Light. — The Undulatory 
 Theory of Light assumes that all space is filled with a 
 very rare medium called the ether, which permeates every 
 substance in the universe. The Molecular Theory of 
 Matter assumes that every body of matter is made up 
 of exceedingly minute particles called molecules, which 
 are in a state of constant vibration, moving back and forth 
 in all directions. If a body is heated, the motion of the 
 molecules increases in rapidity as the temperature rises. 
 When this vibratory motion has attained a certain rapidity, 
 the body becomes luminous. This results from the fact 
 that the motion, originating in the molecules of the body 
 and communicated by these molecules to the particles of 
 ether which permeate the body or lie adjacent to it, pro¬ 
 duces wave motions in the ether, which the ether transmits 
 to the eye. It is known from experimental demonstra¬ 
 tion that, as the temperature of a body is raised, the wave 
 motions become more rapid, and that the wave lengths in 
 the ether are shortened. When the waves are of lengths 
 within certain fixed limits, which have been determined 
 by experiment, they affect the fibres of the optic nerve 
 
 1 
 
XIV 
 
 LIST OF ILLUSTRATIONS. 
 
 PAGE 
 
 Plate XI. Fossil Wood. Silicified Conifer from Yellowstone Park 195 
 
 Transverse section of the stem, showing annual layers of 
 the wood ; the wood tissue appeal’s as trachei'des cut across. 
 
 Plate XII. Fossil Wood. Silicified Conifer from Yellowstone 
 
 Park.195 
 
 Radial section of the stem, showing annual layers, medullary 
 rays, and trachei'des with bordered pits. 
 
 Plate XIII. Burdock, Lappa major .197 
 
 Part of transverse section of the petiole, showing fibro-vascu- 
 lar bundles in different stages of development, parenchyma, 
 and hairs. 
 
 Plate XIV. Cinnamon Fern, Osmunda cinnamommia . . . 197 
 
 Part of transverse section of stipe, showing the fibro-vascu¬ 
 lar bundle, which is made up mainly of scalariform vessels and 
 sieve tubes. 
 
 Plate XV. Male Fern . ..199 
 
 Part of a leaf, showing fructification. 
 
 Plate XVI. Triticum junceum .... ... 199 
 
 Transverse section of the leaf, showing hairs. 
 
 Plate XVII. Petiole of Yellow Water-Lily, Nupliar advena . . 199 
 
 Part of transverse section, showing structure similar to that 
 in Plate I. 
 
PRACTICAL METHODS IN MICROSCOPY. 
 
 CHAPTER I. 
 
 LIGHT AND THE MICROSCOPE. 
 
 The TJndulatory Theory of Light. — The Undulatory 
 Theory of Light assumes that all space is filled with a 
 very rare medium called the ether, which permeates every 
 substance in the universe. The Molecular Theory of 
 Matter assumes that every body of matter is made up 
 of exceedingly minute particles called molecules, which 
 are in a state of constant vibration, moving back and forth 
 in all directions. If a body is heated, the motion of the 
 molecules increases in rapidity as the temperature rises. 
 When this vibratory motion has attained a certain rapidity, 
 the body becomes luminous. This results from the fact 
 that the motion, originating in the molecules of the body 
 and communicated by these molecules to the particles of 
 ether which permeate the body or lie adjacent to it, pro¬ 
 duces wave motions in the ether, which the ether transmits 
 to the eye. It is known from experimental demonstra¬ 
 tion that, as the temperature of a body is raised, the wave 
 motions become more rapid, and that the wave lengths in 
 the ether are shortened. When the waves are of lengths 
 within certain fixed limits, which have been determined 
 by experiment, they affect the fibres of the optic nerve 
 
 1 
 
2 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 spread out to form the retina of the eye, and produce the 
 sensation of light. Light is therefore defined, in a sub¬ 
 jective sense, as a sensation produced on the retina of the 
 eye by ether waves of certain lengths; in an objective 
 sense, it is a form of energy causing wave movements in 
 the ether. 
 
 It is believed that the particles of ether producing these 
 movements vibrate in all directions transversely to the 
 direction in which the light travels, and that the vibra¬ 
 tions are sometimes in straight lines, sometimes in ellipses 
 or circles, the particular kind of vibration depending upon 
 the particular vibration of the molecules of the source of 
 the light, or upon the influence of the molecular structure 
 of the bodies through which the light is transmitted on 
 the original wave movement. 
 
 Any body heated to such a degree that its molecules 
 cause waves in the ether of certain fixed lengths becomes 
 a source of light. Such bodies are said to be incandescent; 
 the sun and stars are examples. Their heat, which depends 
 on the rate of vibration of the molecules, was caused, 
 when these bodies were formed, by the change of the 
 kinetic energy of the molecules of the masses which 
 clashed together into heat energy, and is maintained by 
 some unknown means. It would be out of place here to 
 discuss the different theories which have been proposed 
 to explain how the heat of these bodies is kept up. In 
 the case of ordinary artificial lights, as a gas light, a lamp 
 flame, or an electric light, molecules of carbon made in¬ 
 candescent by chemical or electrical action are the source 
 of the vibratory motions which are transmitted to the eye 
 by the ether. 
 
 If a beam of light is allowed to enter a darkened room 
 through a small aperture, its path through the room is 
 
LIGHT AND THE MICEOSCOPE. 
 
 3 
 
 made visible by the light reflected from the particles of 
 dust in the atmosphere. The path is seen to be straight. 
 The path of light in any homogeneous medium is straight, 
 and its velocity uniform. If, however, the beam of light 
 is made to pass from one medium to another of different 
 density in any other direction than at a right angle to 
 the surface bounding the two media, its path will be bent 
 at the bounding surface, and its velocity diminished or 
 increased according as the second medium is more or less 
 dense than the first medium. 
 
 Refraction of Light. — If we place a triangular prism of 
 any transparent substance in the path of a beam of light 
 entering a darkened room, it is easy to see that the beam 
 
 is bent at the surface of the prism both on entering and on 
 leaving it, unless the light strikes the surface of the prism 
 at right angles. Figure 1 shows the path of such a beam 
 of light. The ray of light AB strikes the surface EG of 
 a glass prism at an oblique angle. It is bent or refracted 
 at the surface EG- towards the base of the prism, passes in 
 a straight line through the glass to the surface GF, and is 
 refracted again towards the base of the prism. 
 
 Figure 2 shows Professor Tyndall’s way of making clear 
 why the ray is bent. As a wave front approaches the 
 
4 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 surface GH of the prism, one end C of the wave reaches 
 the denser medium while the end B is still in the rarer 
 medium. In the denser medium the rate of motion is less 
 than in the rarer medium. The end C moves at a slower 
 rate than the end B until B reaches the glass. A change 
 of direction is the necessary result. When the wave front 
 reaches the surface HM\ the end D of the wave reaches 
 the rarer medium while the end E is still in the denser 
 medium. The end I) moves more rapidly than the end E, 
 until E reaches the air. Another change of direction 
 results at this surface. It is clear that if the ray falls 
 perpendicularly on either surface, it will not be bent, as 
 both ends of the wave strike the surface at the same 
 instant. It is also easy to see that the more obliquely the 
 ray strikes the surface, the greater will be the refraction. 
 These theoretical facts are easily verified by experiment. 
 
 Returning to Fig. 1, the ray, in passing from the air 
 into the glass, is refracted towards the line BH erected 
 perpendicular to the surface bounding the two media at 
 the point B, the point at which the ray enters the prism. 
 The angle ABM is called the angle of incidence for the 
 ray; the angle HBC , the angle of refraction. In passing 
 from the glass into the air, the ray is refracted from the 
 perpendicular ON. In general, a ray of light in passing 
 from a rarer to a denser medium is refracted towards the 
 perpendicular erected on the boundary surface of the two 
 media at the point of incidence of the ray ; and in passing 
 from a denser to a rarer medium, the ray is refracted away 
 from the perpendicular. The greater the difference in 
 density of the two media, the greater the amount of the 
 refraction. 
 
 Dispersion of Light. — It is also to be noted that a ray of 
 white light passing through a prism is dispersed or divided 
 
LIGHT AND THE MICROSCOPE. 
 
 5 
 
 into its primary or rainbow colors. This results from the 
 fact that the waves of white light are compound, each 
 wave being the result of the combination of several waves 
 of different lengths. These different waves are differently 
 retarded by the denser medium, the shortest waves, or 
 those which produce the sensation of violet color, being 
 most retarded and as a consequence most refracted; the 
 longest, or those producing the sensation of red, being 
 least retarded and consequently least refracted. The 
 light, then, in coming from the prism exhibits all the 
 colors of the rainbow. 
 
 The dispersive powers of different substances are very 
 different. Flint glass has nearly twice the dispersive 
 power of crown glass. 
 
 This suggested to 
 Dollond, a London 
 optician, a means of 
 effecting refraction 
 without dispersion. 
 
 He combined a crown 
 glass prism AB C with 
 a flint glass prism 
 CDB , Fig. 3, with 
 
 the edges turned in opposite directions. By a careful 
 adjustment of the size of the angle CBD to the angle 
 ACB , the rays which are separated in the prism ABC are 
 caused to combine in the prism BCD. In this way re¬ 
 fracted light which is nearly but not quite achromatic is 
 obtained. This principle has an important application in 
 the construction of achromatic objectives. 
 
 Index of Refraction. — Let AB , Fig. 4, represent the 
 boundary surface between two media, e.g. glass below, air 
 above. CO is a ray of light entering the glass at 0. It 
 
 Fig. 3. 
 
6 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 is refracted towards the perpendicular OR and passes 
 along the line OR. With 0 as a centre and with any 
 
 radius describe a circle, as 
 AEBF. From the point 
 M where the incident 
 ray cuts the circumference 
 draw MH perpendicular to 
 EF. The angle COE is 
 the angle of incidence, and 
 MH is the trigonometrical 
 sine of the angle of inci¬ 
 dence for the ray CO. 
 From the point P where 
 the refracted ray cuts the 
 circumference, draw PR 
 perpendicular to EF. 
 P OF is the angle of refraction, and PR the sine of the 
 angle of refraction for the ray CO. 
 
 Notwithstanding that it is true that the refraction of 
 the ray increases as the angle of incidence increases, it has 
 been found by experimental demonstration that, as long 
 as the two media remain the same, the ratio of the sine of 
 the angle of incidence to the sine of the angle of refraction 
 
 is constant. This ratio yyyf is called the Index of Refrac- 
 
 JL it 
 
 tion. It is usually expressed decimally. If the ray is 
 passing from a rarer to a denser medium, the index of 
 refraction is greater than unity; if the ray is passing from 
 a denser to a rarer medium, the index of refraction is less 
 than unity. 
 
 The index of refraction for a ray of light passing from 
 a vacuum into any medium is called the absolute index of 
 refraction for that medium. The absolute indices of vari- 
 
 Fig. 4. 
 
LIGHT AND THE MICROSCOPE. 
 
 7 
 
 ous substances have been experimentally determined. I he 
 following table, compiled from several authentic sources, 
 gives the absolute indices of refraction for some of the 
 media that are of importance to the microscopist: — 
 
 Absolute Indices of Refraction. 
 
 Air. 
 
 . 1.000294 
 
 Plate glass (glass slips 
 
 
 Pure water . . . 
 
 . 1.333 
 
 and cover-glasses) . 
 
 1.53 
 
 Canada balsam . . 
 
 . 1.54 
 
 Homogeneous immer- 
 
 
 Oil of cloves . • • 
 
 . 1.53 
 
 sion fluid .... 
 
 1.51 
 
 Oil of turpentine . 
 
 . 1.478 
 
 Iceland spar, ordinary 
 
 1.654 
 
 Oil of cedar . . . 
 
 . 1.51 
 
 ray. 
 
 Crown glass . . . 
 
 . 1.51 
 
 Iceland spar, extraor- 
 
 1.654-1.483 
 
 Flint glass . . . 
 
 . 1.62 
 
 dinary ray . . • 
 
 The relative index of refraction for a ray of light pass¬ 
 ing from one medium to another is found by dividing the 
 absolute index of the medium into which it is passing by 
 the absolute index of the medium from which the ray is 
 coming. If the ray is passing from a rarer to a denser 
 medium, this quotient is greater than unity, and the ray is 
 refracted toward a line erected at the point of incidence 
 perpendicular to the surface bounding the two media. If 
 the ray is passing from a denser to a rarer medium, the 
 quotient is less than unity, and the ray is refracted away 
 from the perpendicular. As the index of refraction shows 
 at a glance which way the ray is bent, and whether it is 
 bent much or little, it is a very important datum. An 
 understanding of the index of refraction is of great prac¬ 
 tical importance in studying immersion objectives and the 
 effects of the cover-glass. 
 
 Total Reflection. — Intimately connected with refraction 
 is total reflection, which, because of its practical bearing 
 in the construction of the polarizing apparatus used on the 
 
8 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 microscope, requires attention here. Place the eye on a 
 level with a gas or lamp flame and raise a glass of water 
 steadily above this level between the eye and the flame. 
 When the water is held in a particular position, the flame 
 is seen reflected as from a mirror from the under part of 
 
 the upper surface of the water. 
 Fig. 5 shows the reason. Let 
 AB be the surface of the water. 
 The ray of light EO in passing 
 from the water into air is re¬ 
 fracted away from the perpen¬ 
 dicular CO along the line OF. 
 The ray GO is refracted along 
 OH, the angles of refraction 
 increasing more rapidly than the 
 angles of incidence. If we con¬ 
 tinue to take the successive 
 rays, a ray will be found, as 
 B'O, which strikes the surface AB at such an angle that 
 the angle of refraction COB is a right angle. The re¬ 
 fracted ray just grazes the surface of the water, passing 
 along the line OB. All rays between B' 0 and AO cannot 
 pass out of the water, but are totally reflected from the 
 surface. The angle of incidence B' OB is called the critical 
 or limiting angle. When the light strikes the surface of 
 a transparent medium at an angle greater than the critical 
 angle, this surface is a perfect mirror. The critical angle 
 for water is about 48° 30'. It is different for different sub¬ 
 stances. For the diamond it is 23° 41'. The brilliancy of 
 the diamond and other gems is due to repeated total 
 reflections of the light within the gem. 
 
 The Double-Convex Lens.—Fig. 6 represents two prisms 
 placed base to base and the paths of rays of light from the 
 
LIGHT AND THE MICROSCOPE. 
 
 9 
 
 point F. It is seen that the rays which diverge from 
 the point F converge, after passing through the prism, to 
 
 H 
 
 the point A. This arrangement of prisms represents very 
 well the principle of the double-convex lens. 
 
 The surfaces of a double-convex lens may he considered 
 as made up of an infinite number of small plane surfaces. 
 Each ray of light coming from a given point, except the 
 
 Fig. 7. 
 
 one passing along the optic axis BA , Fig. 7, is refracted 
 towards the optic axis both on entering and on leaving 
 the lens. The result is that rays coming from the point 
 B are refracted to the point A. 
 
10 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 Spherical Aberration. — The surfaces of convex lenses 
 are usually portions of spheres. As the incident rays 
 approach the edge of the lens, the angles of incidence 
 increase very rapidly, and the refraction of the rays is, of 
 course, correspondingly increased. The result is that 
 those rays which pass through the lens near its edge, as 
 BF and BK , Fig. 7, are brought to a focus sooner than 
 the rays which pass through the central portion of the 
 lens. There results a blurring of the image. This defect 
 of convex lenses is remedied by interposing a diaphragm, 
 a piece of opaque substance with a hole through it of a 
 diameter somewhat smaller than the diameter of the lens, 
 to cut off these objectionable rays. Diaphragms are used 
 in all the optical parts of the microscope, as the eye-piece, 
 objective, condenser. 
 
 Dispersion in Lenses. — Another defect in convex lenses 
 of homogeneous material is dispersion of the light, whereby 
 color effects due to the separation of white light into 
 its primary colors are produced. From the shape of the 
 lens this separation is not so great as in the case of the 
 prism, yet there is enough to give circles of prismatic 
 colors around the edge of the field of view. This defect 
 is remedied by combining, on the principle of Dollond, 
 convex and concave lenses of crown and flint glass so 
 adjusted that the dispersion of the one is corrected by 
 the opposite dispersion of the other, in a manner similar 
 to that already explained under prisms. The correction 
 can, however, be perfectly made for only two colors at 
 a time, so that lenses entirely free from dispersion cannot 
 be made. The skill of the opticians has produced lenses 
 which would seem to be as free from this defect as human 
 ingenuity can make them. Fig. 8 shows such a compound 
 lens. Ordinarily the convex lens is of crown glass and 
 
LIGHT AND THE MICIiOSCOPE. 
 
 11 
 
 Fig. 8. 
 
 the plano-concave lens of flint glass, hut for some purposes 
 the order is reversed. These lenses are called achromatic, 
 and are used in the construction of all optical 
 instruments of high grade. Apochromatic ob¬ 
 jectives are a modern triumph of the opticians 
 in which the correction is practically made for 
 three colors. 
 
 The Simple Microscope. — Every double-convex 
 lens is a simple microscope. The manner in which 
 it acts is illustrated in Fig. 9. A ray of light com¬ 
 ing from the point A. on the arrow AB, placed 
 just within the principal focus of the lens, is refracted on 
 entering the lens and again on leaving it, passing in the 
 line HK to the eye. The eye looking 
 along the line KHA' seems to see the 
 point A at A', the position being deter¬ 
 mined by the intersection of the line 
 KA' and the secondary axis PA'-, simi¬ 
 larly the point B appears at B'. In 
 like manner we could draw lines to 
 show that all the rays coming from all 
 the points of AB would be so refracted 
 that they would appear to come from 
 points on A'B'. We thus see an en¬ 
 larged image of the arrow AB in the 
 position A'B'. It is to be noted that 
 the image is not inverted. 
 
 A simple microscope consists, then, 
 of one or more convex lenses mounted 
 in such a way as to be convenient to 
 use for the purpose for which the micro¬ 
 scope Avas designed. Any lenses of the 
 convex series — double-convex, piano- Fig 9 
 
 A 
 
12 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 Fig. 10. 
 
 Fig. 11. 
 
 convex, or concavo-convex — may be so employed. The 
 manner of action is precisely the same in all, but is 
 
 less in amount in the plano¬ 
 convex and concavo-convex 
 lenses than in the double-con¬ 
 vex. Figs. 10, 11, and 12 show 
 different manners of mounting 
 simple microscopes. 
 
 Fig. 12. 
 
LIGHT AND THE MICROSCOPE. 
 
 13 
 
 The Compound Microscope. — The simplest form of com¬ 
 pound microscope consists of two convex lenses: one 
 small and placed near the object; the other larger and 
 placed in such a position that the image formed by the 
 small lens is in its focus. The small lens is called an 
 
 Fig. 13. Fig. 14. 
 
 objective from the fact that it is placed near the object; 
 the larger lens, an eye-piece or ocular, from the fact that 
 the eye is placed near it to make observation. Fig. 13 
 shows how the objective forms a real, inverted image of 
 the object AB at A'B'. If a screen of oiled paper or 
 delicately ground glass is placed here, the inverted image 
 
14 
 
 PRACTICAL METHODS IN MICROSCOPY, 
 
 of the object will be seen upon it. The eye-piece acts 
 as a simple microscope to magnify this image. 
 
 In practice, an objective consists of two, three, or four 
 achromatic convex lenses placed near together. The lens 
 nearest the object is called the front lens or front com¬ 
 bination; the one farthest from the object is called the 
 back combination. These different lenses act together 
 to magnify the object. Two or more lenses of slight 
 curvature are used rather than one of considerable cur¬ 
 vature, as it is found that spherical aberration and disper¬ 
 sion are thus reduced to a minimum. 
 
 The eye-piece sometimes consists of a single convex 
 lens. It is then known as a posi¬ 
 tive eye-piece and is really a sim¬ 
 ple microscope, as LM in Fig. 13. 
 More commonly a Huyghenian 
 eye-piece is used. This consists 
 of two convex lenses. The one 
 nearest the eye is called the eye- 
 lens ; the other, the field-lens. 
 The field-lens acts precisely as 
 one of the lenses of an objective, 
 and is regarded by many as con¬ 
 stituting a part of the objective. 
 The rays of light from the object, 
 after passing through the objec¬ 
 tive, pass through the field-lens 
 of the eye-piece before coming to a focus. Refracted by 
 the field-lens they form the inverted image of the object 
 between the field-lens and the eye-lens, as A'B\ Fig. 14. 
 If the eye-lens is removed and a piece of oiled paper 
 lowered into the eye-piece, an image of the object will be 
 seen on it when it is carefully held transversely in the 
 
 Fig. 15. 
 
LIGHT AND THE MICROSCOPE. 
 
 15 
 
 tube at the proper distance from the field-lens. The dia¬ 
 phragm of the eye-piece is in the plane where the image 
 is formed. When an eye-piece micrometer is used, it is 
 placed in this position. The eye-lens acts as a simple 
 microscope and magnifies this image. It is thus seen 
 that with the compound microscope the image appears 
 inverted whether the positive eye-piece or the negative 
 Huyghenian eye-piece is used. 
 
 The compound microscope is then an application of con¬ 
 vex lenses. The stand with all its various parts is only 
 a convenient means to render the use of the lenses easy 
 and bring out their full power. In Fig. 16, A is the 
 objective, B the eye-piece, C the tube or body, I) the 
 draw-tube, E the nose-piece which adapts the objective 
 to the body, F the stage, Gr the sub-stage which receives 
 various accessories, II the mirror, K the mirror-bar, J the 
 base, L the flexible pillar, 0 the arm, M the coarse 
 adjustment by rack and pinion, N the fine adjustment. 
 On the stage is seen a microscope slide, three inches by 
 one in dimensions, held in position by spring clips. 
 
 Selection of a Microscope. — Much has been written in 
 regard to the selection of a microscope, but to very little 
 purpose. It is impossible for one person to give another 
 very definite practical advice on such a matter. Tastes, 
 purses, and the object for which the instrument is to be 
 used differ. A few facts should, however, be borne in 
 mind. Very cheap microscopes are useless for practical 
 purposes. It is poor economy for a beginner to suppose 
 that a cheap stand is good enough while learning to use 
 the microscope. It is good economy, although the first 
 outlay will he somewhat greater, to purchase a stand of 
 thorough workmanship, of sufficient steadiness to bear any 
 objective, and of such construction as to receive any of 
 
16 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 the sub-stage accessories. Such a stand admits of the 
 highest character of work and of unlimited development 
 in microscopical skill on the part of the possessor. It 
 must, of course, be used with especial care by the beginner, 
 hut the exercise of this care is the very best means to 
 educate one to a careful and painstaking system of work¬ 
 ing, the secret of success in microscopical manipulation. 
 The prospective purchaser will, of course, consult his 
 microscopical friends and learn what he can about their 
 instruments and receive the benefit of their experience. 
 He will also obtain the catalogues of several of the most 
 reputable makers of microscopes, compare the instruments 
 and prices, and will purchase only of makers of established 
 reputation, who place their names on their apparatus as 
 a guarantee that it is what it is represented to be. 
 
 For a beginning, the stand with two e} r e-pieces, A and C 
 (the latter fitted with an eye-piece micrometer), and two 
 objectives, a three-fourths or two-thirds inch and a one- 
 fifth or one-sixth inch, is ample equipment. As knowledge 
 of the instrument and experience in its use are acquired, 
 other parts may be added. An error that is commonly 
 made in first purchases is to obtain a multiplicity of acces¬ 
 sories that are afterwards found to be useless. It is much 
 the wiser course to purchase accessories as they become 
 actual necessities. 
 
 Care of the Microscope. — The microscope should be kept 
 scrupulously clean in all its parts. The metal portions 
 may be wiped along the grain of the finish with a piece of 
 old, soft linen. The draw-tube should be thus wiped occa¬ 
 sionally. No alcohol or other fluid which dissolves resinous 
 substances should be allowed to come in contact with the 
 metal, or the lacquer finish will be dissolved. 
 
 Dust may be removed from the lenses with a piece of 
 
18 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 old, soft linen or a camel’s-hair brush. One or two folds of 
 the linen over the end of a stick of pine wood, previously 
 chewed until it is quite soft, serves well to reach the 
 smaller lenses. Professor Gage of Cornell University 
 recommends very highly for cleaning lenses the bibulous 
 Japanese filter paper much used by dentists. Do not 
 touch the lenses with the bare fingers. After wiping, 
 breathe upon the lens. If it is optically clean, the moist¬ 
 ure will disappear almost instantaneously. If it is not 
 clean, the moisture will persist. 
 
 To remove the objective from the stand, lightly grasp 
 the lower part, without touching the glass itself, with the 
 thumb and forefinger of one hand to guard against drop¬ 
 ping the objective. Place the thumb and forefinger of the 
 other hand on the milled rim at the upper part of the 
 objective and unscrew it. In replacing the objective on 
 the stand, use the same precautions. The lenses of the 
 objective should not be separated unless one has had con¬ 
 siderable experience with optical apparatus, and in the case 
 of the higher power objectives it is perhaps best to send 
 them to the makers of the instrument if they need to be 
 taken apart for any reason. It will seldom occur that this 
 is necessary. 
 
 In the course of use the front lens of the objective is 
 liable accidentally to come in contact with the reagent 
 used in the study of the specimen. It should be wiped 
 immediately. If it has touched Canada balsam or other 
 resinous substances, it may be wiped with a piece of linen 
 moistened with alcohol, taking great care that the alcohol 
 does not come in contact with the brass mounting of the 
 objective. After using the alcohol, wipe immediately with 
 dry, soft linen. 
 
 Specks in the field of view are most commonly due to 
 
LIGHT AND THE MICROSCOPE. 
 
 19 
 
 dust on the field-lens of the eye-piece. Dust is liable to 
 collect on both the field-lens and the eye-lens. Either 
 lens may be unscrewed from its mounting and wiped, 
 with the precautions already mentioned, which apply 
 to the cleaning of any of the optical parts of the in¬ 
 strument. 
 
 To determine when the dust has been entirely removed, 
 place the eye-piece in the tube, focus sharply on a well- 
 lighted field, and then slowly revolve the eye-piece. If 
 there are any particles of dust on either of its lenses, they 
 will revolve, while dust specks on the slide, mirror, or 
 objective remain stationary. To ascertain if the dust is 
 on the slide or mirror, move first one, then the other. 
 The dust will move with the slide or mirror, if there is 
 any on either. If, after these manipulations of the eye¬ 
 piece, slide, and mirror, there are still dark patches which 
 have not moved, there is dirt on the objective. 
 
 The Glass Slip. — Objects to be examined under the 
 microscope are placed upon glass slips three inches long 
 and one inch wide. These should be purchased ready for 
 use. Only those with ground edges should be used, and 
 it is economy in satisfaction, if not in money, to buy a 
 good quality. Recent improvements in the processes of 
 manufacture, and the increased demand arising from the 
 great advance in the popular use of the microscope in the 
 past few years, have caused large reductions in the prices 
 of the slips, so that the purchase of the best is now no 
 great burden. Slips with unground edges are very liable 
 to leave serious scratches on the stage of the microscope. 
 Slides made from such slips are unsightly and lessen the 
 ambition to produce perfect work. It is a waste of time, 
 which can better be given to something else, to attempt to 
 grind one’s own slips. 
 
20 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 The Cover-Glass. — If the object is to be examined in 
 any fluid medium, it must be covered with a piece of thin 
 glass, known as the cover-glass. This is used in part to 
 protect the objective, in part to flatten the object, but 
 more particularly to prevent the refraction of the light 
 which would occur at the curved surface of an uncovered 
 drop of fluid. This refraction of the light would cause 
 a very distorted image of the object to be formed. The 
 curved surface of the drop would act precisely as the 
 curved surface of a lens. The cover-glass itself exerts 
 a very great influence on the light, but this influence is 
 far less objectionable than that of a distorted drop of 
 water. 
 
 The thickness of the cover-glass is also a matter of 
 importance. A study of Fig. 17 will make plain how the 
 
 thickness of the cover-glass affects 
 the clearness of the image. The 
 figure is drawn for a dry mount. 
 Let A represent a point on an 
 object, and EF the cover-glass. 
 A ray of light, AB , from the 
 object, on striking the lower side 
 of the cover-glass, is passing from 
 a medium, air, whose refractive 
 index is 1.000294, into another 
 medium, plate glass, whose refractive index is 1.53. The 
 relative index of refraction is 1.52. The ray is therefore 
 refracted towards the perpendicular MN erected on the 
 lower surface of the cover-glass at the point where the ray 
 strikes. On passing from the cover-glass into air, the ray 
 is passing from a medium whose refractive index is 1.53, 
 into a medium whose refractive index is 1.000294. The 
 relative index of refraction in this case is .65. The ray is 
 
 Fig. 17. 
 
LIGHT AND THE MICROSCOPE. 
 
 21 
 
 now refracted away from the perpendicular, and as the 
 conditions in the second refraction are the exact reverse 
 of the conditions in the first refraction, its course after 
 leaving the upper surface of the cover-glass is parallel 
 with its course before entering the cover-glass. The ray 
 has suffered displacement. A'B shows the direction from 
 which it appears to come, and the point where the line 
 A'B crosses the optic axis of the microscope, DA , shows 
 the position in which A appears to the observer in so far 
 as this particular ray helps to form its image. It is 
 apparent from an inspection of the figure that the greater 
 the thickness of the cover-glass, the greater the displace¬ 
 ment of the ray. It is also to be remembered that this 
 displacement is magnified when observed through the 
 microscope. The line A"C shows the apparent direction 
 of the ray A 0. Should we, in like manner, construct 
 lines for other rays coming from the point A and trace the 
 directions back, we should see that all are displaced in 
 varying degrees. The result is, of course, the blurring 
 of the image. 
 
 This defect of the cover-glass can to a considerable 
 extent be remedied in the construction of the objective. 
 Except in the case of collar adjustment objectives, which 
 will be considered later, it must be done for a particular 
 thickness of cover-glass. It is accomplished by giving 
 the lenses such shapes, and placing them at such distances 
 from one another, that the rays which have been displaced 
 are refracted back, so that they appear to come from their 
 real source, the point A in Fig. 17. Microscope makers 
 correct their non-adjustable objectives for the particular 
 thickness of cover-glass and for the particular length of 
 tube which they have severally adopted. It is unfortunate 
 that the makers have not agreed upon the same thickness 
 
22 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 of cover and the same tube length. The tube length 
 adopted by opticians of high reputation varies from 125 to 
 254 millimetres. The thickness of covers adopted varies 
 from one-tenth to twenty-five- hundredths of a millimetre. 
 If an objective corrected for a cover one-tenth of a 
 millimetre thick be used on one twenty-five hundredths 
 of a millimetre thick, the image will be blurred, and vice 
 versa. If it is necessary to use cover-glasses that are too 
 thick or too thin, the defect can to a considerable extent 
 he remedied by using a greater length of the draw-tube 
 when the cover is thinner than the standard thickness for 
 which the objective was constructed, and by shortening 
 the draw-tube, if the stand is constructed so as to allow it 
 to be done, when the cover is too thick. In examining 
 
 O 
 
 permanent mounts, when the thickness of the cover-glass 
 is not known and cannot be measured, the proper length 
 of the draw-tube should be found by trial. Focus sharply 
 with the tube of the length for which the objective was 
 constructed. Then lengthen or shorten it until the 
 clearest image possible is obtained. Such intelligent use 
 of the draw-tube aids greatly in securing a clear image 
 and in bringing out the highest capabilities of the ob¬ 
 jective. The foregoing considerations show that it is 
 very important to know the thickness of the cover-glass. 
 
 Cover-Glass Gauge. — Several devices have been con¬ 
 trived for measuring the thickness of the cover-glass. 
 A very commendable form has recently been placed upon 
 the market at a reasonable price by the Bausch and Tomb 
 Optical Company. It is shown in Fig. 18. The cover- 
 glass is placed in the slot provided for it, and the 
 micrometer screw turned until the end touches the cover- 
 glass. The thickness in fractions of a millimetre or of an 
 inch is then read directly from the drum. The drum also 
 
LIGHT AND THE MICROSCOPE, 
 
 23 
 
 shows directly the correct tube length to use for any 
 thickness of cover, when objectives of one-fourth, one-fifth, 
 one-eighth, and one-twelfth inch focus, coirected foi 
 
 Fig. 18, 
 
 cover-glasses .16 of a millimetre thick, and for a tube 
 length of eight and one-half inches, are used. It is a 
 very convenient and useful device. 
 
CHAPTER II. 
 
 SOME ACCESSORIES FOR ADVANCED WORK. 
 
 Immersion Objectives. — For the more advanced histolog¬ 
 ical, and especially for bacteriological work, high power 
 immersion objectives are required. These may be divided 
 into two classes, those with which water is used as the im¬ 
 mersion fluid, and those with which an oil is used, usually 
 cedar oil, or some fluid especially prepared by the opticians, 
 and having a refractive index about the same as the refrac¬ 
 tive index of the front lens of the objective. This fluid is 
 called homogeneous immersion fluid, as it is homog’eneous 
 as respects its refracting and dispersing power with the 
 glass of the front lens. The objective is called a homo¬ 
 geneous immersion objective. Objectives used with water 
 are called water immersion objectives. 
 
 In using an immersion objective, a small drop of the 
 fluid is placed on the front of the front lens or on the 
 cover-glass, and the objective carefully lowered until 
 the fluid f 01 ms a connection between the objective and the 
 covei -glass. The objective is then focussed by means of 
 the fine adjustment, and, if the objective is adjustable, 
 by the collai adjustment. Fhe principle upon which an 
 immersion lens works and its superiority over a dry 
 objective will be apparent from a study of Fig. 19. 
 
 A ray of light starting from the point A of the object 
 on the slide, passes from the balsam to the upper surface 
 of the cover-glass with only a slight change of direction 
 at the lower surface of the cover-glass; for, as will be seen 
 
 24 
 
SOME ACCESSORIES FOR ADVANCED WORK. 
 
 25 
 
 by inspecting the table of Indices of Refraction given in 
 Chapter I., it is passing from a medium, Canada balsam, 
 whose refractive index is 1.54, into another medium, the 
 cover-glass (plate glass), whose index of refraction is 1.53, 
 or from a denser to a slightly rarer medium. The relative 
 index of refraction is .993, which shows that the ray is 
 slightly bent away from the perpendicular. 
 
 If now a dry objective is used, the ray, in passing from 
 the upper surface of the cover-glass into the air, is passing 
 from a medium whose index of refraction is 1.53 into a 
 medium whose index of refraction is 1.000294. The rela- 
 
 o 
 
 tive index of refraction is .653, which shows that the ray 
 is much bent away from the line CTT, erected at the point 
 where the ray strikes the upper surface of the cover-glass 
 perpendicular to that surface. This perpendicular is, of 
 course, parallel to the optic axis of the microscope. The 
 ray takes some such direction as CP and fails to reach and 
 enter the front combination of the objective. 
 
 If, instead of the dry objective, a water immersion 
 objective is used, the ray, in passing from the cover-glass 
 into water, is passing from a medium whose refractive 
 index is 1.53 into a medium whose refractive index is 
 1.333. The relative index of refraction in this case is .871. 
 
26 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 This shows that the ray is still refracted away from the 
 perpendicular to a considerable extent, but by no means to 
 so great an extent as when the dry objective was used. 
 The ray takes some such path as CH, and also fails to 
 reach and enter the front lens of the objective; but it is 
 evident that many more rays will enter a water immersion 
 objective than can enter a dry objective of the same 
 angular aperture and working distance. 
 
 If we use a homogeneous immersion objective, the ray, 
 in passing from the cover-glass into the homogeneous 
 immersion fluid, is passing from a medium whose refractive 
 index is 1.53 into a medium whose refractive index is 
 1.51. The relative index of refraction is .987. The ray 
 is but slightly refracted away from the perpendicular. 
 It takes some such path as CL , reaches and enters the 
 front lens of the objective without refraction, as the re¬ 
 fractive index of the front lens (crown glass) is the 
 same as the refractive index of the homogeneous immersion 
 fluid. It is evident that the homogeneous immersion 
 objective receives a much larger number of rays from the 
 point A than does a water immersion objective of the 
 same angular aperture and working distance. 
 
 The importance of causing the largest possible number 
 of rays from the object to pass through the objective is 
 manifest, when we consider that the perfection of the 
 image depends on the number of rays that help to form it. 
 The gain in light is also of great consequence, for, with 
 high-power objectives having three and four combination- 
 lenses, the absorption of light is considerable. 
 
 Adjustable Objectives. — To remedy the evil effects of 
 changes in the thickness of the cover-glass, and to 
 compensate for variations in the refractive index of dif¬ 
 ferent cover-glasses, and for changes in the tube length, 
 
SOME ACCESSORIES FOR ADVANCED WORK. 27 
 
 which result when different eye-pieces are used, high- 
 power objectives, both dry and immersion, are made with 
 means of adjusting the distance between the front and 
 back combinations of the objective. Theoretically, homo¬ 
 geneous immersion objectives do not require to be made 
 adjustable, and many of them are not so made; but 
 because the conditions for which they are made can 
 seldom be perfectly met, because of the variation in the 
 refractive indices of different cover-glasses, and because 
 the immersion fluid and other factors which influence 
 the working of the objective are liable to vary, the 
 highest grade homogeneous immersion objectives are 
 made adjustable: water immersion objectives are always 
 made adjustable. 
 
 An objective is made adjustable by mounting either the 
 back or the front combination so as to be moved up or 
 down by means of a milled collar. This collar is graduated 
 arbitrarily, and when the adjustment is at the medium 
 point, the objective is corrected for the standard thickness 
 of cover-glass, and the standard length of tube used for 
 all objectives by the maker. When the adjustment is 
 at the lowest point, the front and back combinations 
 are separated and adjusted for the thinnest cover-glasses. 
 When the adjustment is at the highest point, the lenses 
 are brought nearest together and adjusted for thick covers. 
 In some objectives the front combination is movable; in 
 others, the back combination. The latter arrangement is 
 the one now in common use, as the objective is much 
 more easily handled. 
 
 The manipulation of adjustable objectives calls for the 
 highest skill of the microscopist. Only practice gives 
 proficiency in handling them. General directions can be 
 given, but much must be left to the patient perseverance 
 
28 
 
 PRACTICAL METHODS IH MICROSCOPY. 
 
 of the worker. Some useful hints on their manipulation 
 are given in Chapter IV. 
 
 Angular and Numerical Aperture. — Since the perfection 
 of the image depends upon the number of rays which 
 help to form it, the efficiency of an objective depends 
 upon the number of rays it can transmit. Dry objectives 
 were long compared with one another by comparing the 
 angles formed by the most divergent rays which can 
 pass through the objective from a point on the object 
 situated on the optic axis of the microscope. These 
 angles are called angles of aperture. In a similar way, 
 water immersion objectives were compared with one 
 another, and also homogeneous immersion objectives were 
 compared among themselves. There was even confusion, 
 and objectives of one kind were compared with those 
 of another kind by the angular apertures. Even when 
 objectives of the same kind are compared, only rude ideas 
 of the relative efficiencies are obtained. The efficiencies 
 are really to one another as the sines of one-half the 
 angles of aperture. 
 
 If, however, we wish to compare dry, water immersion, 
 and homogeneous immersion objectives with one another, 
 we must find some means of comparing the number of 
 rays which can pass through these different kinds of 
 objectives. Professor Abbe of Jena first demonstrated 
 the fact that the relative efficiencies of objectives could 
 be expressed by the numbers obtained by multiplying 
 the sine of one-half the angle of aperture by the index 
 of refraction of the immersion fluid employed. The 
 result is called the numerical aperture. It is calculated 
 by the formula N.A. = wsinw, where N.A. stands for 
 numerical aperture, n for the index of refraction of the 
 immersion fluid employed, if the objective is an immersion 
 
SOME ACCESSORIES FOR ADVANCED WORK. 
 
 29 
 
 objective, or of air, if the objective is used dry, and sin u, 
 for the sine of one-half of the angle of aperture of the 
 objective. 
 
 If, for example, we wish to compare three objectives 
 each 1-in. focus, the first, dry, angular aperture 135°; 
 the second, water immersion, angular aperture 170°; 
 the third, homogeneous immersion, angular aperture 138°; 
 they do not compare as the angles 135°, 170°, and 138°, 
 but obtaining the sines of one-half of each of these angles 
 from a table of natural sines, and making the substitutions 
 in the above formula, we have for the dry objective, N.A. 
 = 1.000294 x .9239 = .924; for the water immersion 
 objective, N.A. = 1.33 x .9962 = 1.32; for the homo¬ 
 geneous immersion objective, N.A. =1.51 x .9336 = 1.41. 
 The efficiency of these three objectives is then as .924, 
 1.32, and 1.41. 
 
 By a similar application of the formula, it may be seen 
 that objectives of 82° homogeneous immersion, 96° water 
 immersion, and 180° dry, are practically of the same 
 efficiency in transmitting the light from the object. 
 
 Sub-stage Condenser. — On account of the great ab¬ 
 sorption of light by high-power objectives, a sub-stage 
 condenser must be used in the more advanced studies 
 with the microscope. The condenser is a lens or system 
 of lenses which converge the light upon the object. A 
 medium-power objective makes a very good condenser 
 when the sub-stage is so arranged that the objective can 
 be attached with its front lens towards the object, and 
 moved up and down until the object on the slide is in its 
 correct focus; for, like any other lens, the sub-stage 
 condenser, whatever its construction, must be focussed, 
 and upon the accuracy with which this is done depends its 
 usefulness. 
 
30 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 For fine work, however, the condenser must be of 
 special construction. Figs. 20 and 21 show the con¬ 
 struction of different forms of the Abbe condenser as 
 made by the Bausch and Bomb Optical Company. The 
 working distance of these condensers is very short, so 
 they must be placed close up to the slide; and as the 
 thickness of the glass slip is variable, the condenser must 
 be specially focussed for each new slide used. The better 
 classes of sub-stages are provided with rack and pinion to 
 
 Optical part of Condenser 1.20 N. A. Optical part of Condenser 1.42 N. A. 
 
 Fig. 20. * Fig. 21. 
 
 accomplish this. Centring-screws are also provided in 
 the sub-stage to centre the condenser correctly. In 
 centring, a pin-hole cap, provided for the purpose, is 
 placed over the front lens, and the centring-screws 
 adjusted until the opening is in the centre of the field. 
 In using the condenser, it is very important to see that it 
 is correctly centred and correctly focussed. 
 
 These condensers may be converted into immersion 
 condensers by placing a drop of water or of immersion 
 fluid on the front lens, and making contact with the 
 lower side of the slide. 
 
SOME ACCESSORIES FOR ADVANCED WORK. 
 
 31 
 
 
 The Camera Lucida. — The camera lucida is an attach¬ 
 ment to the eye-piece of the microscope, whereby the 
 image may be seen 
 projected upon the 
 surface of a sheet 
 of paper. One of 
 the simplest forms 
 is shown in Fig. 22. 
 
 To use this form, the 
 microscope is in¬ 
 clined. The draw¬ 
 ing surface must be 
 placed parallel with 
 the tube. The im¬ 
 age of the object is 
 then seen without 
 distortion when the 
 eye is placed so that 
 the axial ray from it to the drawing surface is perpendicu¬ 
 lar to the latter. The principal difficulties to be over¬ 
 come arise from the indistinct¬ 
 ness of the pencil point, and 
 from the fact that the image is 
 inverted. 
 
 Fig. 23 shows the principle 
 upon which the Abbe camera 
 lucida is constructed, and Fig. 
 24 shows an improved form. 
 These are the best forms of the 
 camera lucida made. They 
 are designed primarily to be 
 used with the microscope in the upright position, but may 
 also be used with the instrument inclined. To render the 
 
 Fig. 22. 
 
 Fig. 23. 
 
32 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 pencil point more distinct, a prism made up of a wedge of 
 smoked glass and a counterbalancing wedge of clear 
 glass is provided to be mounted in spring clips. By 
 means of this prism a varying thickness of the smoked 
 glass may be placed in the path of the rays which form 
 the image, until the pencil point is distinctly visible. 
 
 The drawing: surface should be shaded from all extraneous 
 
 O 
 
 light. 
 
 The educational value of drawing the objects which are 
 being studied cannot be too highly estimated. In no 
 other way can the structure be so easily and clearly fixed 
 in the mind. Points of structure are thus frequently 
 called to the attention which otherwise would be entirely 
 overlooked. The process is slow and tedious, but 
 measured by the knowledge gained the results are all 
 valuable. 
 
SOME ACCESSORIES FOR ADVANCED WORK. 
 
 33 
 
 Micrometers and Micrometry. — There are two simple 
 ways of measuring the size of an object under the 
 microscope. The first is by means of a stage micrometer. 
 A stage micrometer is simply a scale ruled on glass by a 
 dividing engine. The divisions are usually yygy, tto’ 
 and yq of an inch, or, in the metric system, yy y and yy 
 of a millimetre. The stage micrometer is placed on the 
 stage, and the object placed above it. The size is read 
 directly. Theoretically this is the simplest way to 
 measure an object, but practically there are almost in¬ 
 surmountable difficulties in the way of accurate work. 
 Unless the object is a perfect circle it cannot be made 
 to coincide with the micrometer in more than one direction, 
 and it is probable that it will not coincide even in that 
 one direction, for neither the micrometer nor the object 
 can be conveniently revolved so that the graduations may 
 coincide with the dimension to be measured. It is, then, 
 usually the case that the measurements made with the 
 stage micrometer are crude and lack much in accuracy. 
 
 The second way of measuring is by means of an eye¬ 
 piece micrometer. By this method the object is not 
 directly measured, but the image of it formed in the 
 eye-piece. The eye-piece micrometer is also a scale ruled 
 on glass. The rulings are generally made Jy of a 
 millimetre from one another. The slip of glass is inserted 
 in the eye-piece just above the diaphragm, and in such 
 a position that the scale is exactly in the plane in which 
 the image of the object is formed. As it is the image 
 of the object and not the object itself that is measured, 
 it becomes necessary to know how much the image has 
 been magnified. This differs with objectives of different 
 focal lengths, and also to some extent even with objectives 
 marked as of the same focal length. It is necessary, then, 
 
34 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 to determine this magnification for every objective used. 
 This is done by placing an accurate stage micrometer 
 on the stage, and using it as an object. Observe how 
 many of the divisions of the eye-piece micrometer corre¬ 
 spond to one division of the stage micrometer. Naturally 
 the way to do this is to observe some number of entire 
 divisions of the stage micrometer that correspond to some 
 number of entire divisions of the eye-piece micrometer, 
 and calculate the value of a single division of the eye-piece 
 micrometer from these data. The figures obtained are 
 the valuation of each division of the eye-piece micrometer 
 scale in terms of the divisions on the stage micrometer. 
 These figures must be determined for each objective used. 
 
 These determinations are made by the manufacturers 
 before the microscope leaves the factory, so that practi¬ 
 cally the user of the microscope, in measuring an object, 
 has simply to observe how many of the divisions of the 
 eye-piece micrometer are covered-by the object; divide 
 the result by the number furnished by the manufacturers 
 for the objective used, and multiply the value of one space 
 on the micrometer scale, usually of a millimetre, by the 
 quotient. The draw-tube must be placed at the standard 
 length. 
 
 As the eye-piece can be rotated,*• the micrometer scale 
 may be made to coincide in direction with any dimension 
 of the object, so that the difficulty experienced with the 
 stage micrometer is wholly obviated. 
 
 The unit of measurement adopted by microscopists is 
 the micro-millimetre, or part of a millimetre. It 
 
 is sometimes represented by nnnm; more commonly, and 
 better, by the Greek letter fi. An object which measures 
 .001 of a millimetre in length is said to be 1 /x long; 
 similarly .015 mm. = 15 p,; etc. 
 
SOME ACCESSORIES FOR ADVANCED WORK. 
 
 35 
 
 To measure the magnification of a camera lucida 
 drawing, place a stage micrometer on the stage with the 
 microscope arranged for drawing. Place two dots on 
 the drawing-paper to mark exactly the limits of the 
 image of one division of the micrometer scale. With an 
 accurate rule measure the distance between the dots. If 
 a micrometer, with an English scale, divided in thousandths 
 of an inch is used, and the image of one division measures 
 one-fourth inch, the magnification is 250; similarly, if a 
 micrometer, with a metric scale, divided in hundredths of a 
 millimetre is used, and the image of one division measures 
 one millimetre, the magnification is 100. 
 
 The Dissecting-Microscope. — This accessory is shown in 
 Fig. 12. It is used mainly in the preparation of objects 
 for examination under the compound microscope, or for 
 permanent mounting. As the dissecting-microscope is a 
 simple microscope, the image is not inverted, as is the case 
 with the compound microscope. It is, therefore, easier, 
 especially for a beginner, to work with a specimen under 
 the dissecting-microscope. 
 
 Dissecting-needles are a necessary accompaniment of 
 the instrument. They are easily prepared by thrusting 
 ordinary sewing-needles, eye first, into wooden handles 
 about the size and shape of a penholder. 
 
 In using the dissecting-microscope, the object to be 
 studied is placed on the stage of the microscope in a watch 
 glass, or other shallow dish with a clear glass bottom, or 
 on a wide slip of glass, either in its natural state, or in a 
 drop of water, or in a drop of some other fluid. It may 
 then be conveniently dissected, treated with staining fluids 
 and reagents, or otherwise specially prepared. 
 
 Some workers find the dissecting-microscope a neces¬ 
 sity ; others accustom themselves to the use of the com- 
 
36 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 pound microscope for this work, using a very low power. 
 With practice the difficulties which are at first encountered 
 because of the inversion of the object are overcome, and 
 there is no longer inconvenience. 
 
 The Turn-Table. —If one is to mount many objects, and 
 particularly if he has an ambition to do neat work, a turn¬ 
 table is a necessity. The instrument should be self¬ 
 centring. It is the most convenient means of centring 
 the object in mounting. The glass slip is placed on the 
 
 Fig. 25. 
 
 turn-table, the prepared object placed on the centre, and 
 the mounting medium and cover-glass applied before the 
 slip is removed from the turn-table. The method of using 
 the turn-table in finishing and ornamenting mounts is 
 described in Chapter VI. 
 
 The Section-Cutter. — While, for purposes of studying 
 fresh objects, sufficiently good sections may be made free 
 hand by the method described in Chapter V., if many sec¬ 
 tions of uniform evenness and thickness are required, a 
 section-cutter, or microtome, must be employed. There 
 are many forms of both. Fig. 26 shows a convenient 
 form of section-cutter to be clamped to the table. If the 
 object is of a suitable degree of firmness to be cut with- 
 
SOME ACCESSORIES FOR ADVANCED WORK. 
 
 37 
 
 out special preparation, as, for instance, many young plant 
 stems, it is placed in the jaws of the clamp with its upper 
 end even with the upper surface of the section-cutter. 
 By means of the micrometer screw any desired measured 
 thickness of section may be obtained. The sections are 
 cut with a razor, ground Hat on one side, held squarely on 
 the glass plates on the top of section-cutter, and drawn 
 across the specimen obliquely. In this instrument, one 
 
 Fig. 26. 
 
 complete revolution of the micrometer screw raises the 
 specimen of an inch. The head of the micrometer screw 
 is graduated into ten divisions. If it be turned through 
 one of these divisions, the object is raised ^ J s of an inch. 
 By turning the micrometer screw through a fraction of one 
 of these divisions, sections of greater fineness may be cut. 
 
 The Microtome. — Fig. 27 shows the laboratory micro¬ 
 tome of the Bausch and Bomb Optical Company. The 
 prepared specimen is placed in the jaws of the clamp, 
 
38 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 which may be adjusted at any height and at any angle by 
 means of the adjustments, H and G-. The lever, J, also 
 
 furnishes means of raising and lowering the carriage, F, 
 which carries with it H , Cr, and the clamp. The gradu¬ 
 ated disk, K, is mounted upon a delicately cut screw, 
 
SOME ACCESSORIES FOR ADVANCED WORK. 39 
 
 which, by turning K, may be brought up to support F. 
 The pitch of the screw is of an inch. The disk has 
 eighty divisions in its graduation. By turning the disk 
 through one of these divisions, the clamp carrying the 
 specimen is raised ^Vo an inc ^ 1 - amoimt which 
 
 the disk, K , is turned is indicated to the eye by an index, 
 Z, and to the ear by a piece of spring metal, A, which 
 may be adjusted to give a pronounced click as each of the 
 divisions on the rim of the disk passes it. 
 
 The knife, A , is mounted on a block, (7, which slides 
 the entire length of the instrument. The knife may be 
 placed so as to strike the specimen at any angle. It is 
 usually adjusted to cut the specimen obliquely. 
 
 Methods of preparing hard and soft tissues for cutting 
 with the microtome and the section-cutter are given in 
 subsequent chapters. 
 
 The Water-Bath and Oven. — In the processes of prepara¬ 
 tion of objects for imbedding in paraffin a water-bath is 
 indispensable. A bath may, of course, be improvised. 
 The form represented 
 in Fig. 28 is very 
 convenient. The ves¬ 
 sel is capacious, hold¬ 
 ing a large volume of 
 water. This is a 
 great advantage, as 
 the temperature is 
 under much better 
 control than when a 
 small amount of 
 water is used. The 
 paraffin cup sits in a 
 
 compartment extend- Fig. 28. 
 
40 PRACTICAL METHODS IN MICROSCOPY. 
 
 ing deeply into the water. A thermometer indicates the 
 temperature. The oven is useful in evaporating volatile 
 reagents from a section, as in removing the clove oil from 
 the collodion fixative used in fixing sections to the glass 
 slip; also in hardening balsam mounts and some kinds of 
 finishing cements. The bath is heated over a laboratory 
 burner or an oil stove. A temperature of between 130° 
 and 140° F. is suitable for most purposes for which the 
 bath is used. After this temperature is reached, the flame 
 is turned low, and with a little experience it is not difficult 
 to adjust it so that the temperature will not vary more 
 than three or four degrees in several hours. 
 
CHAPTER III. 
 
 POLARIZED LIGHT AND THE POLARIZER. 
 
 The elasticity of the ether in space is believed to be 
 equal in all directions. The same is true of the ethei in 
 non-crystalline substances and in crystalline substances of 
 the cubical system. The particles of ether are conse¬ 
 quently free to vibrate equally in all directions. In other 
 crystalline substances the elasticity of the ether is modified 
 by the crystalline structure. In some crystals there is 
 one axis or direction about which the molecules are 
 arranged in a uniform manner; such crystals are said to 
 be uniaxial. In other crystals there are two such axes. 
 If a ray of light passes through a crystal along one of the 
 axes, the ether particles are as free to move in one 
 direction as in another; and, except that the light is 
 retarded and to some extent absorbed, which is the case in 
 all dense media, the ray passes through unmodified. If, 
 however, the ray passes through a crystal in any other 
 direction than along an optic axis, the particles of the 
 ether are restricted in their motion in some directions 
 more than in other directions. The result is that the 
 wave movements are mechanically resolved into two sets 
 of vibrations at right angles to each other, and still at 
 right angles to the direction of motion of the ray; but 
 as there is greater freedom of motion in one direction 
 than in the other, the vibrations in this direction are 
 longer than those in the other direction. The effect on 
 the ray is that it is divided into two rays, each caused by 
 vibrations in a single direction. 
 
 41 
 
42 PRACTICAL METHODS IN MICROSCOPY. 
 
 These rays are both refracted; but as the density of the 
 crystal is different in different directions on account of 
 the difference in structure, the rays are refracted to 
 different degrees, so that they leave the crystal as two 
 separate rays. In some crystals one of these rays is 
 lefi acted like an ordinary ray of light, and is therefore 
 called the ordinary ray. The other ray is differently 
 refracted in different positions of the crystal, and is 
 called the extraordinary ray. By the unaided eye these 
 rays cannot be distinguished from ordinary light, but 
 when examined by special means they are found to be 
 quite different. Light when thus separated is said to 
 be polarized. 
 
 The number of substances that exhibit double refraction 
 is very large; in fact, it is the rule for crystalline sub¬ 
 stances other than those of 
 the cubical system. Many 
 organic substances also 
 have such differences of 
 structure in different direc¬ 
 tions that they act in a simi¬ 
 lar way upon light. The 
 substance that exhibits this 
 property in the highest de¬ 
 gree, and the one that is of 
 the most consequence to the 
 microscopist from the fact that the polarizer and analyzer 
 of the microscope are made of it, is Iceland spar, or the 
 crystallized carbonate of lime. This is a uniaxial crystal, 
 the axis joining the blunt angles. If an object is viewed 
 through a crystal of Iceland spar in any other direction 
 than that of the optic axis, it appears double, and if the 
 spar is turned around, one of the images, that formed by 
 
POLARIZED LIGHT AND THE POLARIZER. 
 
 43 
 
 the extraordinary rays, revolves about the other. The 
 ray which is most refracted is the ordinary ray. 1 he 
 index of refraction is 1.654. The index of refraction for 
 the extraordinary ray varies from 1.654 to 1.483. 
 
 For use in optical instruments, it is desirable to obtain 
 one of these polarized rays entirely apart from the other. 
 This is very ingeniously done in the Nicol prism. In the 
 construction of this prism, advantage is 
 taken of the principle of total reflection, 
 and of the fact that the index of refraction 
 of Canada balsam (1.54) is less than the A 
 index of refraction of the ordinary ray, and 
 more than the minimum index of the ex¬ 
 traordinary ray. We have already seen 
 in Chapter I. that when a ray of light, in 
 passing from a dense into a rarer medium, 
 strikes the surface at certain angles, it is 
 totally reflected. A rhombohedron of 
 Iceland spar, Fig. 30, three times as long 
 as thick, is cut by a plane AB, joining the 
 obtuse angles, the ends being cut off so as 
 to form right angles with the dividing 
 plane. The parts, after being polished, 
 are cemented together with Canada bal¬ 
 sam. A ray of light entering the prism from the direction 
 EF is doubly refracted at F. The ordinary ray, FK, 
 meets the surface of the balsam at an angle greater than 
 the critical angle, and is totally reflected along the line 
 KL , and thus thrown out of the field of view. The 
 extraordinary ray, FGr , passes straight through the prism, 
 and after refraction on passing into the air, takes a course 
 parallel to its course EF before entering the Iceland spar. 
 By using the Nicol prism, then, we obtain a beam of 
 
 B 'L 
 
44 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 polarized light, or light caused by vibrations in a single 
 direction at right angles to the direction in which the ray 
 is travelling. 
 
 Two Nicol prisms appropriately mounted constitute the 
 polarizer and analyzer of the microscope. One is mounted 
 to be attached to the sub-stage, and is called the polarizer, 
 Fig. 31. The other, the analyzer, Fig. 32, is screwed into 
 the body of the tube, in the place usually taken by the 
 objective, and the objective is screwed into the lower part 
 of the mounting of the analyzer, so that the analyzer is 
 
 Fig. 31. 
 
 Fig. 32. 
 
 between the objective and the body of the microscope. 
 The analyzer is sometimes mounted in a double com¬ 
 partment box arranged to slide in and out in the body 
 over the objective. In one position of the box the light 
 passes through the analyzer. In the other position the 
 light passes through an empty opening. This is a very 
 convenient way of mounting. The analyzer is also 
 mounted to be placed over the eye-piece in some instru¬ 
 ments. The only conditions that must be fulfilled are 
 that objects to be examined by polarized light must be 
 
POLARIZED LIGHT AND THE POLARIZER. 45 
 
 placed between the polarizer and analyzer, and the Nicols 
 must be so placed that the whole of the light can pass 
 through them. 
 
 Either the polarizer or analyzer is mounted so as to be 
 rotated about the optical axis of the microscope. In the 
 better grades of microscopes, the mounting of the rotating 
 Nicol has a graduated circumference. When the rotating 
 Nicol is in the same position as the fixed Nicol, the axes 
 of the Nicols are said to be parallel. When the rotating 
 Nicol has been turned through an angle of 90°, the axes 
 are said to be crossed. The effects produced are precisely 
 the same whether it be the polarizer or the analyzer that is 
 rotated. For purposes of explanation, it is less confusing 
 to consider that the analyzer is turned. In the following 
 pages, then, the analyzer will be understood to be the 
 rotating Nicol. 
 
 Place the polarizer and analyzer on the microscope, 
 using a low-power objective and eye-piece, and turn the 
 rotating Nicol until the field is well lighted. Observe 
 the reading of the graduated circumference, and turn 
 the Nicol through 90°. The field is now dark. Turn the 
 Nicol through another 90°, and the field is again light. 
 Turn it another 90°, and we have darkness again. Turn 
 it still another 90°, and we have completed the circle and 
 come back to the original position of light. 
 
 We will trace a ray of light in its passage from the 
 mirror through the Nicols. As we have seen, the ray is 
 doubly refracted by the Nicol, the ordinary ray being 
 thrown out of the field of view, and the extraordinary ray, 
 which is caused by vibrations in a single direction at right 
 angles to the direction in which the light is travelling, is 
 alone left to pass on. The vibrations which produce 
 this ray are in a definite plane, one which permits them 
 
46 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 to pass through the Nicol prism in planes lying in a par¬ 
 ticular direction. If the analyzer is in exactly the same 
 position as the polarizer, or if it is turned 180° from the 
 position of the polarizer, these particular planes lie in the 
 same direction in both polarizer and analyzer. The vibra¬ 
 tions can pass, and the field is light. Turn either Nicol 
 from this position, and the particular planes which allow 
 the vibrations to pass do not correspond in direction in 
 the two Nicols. As the rotating Nicol is turned from the 
 position parallel with the other Nicol, the field gradually 
 grows dark. This is due to the fact that the vibrations 
 strike the second Nicol at an acute angle with its planes 
 of vibration, and are mechanically resolved into two sets 
 of rays at right angles to each other. The rays of one 
 set are in the planes of vibration of the second Nicol, and 
 pass, lighting the field to some extent; the rays of the 
 other set are at right angles to the planes of vibration in 
 the second Nicol, and are suppressed. As the angle at 
 which the rays strike the planes of vibration of the ana¬ 
 lyzer increases in size, the resolution of the rays becomes 
 less and less, until, when the analyzer has been turned 90°, 
 the rays strike the planes of vibration of the analyzer at 
 right angles. There is now no resolution of the rays, the 
 light cannot pass at all, and the field is dark. Continue 
 to turn the analyzer. The rays now strike its planes of 
 vibration at an acute angle, and are resolved into two sets. 
 At first the amount of the resolution is small, but it in¬ 
 creases until the analyzer is turned 90° from the position 
 of darkness. The rays now strike the planes of vibration 
 of the analyzer at an angle of 0°, or parallel to these 
 planes. They are wholly transmitted, and the field is 
 light. Note that this is the position 180° from the first 
 position of light. Again continue to turn the analyzer, 
 
POLARIZED LIGHT AND THE POLARIZER. 
 
 47 
 
 and the field gradually grows dark for the same reasons 
 as in the first case. 
 
 Colors in Thin Plates. — Cross the Nicols so as to produce 
 a dark field. Place on the stage of the microscope a thin 
 piece of mica, and slowly revolve it. Four positions of 
 the mica, or two directions at right angles to each other, 
 will he found in which the field remains dark. For all 
 other positions of the mica, the field will be more or less 
 lighted, and if the thickness of the mica plate is right, the 
 light will he colored. The brightest colors are seen when 
 the mica is turned 45° from the position of darkness. 
 
 Mica is a doubly refracting substance. When in the 
 positions of darkness, one set of the planes of vibration 
 by which light can pass is parallel with the planes of 
 the polarizer, and the other set with the planes of vibra¬ 
 tion of the analyzer. It is as though one set of these 
 planes of vibration in the mica were a continuation of the 
 same planes in the polarizer, and the other set a part of 
 the corresponding planes of the analyzer. The light is 
 not further modified, and the field remains dark. 
 
 In any other position of the mica plate, the ray coming 
 from the lower Nicol, striking against the mica, is doubly 
 refracted, and passes out of the mica as two rays in direc¬ 
 tions parallel to each other. As in the case of Iceland 
 spar, one of these rays is more refracted than the other, 
 and must pass through a greater thickness of mica before it 
 emerges. It is consequently more retarded than the other 
 ray during the passage through the dense mica, and comes 
 out a short distance behind the other. If, now, the thick¬ 
 ness of the thin plate of mica is right, the retardation may 
 be of such an amount that if the two rays can be brought 
 together so as to interfere, the waves resulting from the 
 interference will be of the right length to produce the sen- 
 
48 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 sation of color. But observe that the rays are each caused 
 by vibrations in a single direction, and the directions for 
 the two rays are at right angles to each other. There can 
 be no interference unless the rays can be brought into the 
 same plane. This is what actually happens in the ana¬ 
 lyzer. Suppose the mica to be in one of the positions 
 which give the brightest color. The two sets of vibration 
 at right angles to each other, coming from the mica plate, 
 strike the analyzer at an angle of 45° with its planes of 
 vibration. Each of the two rays is mechanically resolved 
 into two sets of vibrations at right angles to each other, 
 one set of vibrations from each of the original rays lying 
 in the planes of vibration of the analyzer, the other set 
 from each lying at right angles to the planes of the ana¬ 
 lyzer. The latter are suppressed; the former pass, and 
 if one is behind the other by half of the wave length of 
 any of the colors composing the white light of which the 
 rays are made up, that color is suppressed through inter¬ 
 ference, and the color of the transmitted rays is comple¬ 
 mentary to the suppressed color. The particular color 
 produced depends on the thickness of the mica plate, for, 
 as is well known, the waves that produce the sensation of 
 red light are nearly twice as long as those that produce 
 the sensation of violet light, and the other colors are pro¬ 
 duced by waves of lengths intermediate between these 
 extremes. Different thicknesses of mica, therefore, cause 
 interference of the waves of different colors, and the 
 transmitted complementary colors are different. If, how¬ 
 ever, the mica is quite thick, the retardation is such as to 
 suppress by interference portions of the several colors of 
 which the white light is composed, so that the effect is 
 simply to reduce the intensity of the white light that 
 is transmitted. 
 
POLARIZED LIGHT AND THE POLARIZER. 
 
 49 
 
 Without changing the positions of the polarizer and 
 analyzer, place pieces of mica of different thicknesses upon 
 the stage, and in each case turn the piece until the bright¬ 
 est color is produced. It is at once seen that, within cer¬ 
 tain limits of thickness, each different piece produces a 
 different color, but that pieces beyond a certain thickness 
 do not cause color. 
 
 Complementary Colors. — Cross the Nicols, place a plate 
 of mica on the stage, and turn it until the field is dark. 
 If the microscope has a graduated revolving stage, the 
 next step is easy; if not, the measurements may be taken 
 with sufficient accuracy with a protractor. Revolve the 
 mica until the field is brightest. It will be found, by 
 accurate observation and measurement, that the mica has 
 been turned through 45°. The two rays coming from the 
 mica now strike the analyzer so that the vibrations of 
 each form an angle of 45° with the planes of the analyzer. 
 As we have before seen, each vibration is mechanically 
 resolved into two at right angles with each other, one set 
 of vibrations from each of the original rays lying at right 
 angles with the' planes of vibration of the analyzer, and so 
 not able to pass, the other set of vibrations from each of 
 the original rays lying in the same planes with the planes 
 of vibration of the analyzer. The latter pairs of vibrations 
 pass, interfere, and produce waves of the right length to 
 cause the sensation of color. Observe what happens 
 when the analyzer is turned through 90° from this position. 
 The pairs of vibrations, which before could pass, now 
 strike the analyzer at right angles to its planes of vibration, 
 while the pairs, which before were suppressed, now lie in 
 the same planes with the planes of vibration of the 
 analyzer, pass, interfere, and cause a color. 
 
 Observe that the sum-total of the vibrations which 
 
50 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 caused the last pairs of rays, plus the total vibrations 
 which caused the former pairs, originally produced white 
 light. We should, therefore, expect that the color pro¬ 
 duced in the last instance, added to the color produced in 
 the first, would produce white light; in other words, that 
 the colors would be complementary. We find that this is 
 the case. 
 
 If we turn the analyzer through another 90°, its planes 
 of vibration lie in the same directions as in the first 
 instance, and the same color is seen. Turn the analyzer 
 through another 90°, and the complementary color must 
 again result. 
 
 The Dark Cross. — Cross the Nicols, and place upon the 
 stage a slide of sulphonal crystals prepared by the method 
 described in Chapter X. The light is more or less 
 restored, and black crosses, the arms at right angles to 
 each other, are seen. Bring the point at which the arms 
 of one of these crosses intersect to the centre of the field, 
 and revolve the specimen on the stage. The cross will be 
 seen to be stationary. It shows the position of the planes 
 of vibration in the Nicols, one arm of the cross lying 
 in the planes of the polarizer, the other in the planes 
 of the analyzer. It appears because there is no polari¬ 
 zation in these two directions, the light being simply 
 absorbed. Other portions of the crystal are colored 
 because there is polarization in all other directions. 
 
 Turn the analyzer 90°, and a white cross takes the place 
 of the black one, the colors of the intervening spaces at 
 the same time changing to their complementaries, so 
 that there is an appearance of a revolving cross changing 
 color as it revolves. There is still no polarization in these 
 two directions, but the light is now transmitted. 
 
 A full explanation of these phenomena would involve a 
 
POLARIZED LIGHT AND THE POLARIZER. 
 
 51 
 
 discussion of elliptical polarization, which would require 
 too much space for a proper presentation here. Suffice it 
 to say that these appearances are similar to the phenomena 
 produced by uniaxial crystals cut perpendicular to their 
 optic axes, and viewed in convergent polarized light. In 
 the crystals of sulphonal the point of intersection of the 
 arms of the cross is a point on the axis of the crystal. 
 From this point the crystallization proceeded laterally in 
 all directions. This may easily be verified by watching 
 the formation of the crystals when the slide is prepared. 
 These crystals are, then, similar in structure to sections of 
 uniaxial crystals cut perpendicular to the principal axis. 
 Such sections of quartz, emerald, mica, and other uniaxial 
 crystals show the cross when viewed in convergent 
 polarized light. Crystals of salicine and other chemical 
 salts which form from centres, as do the sulphonal crystals, 
 on account of the peculiar arrangement of their molecules 
 around the axis, show the cross in plane polarized light. 
 Starches, for a similar reason, show a more or less distorted 
 cross, very characteristic and interesting, radiating from 
 the hilum. 
 
 Uses of Polarized Light. — As has already been seen, the 
 particular color produced by the interposition of a thin 
 film depends upon the thickness of the film. If the film 
 is of varying thickness, or of varying composition, different 
 colors are produced simultaneously. In the case of many 
 chemical crystals the slight differences in thickness give 
 rise to most beautiful combinations of colors, rendered 
 still more striking by the changes to complementary 
 colors as the analyzer is turned. Many crystals that are 
 almost, if not quite, invisible in ordinary light are made 
 plainly visible by using polarized light; in some cases 
 revealing most delicate and beautiful forms. It is hardly 
 
52 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 " possible to use too strong language in describing the 
 fascination felt in watching the process of crystallization 
 under polarized light, or even in examining crystals 
 already formed and permanently mounted. 
 
 In the case of rock sections the different minerals of 
 which the rock is composed affect the light differently, 
 and the different effects produced are important aids in 
 distinguishing and determining the composition of the rock. 
 
 So, too, in the case of many common objects, there are 
 sufficient differences in structure to polarize the light in 
 greater or less degree. The structure of the object is usu¬ 
 ally made much plainer in some positions of the analyzer. 
 Polarized light is, therefore, a valuable means of effect¬ 
 ing special illumination of some classes of objects. It is 
 always well to try polarized light on any very transparent 
 object. The results thus obtained are in many cases 
 equally surprising and interesting. 
 
 A few of the large number of polarizing substances are 
 the following: thin and transparent cellular tissue of 
 plants; plant hairs, which often yield most striking 
 effects; starches; the silicious cuticle of plants ; fibres of 
 cotton, wool, and hemp; crystalline deposits in plant tis¬ 
 sues and sections; muscular tissues of animals; animal 
 hairs; sections of bone, horn, and similar substances; all 
 crystalline substances except those of the cubical system. 
 It would not be possible, nor is it desirable, to give a com¬ 
 plete list. Even when an object does not distinctly polar¬ 
 ize, the modification of the light effected by turning the 
 analyzer aids materially in bringing out the less distinct 
 portions of the structure. 
 
 Substances which are not of the right thickness of 
 themselves to polarize often give good effects when a 
 film of selenite or mica is used with them to increase 
 
POLARIZED LIGHT AND THE POLARIZER. 
 
 53 
 
 the thickness. Such a film should form a part of the 
 polarizing outfit. It may be introduced anywhere between 
 the polarizer and analyzer. A single film is sometimes 
 mounted in the fitting of the polarizer, or several films of 
 different thicknesses are so mounted on the polarizer fit¬ 
 ting that any one may be interposed at will, as is shown in 
 Fig. 31. The film is sometimes mounted in a brass plate 
 to be laid upon the stage. It is then called a selenite 
 stage. Sometimes it is mounted in a small metal plate to 
 be inserted in an opening made for it in the body of the 
 microscope just above the objective, but, of course, below 
 the analyzer. 
 
 A well-selected mica film, mounted in Canada balsam on 
 an ordinary glass slip, and covered with a thin cover-glass 
 to prevent its becoming scratched, serves the purpose well. 
 
 Single-Refracting and Double-Refracting Objects. — It is 
 evident from what has already been said that polarized 
 light affords means of determining whether a substance is 
 singly refractive or doubly refractive. If a singly refrac¬ 
 tive substance, as, for instance, a piece of ordinary glass, 
 or a crystal belonging to the cubical system, as crystals of 
 potassium iodide, potassium bromide, or common salt, is 
 placed upon the stage with the Nicols crossed, the field 
 remains dark, nor are color effects produced when the 
 analyzer is turned. Such substances are said to be opti¬ 
 cally isotropic or monorefringent. 
 
 If a doubly refractive substance, as, for instance, any 
 chemical crystal not belonging to the cubical system, or 
 any other of the substances already mentioned as polar¬ 
 izing substances, is placed upon the stage, the Nicols 
 being crossed, the light is restored and color effects are 
 produced, when the analyzer is turned. Such substances 
 are said to be birefringent or anisotropic. 
 
CHAPTER IV. 
 
 INSTRUCTIVE PRACTICE IN MANIPULATION. 
 
 The beginner will do well to make a careful study of 
 the microscope before attempting practical work. With this 
 end in view, he may put himself through a course of train¬ 
 ing in microscopical gymnastics. The knowledge gained 
 will be ample reward for the trouble. One or two slides 
 of diatoms, to be purchased at twenty-five cents each of 
 various dealers, will afford means for very instructive study 
 of the instrument. A slide of the diatoms called Arach- 
 noidiscus Ehrenbergii and one of Pleurosigma angulatum 
 will serve the purpose. In the lack of these slides the 
 scales of a moth, obtained by pressing the wing of a moth 
 to a cover-glass, or a little potato starch dusted on a cover, 
 may be used. Invert the cover on a glass slip. 
 
 Place the slide of Arachnoidiscus Ehrenbergii on the 
 stage of the microscope and focus sharply upon it, using 
 the A eye-piece and the f-in. objective. In focussing, do 
 not at first look through the instrument, but place the eye 
 in position to watch the approach of the objective to the 
 glass slip on the stage, and slowly move the body of the 
 microscope down by means of the coarse adjustment until 
 the front lens of the objective is as near its working dis¬ 
 tance from the glass slip as can be estimated; in this case 
 about three-fourths of an inch. Now look through the 
 instrument, and the outlines of the object should be more 
 or less visible if the object is properly centred on the 
 stage. If nothing is seen, move the glass slip about to 
 
 54 
 
INSTRUCTIVE PRACTICE IN MANIPULATION. 
 
 55 
 
 centre the object more perfectly. If it is now necessary 
 to use the coarse adjustment to change the focus, focus up 
 only. If the object is not thus brought into view, turn 
 the body down again with the coarse adjustment while 
 watching the approach of the objective to the object as 
 before. When the object is brought into view, focus up 
 and down with the fine adjustment to obtain a more 
 perfect image. Recall what was stated in Chaptei I. about 
 the influence of the thickness of the cover-glass on the 
 clearness of the image. If the thickness of the cover-glass 
 is known, adjust the draw-tube to the required length. If 
 the thickness of the cover-glass is not known, aftei getting 
 the sharpest image possible with the draw-tube at the 
 standard length, try the effect of longer and shorter 
 lengths until the image is clearest. 
 
 See that the field is properly lighted. Direct sunlight 
 must not be used. The diffused sunlight from a north 
 window, or the light reflected from a cloud or white wall, 
 is regarded as the best light. The light from an ordinary 
 kerosene lamp is a very suitable artificial light. 
 
 Place the mirror in central position, and use first the 
 plane and then the concave side. Use each of the open¬ 
 ings in the diaphragm in succession with each mirror. 
 Focus up and down occasionally with the fine adjustment. 
 Practise these manipulations until the arrangements are 
 found which show the markings on the diatom most 
 perfectly. It will be found that the best effects are 
 obtained when the field is lighted with a subdued light 
 that is very pleasant and comfortable for the eye. A 
 glare of light should always be avoided. The light should 
 be sufficient, but not too intense. If the light is thus 
 properly regulated, and suitable precautions are exer¬ 
 cised not to use the eyes to the point of fatigue, the 
 
56 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 eyes will be strengthened rather than injured by Micro¬ 
 scopical work. Just as judicious exercise strengthens the 
 body, so judicious use of the eyes improves them. If one 
 makes a practice of reading in the glare of the sun or 
 other light, his eyes will soon be injured. So too, if he 
 uses the microscope with glaring light, his eyes will be 
 injured. The correct modulation of the light is a matter 
 of very great importance, and one that is too often over¬ 
 looked even by those who have had considerable experience 
 with the microscope. The light from the concave mirror 
 is moie intense than that from the plane mirror, and is 
 better adapted to the higher power objectives and eye¬ 
 pieces, which absorb more light than do the lower powers. 
 In using the concave mirror, slide it up and down on the 
 mirror bar until a bright circle of light from half an inch 
 to an inch in diameter is projected upon the slide. This 
 circle of light is best seen when the slide is shaded from 
 other light. 
 
 After noting the effects obtained from each of the above 
 arrangements, move the mirror about 10° to the side away 
 fiom the light, adjust it to the light, and again go through 
 the same practice manipulations, carefully observing differ¬ 
 ences of effects. Then move the mirror another 10° to the 
 side, and again repeat the manipulations. Continue to 
 move the mirror to one side 10° at a time, and to repeat 
 the above manipulations as long as good results can be 
 obtained. Having observed the effects for each position 
 of the mirror with the draw-tube closed, place the draw- 
 tube at different lengths. Notice the slight change of 
 focus necessary and the effect on the image. Determine 
 what length of draw-tube gives the best results. 
 
 Remove the A eye-piece and put in its place the O 
 eye-piece, and practise the manipulations again. When 
 
INSTRUCTIVE PRACTICE IN MANIPULATION. 
 
 57 
 
 some degree of skill has been acquired in manipulation, 
 the -jf-in. objective may be substituted for the f-in., and the 
 slide of Pleurosigma angulatum for the Arachnoidiscus 
 Ehrenbergii as a more suitable object for this objective. 
 In focussing the jb-in. objective the precaution to watch it 
 as it is brought to its proper working distance, about \ of 
 an inch from the slide, must be carefully observed. On 
 no account allow the front lens to come in contact with 
 the cover-glass, or both objective and cover-glass may be 
 injured. With both the A and the C eye-pieces repeat 
 the manipulations described above until the best results 
 possible are obtained. 
 
 Patient practice of these manipulations at intervals for 
 a few weeks will give one a knowledge of the instrument, 
 and an ability to handle it in practical work that cannot be 
 gained by years of heedless use. 
 
 Examination of Opaque Objects. — Place a glass slip upon 
 the stage, and on it any opaque object, as, for instance, 
 seeds of grasses or flowers, a plant leaf, grains of sand, or 
 an insect. If the mirror can be swung above the stage, 
 arrange the concave mirror so as to focus the light upon 
 the object. This should be done roughly before looking 
 into the microscope. With the coarse adjustment move 
 the objective to about its working distance. Then look¬ 
 ing at the object through the microscope, focus up and 
 down with the coarse or fine adjustment, as occasion may 
 require, always remembering to guard against bringing 
 the objective too near the slide or specimen. Change the 
 position of the mirror to illuminate the object better. If 
 the mirror cannot be used thus, a bull’s-eye condenser may 
 be used to focus the light upon the object, the mirror 
 being put in such position that light reflected from it will 
 not interfere with the illumination. 
 
58 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 Air Bubbles. — Many curious and ridiculous blunders 
 have been made from misinterpretations of the appear¬ 
 ances of air bubbles. To guard against such errors, air 
 bubbles should be made the object of special study. A 
 drop of saliva, or soapsuds, or of mucilage well beaten 
 up on a slide with the point of a knife blade, have been 
 suggested as suitable means of easily obtaining the bub¬ 
 bles for study. Study the bubbles both without and with 
 a cover-glass, which should be placed lightly over the 
 mass. Focus up and down, using both central and oblique 
 light. Once studied in this way, the air bubble will ever 
 after easily be recognized. Observe that air bubbles afford 
 a most excellent means of determining when the light is 
 central. 
 
 Oil Globules. — Oil globules have also led to errors of 
 interpretation. They are liable to occur in prepara¬ 
 tions of animal or vegetable tissues. To form them for 
 study, beat up a small drop of any oil with a drop of 
 water on a slide, cover lightly, and study them in the 
 same manner as the air bubbles. 
 
 Fibres and Hairs. — Fibres of cotton, wool, silk, and 
 hemp, and hairs of different kinds, are liable to find their 
 w T ay into preparations from the air of the room, or from 
 lack of care during the processes of preparation. It is 
 well to make a special study of these and similar objects. 
 Examine them dry, also in water or glycerine, or in some 
 oily medium, as oil of turpentine, or oil of cloves, covering 
 with a cover-glass. Examine in a similar manner any 
 other objects which may suggest themselves as likely to 
 find their way adventitiously into a microscopical prepa¬ 
 ration. 
 
 Currents and the Brownian Movement. — Grind up a little 
 solid carmine, or any very finely powdered mineral sub- 
 
INSTRUCTIVE PRACTICE IN MANIPULATION. 
 
 59 
 
 stance, in a drop of water on a glass slip, cover with a 
 cover-glass, and place the slip on the stage of the micro¬ 
 scope. Incline the stage, and watch the current move¬ 
 ment of the particles. Then place the stage in the 
 horizontal position, and wait a few minutes for the cur¬ 
 rents to cease. A peculiar, jerky motion of the finer 
 particles may now be observed as they dart about among 
 the larger masses. This is the so-called Brownian move¬ 
 ment, or Pedesis. The study of these phenomena may 
 prevent serious errors of interpretation. 
 
 Practice with Immersion Objectives. — On beginning to 
 use an immersion objective, practice work is of especial 
 importance. The slide of Pleurosigma angulatum is still 
 a good object to use, as the |-inch objective has left much 
 unseen. With this slide on the stage, place a small drop 
 of water, if the objective is a water-immersion objective, or 
 of homogeneous immersion fluid, if the objective is a homo¬ 
 geneous immersion objective, on the front lens, or on the 
 centre of the slide, the microscope being placed in the 
 upright position. Watching the objective, run it down by 
 means of the coarse adjustment in the manner previously 
 described, until the drop of fluid forms a connection 
 between the front lens and the cover-glass. The drop 
 should be just large enough to fill the space between the 
 lens and the cover-glass. After the connection is made, 
 the microscope may be inclined, if desired. If a non- 
 adjustable objective is used, and the thickness of the 
 cover-glass is known, the draw-tube should be adjusted to 
 give the correct tube length according to the cover-glass 
 gauge. When the tube length can thus easily be obtained, 
 much has been gained. We have now simply to light the 
 field well by means of the sub-stage condenser, remember¬ 
 ing that as much depends upon correctly focussing the con- 
 
60 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 denser as on focussing the objective, and obtain the correct 
 focus of the objective by means of the fine adjustment. 
 One should remember always to focus up and not down, 
 or both the cover-glass and objective will be endangered. 
 
 If the objective has collar adjustment, and the thickness 
 of the cover-glass is known, the collar may be set at once 
 at the proper mark, remembering that the objective should 
 be set at the medium point for cover-glasses of the standard 
 thickness adopted by the makers of the objective. If the 
 cover-glass is thinner than the standard thickness, the 
 milled collar should be turned towards the lowest point. 
 If the cover-glass is thicker than standard thickness, the 
 milled collar should be turned towards the highest point, 
 the amount of the turning being estimated by the amount 
 of deviation from the standard thickness of cover-glass. 
 
 If the thickness of the cover-glass is not known, the 
 collar should be set at the highest point, that is, for thick 
 covers, when the connection is first made with the cover- 
 glass. Now with one hand on the milled ring of the collar 
 adjustment, and the other on the fine adjustment, and 
 while looking into the microscope, turn the collar adjust¬ 
 ment through part of one of its divisions, and gently focus 
 up and down with the fine adjustment. Repeat this 
 manipulation until the most perfect image attainable has 
 been obtained, remembering to test the focussing of the 
 sub-stage condenser from time to time by moving it up and 
 down. 
 
 After obtaining the best effects possible with central 
 light, oblique light should be used. If the sub-stage 
 condenser is provided with a diaphragm, make the dia¬ 
 phragm eccentric; that is, move it a little to one side. 
 After noting the effects, by successive steps make the 
 diaphragm more and more eccentric as long as good results 
 
INSTRUCTIVE PRACTICE IN MANIPULATION. 
 
 61 
 
 can be obtained. Then, in like manner, move the dia¬ 
 phragm in the opposite direction. If there is no diaphragm, 
 obtain oblique light by means of the mirror by moving it 
 away from the light a few degrees at a time in the same 
 manner as when using the low power objectives. 
 
 It cannot be stated too emphatically that a large amount 
 of this practice manipulation should precede any attempts 
 to make practical use of immersion objectives. The ability 
 to bring out the full power of these objectives can be 
 attained only by patient perseverance and the intelligent 
 study of the effects produced by manipulation. It hardly 
 seems necessary to waste time in describing the results 
 which should be obtained. The observer will have very 
 little doubt about his success when he obtains clear-cut 
 images, and he should not be satisfied until he does this. 
 
CHAPTER V. 
 
 METHODS OF STUDYING FRESH OBJECTS. 
 
 The microscope is but an aid to the eye, and the eye an 
 aid to the brain behind it. Something more than the 
 mere collection of objects and multiplying the number of 
 slides in a cabinet should be the aim of every owner of a 
 microscope. The highest use of the instrument is in 
 following out definite courses of investigation. In the 
 pursuit of such investigations the examination of a very 
 large number of different specimens is necessary to obtain 
 clear ideas of structure, and give the brain material with 
 which to work. It would not be possible, nor would it be 
 desirable, permanently to preserve all the specimens ex¬ 
 amined. The methods of working will of course differ 
 with different lines of investigation. 
 
 It is not The purpose here to furnish directions and 
 descriptions for detailed and comprehensive work in any 
 department. Volumes would be required for this, and 
 excellent manuals already exist for the different depart¬ 
 ments of investigation. It will be convenient for the 
 purposes of this chapter to consider some methods of 
 working with vegetable and animal specimens, and to 
 illustrate the methods by particular descriptions in a few 
 cases. For methods of preparing vegetable and animal 
 specimens for permanent mounting the reader is referred 
 to Chapters VIII. and IX. 
 
 In the investigation of these specimens, a razor ground 
 flat on one side, a supply of glass slips and cover-glasses, 
 
 63 
 
PRACTICAL METHODS IN MICROSCOPY. 
 
 64 
 
 some dissecting-needles, and various inexpensive chemicals 
 and stains, formulas for which will be found in the 
 Appendix, are necessary. Glass slips three inches by one 
 and a half are to be preferred for this purpose to the 
 ordinary slips, as they present a broader surface to hold 
 the reagents used. For many of the processes a dissecting- 
 microscope is a great convenience. This is most needed 
 when many dissections are to be made. If this instrument 
 is not at command, use the lowest power of the compound 
 microscope. 
 
 The microscope is best kept in the upright position 
 to prevent the specimen from slipping out of the field 
 of view, to prevent currents due to gravity in the fluid 
 media employed, and to prevent the reagents from injuring 
 the microscope. Neatness in all operations is the mark of 
 a skilful workman. In case any of the reagents used 
 accidentally come in contact with the microscope, they 
 should immediately be removed. The reagents should not 
 be allowed to flow over the upper surface of the cover- 
 glass, as they are liable to come in contact with the front 
 lens of the objective. Especial pains should be taken to 
 prevent this, and in case they do touch the objective, it 
 should immediately be cleaned with great care. 
 
 In these practical studies air bubbles and oil globules are 
 very sure to be met with, and it may be well to recall 
 or repeat the studies already made of these objects. 
 
 Vegetable Specimens. 
 
 For purposes of study vegetable substances are usually 
 examined in water. Some substances require no special 
 preparation. Any of the minute and transparent plants, 
 either those growing on land or those growing in the 
 
 
METHODS OF STUDYING FRESH OBJECTS. 
 
 65 
 
 water, may be placed on tlie centre of a glass slip in a 
 small drop of water, a cover-glass laid over the specimen, 
 and the examination made. Care should be exercised not 
 to use so large a drop of water that it will run out from 
 under the cover-glass. Enough only should be used to fill 
 the space between the cover-glass and the slip. If more is 
 added, it is liable to run on to the microscope. In any case 
 the work is slovenly. An excess may be removed by 
 touching it with a piece of blotting paper or of filter 
 paper. 
 
 Microscopic Water Plants. — The light green stringy 
 masses found floating in the water or attached to sub¬ 
 merged objects in ponds and ditches in summer afford 
 good material for examination. These plants belong to 
 the family of Zygnemacese. They are unicellular, but the 
 cells are joined in large numbers so that the plant appears 
 to be multicellular. It is easily recognized by its slippery 
 feeling when a mass of it is taken in the hand, and from 
 the fact that each filament is without branches. In young 
 cells the greenish yellow chlorophyll granules are quite 
 evenly distributed throughout the cell. At more advanced 
 stages the chlorophyll collects in some species in a line 
 running lengthwise through the centre of the cell, often 
 terminating in star-shaped or globular knobs, so that each 
 cell contains a dumb-bell-like figure. These knobs are 
 sometimes left isolated by the breaking down of the con¬ 
 necting line of chlorophyll granules. 
 
 In other cases the chlorophyll collects into spiral bands, 
 differing in number in different species, the bands giving 
 these species their name, Spirogyra. In favorable cases 
 conjugation between the cells of two adjacent-lying fila¬ 
 ments affords opportunity for much interesting study. 
 June or July is the most favorable time to observe the 
 
66 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 conjugation. From this yoking together of the cells in 
 conjugation the whole family takes its name, Zygnemacese. 
 There are many varieties in the family, any one being an 
 interesting object for study. 
 
 In examining this plant, observe that the cells are all 
 alike, and, being joined end to end, form filaments of great 
 length, but of uniform diameter throughout. As the 
 plant approaches maturity, the bright yellow-green changes 
 to a dirty brown. When at this stage, it is in a particu¬ 
 larly favorable condition to exhibit some of its distinguish¬ 
 ing features. 
 
 Attached to the cells of the Spirogyra are frequently 
 found numerous spherical, or nearly spherical, bodies of 
 a reddish color. They are the Vavipyrella spirogyrce , 
 belonging to the Monerozoa, one of the lowest types of 
 animal life. They have attacked the Spirogyra to feed on 
 its chlorophyll. 
 
 Yeast. — Yeast, the Torula cerevisice, is another plant 
 which furnishes interesting material for examination. 
 Dissolve some sugar in water, and add a little yeast. 
 When it is seen that the yeast is working, place a drop on 
 a glass slip without a cover-glass, and examine with a low 
 power. Then cover, and examine with a power of four or 
 five hundred diameters. The yeast is seen to be made up 
 of very transparent spherical and oblong cells of the 
 same kind, but of varying sizes. Some will no doubt be 
 seen with minute cells attached, showing the character¬ 
 istic mode of propagation by budding. 
 
 Other Studies. — In a similar manner, any other sub¬ 
 stances which are sufficiently thin to allow light to pass 
 through them may be examined. Pollen grains are thus 
 easily examined, and are very attractive objects. Their 
 forms are almost infinite in number and variety. The 
 
METHODS OF STUDYING FRESH OBJECTS. 
 
 67 
 
 spores of ferns and mosses, and the numerous varieties of 
 fungi, afford abundant material for examination. Of the 
 fungi, mention may be made of the green mould which 
 forms on bread, old leather, and numerous other substances. 
 It is the Penicillium glaucum. Another mould, the Mucor 
 stolovitfer , is easily grown on a piece of bread kept moist 
 under a bell-jar or tumbler for two or three days. It 
 will be recognized by its fine, thread-like stalks, each hav¬ 
 ing a round head, and by the stolons connecting the stalks. 
 In examining either of these fungi, treat different speci¬ 
 mens with hsematoxylin and carmine stains, and with iodine. 
 
 The epidermis of leaves and stems may in many cases 
 be scraped off with a knife, or torn off with forceps, and 
 without further preparation be placed under the micro¬ 
 scope in a drop of water. The epidermal cells, stomata, 
 and hairs, are thus readily obtained for examination from 
 many plants. 
 
 Maceration and subsequent teasing or tearing the spec¬ 
 imen to pieces by means of dissecting-needles is a very 
 important means of reaching the minute internal structure 
 of plants. The maceration may be effected by soaking 
 the specimen for some time, or by boiling it a few minutes, 
 in water. It may be effected more quickly and thoroughly 
 by treating with a warm, strong solution of caustic potash, 
 or by placing the specimen in a test-tube with a few crys¬ 
 tals of potassium chlorate and enough strong nitric acid 
 to cover them (Schultze’s Macerating Mixture). Strong 
 hydrochloric acid is also a good macerating fluid. After 
 the action has continued for a short time, the specimen 
 should be washed thoroughly in water, transferred to a 
 glass slip, and torn to pieces with the dissecting-needles 
 under the dissecting-microscope or a low power of the 
 compound microscope. Spiral and scalariform vessels are 
 
68 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 easily obtained by this process from the leaf stalks of 
 rhubarb and of ferns, the stem of banana, and other 
 sources. The cell structure of some tissues is also re¬ 
 vealed by this treatment much better in some respects 
 than can be done by sections. 
 
 Vegetable Sections. — To obtain sections of vegetable 
 specimens, a piece is held firmly in the left hand between 
 the thumb and second joint of the forefinger, the blade of 
 the razor is rested on the second joint of the forefinger as 
 a guide, and drawn towards the person, resting both hands 
 against the breast to steady them. The thumb of the 
 left hand should be kept depressed to guard against 
 injury. Keep the specimen and the blade of the razor 
 wet with water, and when the section is cut, dip the razor 
 in a glass of water to float the section off. Small or soft 
 objects may be held between pieces of pith, carrot, or 
 potato. Hard specimens may be made soft enough to cut 
 by boiling for a few minutes in water. The hard woods 
 yield to this treatment. For purposes of study it is not 
 always necessary that large, even sections be cut. Indeed, 
 an uneven section is sometimes more valuable than an 
 even one for this use. Some parts near the edges will 
 show extreme thinness, while other parts often give a 
 better idea of the relations of the cells to one another. 
 Ordinarily the rule for thinness is to cut to the thickness 
 of one row of cells. It is best to cut quite a number of 
 sections of each specimen. 
 
 For study, a section should be transferred from the 
 glass of water to a glass slip with a small drop of water. 
 A camel’s-hair brush is the best section-lifter. The section 
 is covered with a cover-glass and is ready for examination. 
 While water is ordinarily the best medium for the exami¬ 
 nation of vegetable substances, there are some cases where 
 
METHODS OF STUDYING FRESH OBJECTS. 
 
 69 
 
 it produces an undesirable change in the tissues of the 
 specimen, causing the cell contents or the cell walls to 
 swell. Other reagents then become desirable. Strong 
 alcohol, by extracting water, causes a contraction of the 
 cell wall and contents. More dilute alcohol is, in many 
 cases, a suitable medium to maintain the tissue in its 
 normal condition. 
 
 It is often desirable to watch the changes which the 
 specimen undergoes under the action of a reagent. It 
 may be desired, for instance, to remove water from the 
 specimen and introduce alcohol. This is best done without 
 removing the specimen from the slide, or even removing 
 the cover-glass from the specimen. The slide still re¬ 
 maining under the microscope, by means of a pipette (a 
 glass tube drawn out to a fine point) hold a little alcohol 
 near one side of the cover-glass, and at the opposite side 
 hold a piece of blotting paper, or better, good filter paper. 
 The water is drawn out from under the cover-glass by 
 capillary attraction, and the alcohol takes its place. By 
 repeating the operation several times the water is entirely 
 removed and alcohol substituted. In the same way any 
 reagent or stain may be introduced, or the specimen 
 may be washed free of a reagent by means of water. 
 
 It often happens that a specimen is too opaque to be 
 well studied, even when the section is very thin. In such 
 cases glycerine is a valuable medium for the examination. 
 If the specimen is already mounted in water, the water 
 should be removed and the glycerine introduced in the 
 manner just described. The changes which the specimen 
 undergoes during the process afford excellent oppor¬ 
 tunities for studying the object. Opaqueness may also be 
 remedied by bleaching the specimen before it is mounted. 
 Sodium or calcium hypochlorite is a good bleaching agent. 
 
70 PRACTICAL METHODS IN MICROSCOPY. 
 
 A considerable volume of tlie fluid should be used, and the 
 specimen should remain in it for some hours. If it is 
 desired to render the specimen more transparent during 
 the progress of the examination, a moderately strong 
 solution of caustic potash may be run under the cover- 
 glass. The first effect of the caustic potash in some cases 
 is to turn the section black. This may be remedied by 
 washing out the caustic potash with water, and running 
 under a very weak solution of hydrochloric acid. The 
 specimen soon becomes transparent. The acid should 
 now be washed out with water. In the more obstinate 
 cases it may be necessary to repeat the treatment with 
 caustic potash several times before the desired effect is 
 produced. 
 
 In similar ways the changes which the specimen under¬ 
 goes when treated with different chemical solutions may 
 be studied. It is usually best to add the reagent in small 
 amounts at first, and to watch the changes as the strength 
 is increased. Any of the staining solutions for which 
 formulas are given in the Appendix may be thus employed. 
 The use of different stains on the same section, or on 
 different sections of the same specimen, tends to reveal 
 varied points of structure. The study of fresh sections in 
 this way is a valuable preparation for the correct use of 
 stains for permanent mounts. 
 
 Starch. — Under the microscope starch granules show a 
 distinct point, usually situated near one end, called the 
 hilum, and an appearance of being made up of lamellae, 
 or plates. The test for starch is a solution of iodine. 
 Iodine dissolved in absolute alcohol gives a brown color to 
 dry starch. If any moisture is present, the color is blue. 
 As an ordinary test for starch, iodine dissolved in a watery 
 solution of potassium iodide may be used. If a little 
 
METHODS OF STUDYING FRESH OBJECTS. 
 
 71 
 
 of this is run under the cover, the characteristic blue color 
 of the iodide of starch appears. 
 
 The changes caused in the starch granule by the gradual 
 addition of a strong solution of caustic potash are in¬ 
 structive. As the caustic potash is run under the cover- 
 glass, the starch granule is seen to swell, and the hilum 
 and laminations become more distinct. Soon the lami¬ 
 nation becomes less distinct as the granule becomes more 
 glassy and transparent. Dilute chromic acid is another 
 reagent which renders the stratification more distinct. 
 
 Under polarized light the starch granules are brought 
 distinctly into view, each with its characteristic black cross. 
 A good specimen for the study of starch granules in situ is 
 a section of a potato taken from just below the skin. A 
 little of the juice of a potato, or the flour obtained by 
 grinding to a powder a pea, bean, kernel of corn, wheat, 
 or oats, also affords good material for study. 
 
 Cellulose. — Iodine imparts a yellowish brown color to 
 cellulose, the fundamental material of the cell wall. If a 
 little strong sulphuric acid is run into the specimen, the 
 color is changed to blue. The best reagent for detecting 
 cellulose, however, is Scliultze’s reagent or chlorzinc iodine. 
 This is made by dissolving metallic zinc in hydrochloric 
 acid, evaporating to the consistency of syrup with a piece 
 of undissolved zinc remaining, and adding as much potas¬ 
 sium iodide as will dissolve. Finally iodine is added until 
 a deep reddish brown color is produced. This reagent 
 colors cellulose violet. 
 
 Another useful reagent for the study of cellulose is 
 ammoniacal cupric oxide. It is prepared by adding caustic 
 soda or caustic potash to a strong solution of copper sul¬ 
 phate as long as a precipitate is formed. Filter, and wash 
 the precipitate in water that has been boiled. Place the 
 
72 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 greenish mass in a bottle, and add just enough strong 
 ammonia to dissolve it. When properly prepared, fibres 
 of cellulose swell up and dissolve in it. To test the 
 reagent, see if fibres of cotton are so acted on by it. It 
 must be kept in the dark. 
 
 Lignified Cellular Tissue.—Aniline chloride dissolved in 
 ten parts of alcohol and one part of hydrochloric acid 
 imparts a yellow tinge to lignified cellular tissue. Ani¬ 
 line sulphate dissolved in ten parts of alcohol, or of water, 
 and one part of sulphuric acid also gives a yellow color to 
 lignified cellular tissue. Lignified cellular tissue turns 
 yellowish green if treated with a solution of carbolic acid 
 crystals in warm hydrochloric acid and subsequently 
 exposed to the direct sunlight. A strong solution of 
 chromic acid readily dissolves cell walls. In weaker solu¬ 
 tions it causes the cell wall to swell. It is therefore a 
 valuable reagent in studying cellular tissues. Schultze’s 
 reagent imparts a clear yellow color to lignified cellular 
 tissue, and brings it into prominent view. If this reagent 
 is allowed to act on the specimen for some hours, the 
 result is more decided. Objects will keep in good con¬ 
 dition in this solution for a long time. 
 
 Protoplasm. — Protoplasm is a viscid, transparent fluid 
 often containing finely divided dark particles, occurring in 
 the younger cells of plants. It is the living part of the 
 plant. It is best studied in the hairs of nettles, the hairs of 
 very young shoots of the pumpkin, squash, or cucumber, 
 and the hairs upon the stamens of the cultivated spider- 
 wort, Tradescantia Virginica. If some of these hairs are 
 examined in a drop of water under high power, the moving 
 currents of the protoplasm are visible. It is often neces¬ 
 sary to warm the slide slightly to cause the movement to 
 start. In studying the protoplasmic movements, remove 
 
METHODS OF STUDYING FRESH OBJECTS. 73 
 
 the water, and run a little glycerine under the cover-glass 
 in the usual manner, and observe the effects on the proto¬ 
 plasm. The action -of alcohol may, in like manner, be 
 observed. Strong alcohol kills the protoplasm, which is 
 then much more readily acted upon by staining fluids. 
 A nucleus, containing a nucleolus, may be seen with a 
 high power, in the cells of successful preparations, sus¬ 
 pended by threads, and carried about in the cell as the 
 protoplasm is acted upon by the reagents. If tissues con¬ 
 taining protoplasm are treated with a thick syrup of cane 
 sugar, and afterwards with strong sulphuric acid, the pro¬ 
 toplasm is slightly colored red. Protoplasm also absorbs 
 iodine from solution, and becomes reddish brown. 
 
 After some experience has been acquired in the study 
 of protoplasm in plant hairs, the various reactions for it 
 may be applied to the young cellular tissue found at the 
 growing points of stems, roots, and leaves. It is to be 
 remembered that protoplasm can be studied best in young 
 cells. As the cells increase in age, cavities, or vacuoles, 
 are formed, and the protoplasm is pushed close up to the 
 wall. If such a tissue be treated with a somewhat strong 
 aqueous solution of common salt, the protoplasm is caused 
 to contract and becomes more easily visible. In still older 
 cells the protoplasm wholly disappears. In very young 
 tissues the application of a stain — hsematoxylin is espe¬ 
 cially good — often brings out a network of delicate fibres 
 in the protoplasm. 
 
 Chromatophores. — The green color of plants is due to 
 minute spherical or polygonal shaped particles imbedded 
 between layers of protoplasm. When viewed under the 
 microscope, they appear to be suspended between the cell 
 wall and the vacuole, or sap cavity, in the central part 
 of the cell. When treated with alcohol for some time, 
 
74 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 the color disappears, but the forms of the granules are 
 still faintly visible as colorless bodies. The green pigment 
 is called chlorophyll; the granules that bear the pigment 
 are known as chlorophyll bodies. Other pigments give 
 the characteristic colors to colored plants. Chromato- 
 phore is a general name for the bodies that bear the various 
 colors. It is characteristic of chromatophores that starch 
 granules are developed in them. It is not always easy to 
 see these without special treatment. Cut a green leaf 
 into several small pieces, and place the pieces in a drop of 
 water under the microscope. The chlorophyll bodies are 
 liberated, and from some of them the enclosed starch 
 granules are set free. Exposure to direct sunlight for 
 several hours previous to the examination will render suc¬ 
 cess more certain. Treat with iodine solution, and the starch 
 shows the characteristic blue color, while the uninjured 
 chlorophyll bodies take on a brown color. The enclosed 
 starch granules may also be demonstrated by treating 
 material which has lain in alcohol for some time with 
 caustic potash to cause the starch granules to swell, and 
 afterwards treating with iodine solution to produce the 
 blue color. It is to be noted that the origin of starch 
 granules is in the chlorophyll bodies. As the chlorophyll 
 bodies increase in age, they gradually become more and 
 more transparent until they disappear, and the matured 
 starch granules are left uncovered in the cells of the plant. 
 
 The piothallia of ferns, which are always found grow¬ 
 ing on the plant-pots in hot-houses where ferns are culti¬ 
 vated, are particularly favorable specimens in which to 
 study chlorophyll. The Zygnemacese are also good mate¬ 
 rial for study. Keep the specimens in the sunlight several 
 hours, treat with iodine, and the masses of chlorophyll 
 will be seen to be surrounded with starch granules. 
 
METHODS OF STUDYING FliESH OBJECTS. 
 
 75 
 
 Plant Crystals. — Deposits of crystalline earthy matters 
 are of very common occurrence in the cells of plants. 
 The crystals most commonly take the shape of a prism, an 
 octahedron, or of needles. Some can be found in almost 
 any plant. The cuticle of the onion shows fine examples 
 of the prismatic and octahedral crystals of the oxalate of 
 lime. Similar crystals may also be found in the bark of 
 the rose around the bases of the prickles. Raphides, or 
 needle-shaped crystals, of oxalate of lime are abundant 
 in rhubarb, especially in the root, and in the epidermis of 
 the bulbs of tulips and hyacinths. The petioles of begonia 
 contain octahedral crystals of the same salt. Tubers of 
 the dahlia which have been in alcohol for some days show 
 somewhat spherical-shaped crystals of inulin. During the 
 examination for inulin allow nitric acid slowly to replace 
 the water. 
 
 Owing to the difficulty of isolating the crystals from 
 the cellular tissues the chemical composition is not known 
 in many cases. It is known, however, that crystals of the 
 oxalate of lime are most common. The carbonate of lime 
 is the second most common crystal found. These are 
 easily distinguished by means of acetic and hydrochloric 
 acid. If acetic acid is applied to the section, crystals of 
 calcium oxalate are not affected; crystals of calcium car¬ 
 bonate dissolve with effervescence. If hydrochloric acid is 
 applied to the section, crystals of calcium oxalate dissolve 
 without effervescence; crystals of calcium carbonate dis¬ 
 solve with effervescence. 
 
 Polarized light affords a very delicate means of detect¬ 
 ing the presence of crystalline or other earthy deposits in 
 vegetable sections. Even if the crystals do not distinctly 
 polarize, their forms, especially when quite transparent, 
 are brought out in some positions of the Nicols. 
 
76 
 
 PRACTICAL METHODS IN MICEOSCOPY. 
 
 Animal Specimens. 
 
 Many of the directions already given for the study of 
 vegetable specimens might be repeated for animal speci¬ 
 mens. The methods described for the manipulation of 
 the object under the microscope apply equally well to the 
 study of both classes of objects. 
 
 Animalcules. hlo attempt will be made here to describe 
 the microscopic forms of animal life. The subject is too 
 extensive for cursory treatment. Some good manual should 
 be consulted for their identification. Any of these forms 
 may be examined under the microscope if placed in a drop 
 of water on the slide, and a cover-glass laid over the speci¬ 
 men. Ihe cover-glass should be supported by bits of other 
 thin glass. A shallow cell made as described in the fol¬ 
 lowing chapter makes a good live-box for the temporary 
 examination of these specimens. The specimens are trans¬ 
 ferred by means of a piece of glass tubing used as a pipette. 
 
 The stagnant water of swamps furnishes countless 
 varieties of microscopic animal life. The more quiet the 
 watei, the more abundant the animalcules. Seek out small, 
 isolated pools. Collect some of the water around sub¬ 
 merged objects. In collecting, it is best to place some of 
 the aquatic plants in the collecting vessel with the water, 
 distuibing the plants as little as possible. The animalcules 
 cling in large numbers to the plants. A jar of this swamp 
 water will furnish instructive entertainment for many an 
 hour. 
 
 Water Sediment. Allow a considerable amount of water 
 to stand in a tall glass vessel for several hours. With a 
 piece of rubber tubing, siphon off all but a small remnant 
 of the water. With a pipette, place a very small drop of 
 
METHODS OF STUDYING FRESH OBJECTS. 
 
 77 
 
 the sediment on a glass slip, and put a cover-glass over it. 
 Examine with objectives of different powers, beginning 
 with a low power. Solid particles of an earthy character 
 will probably be found together with some microscopical 
 forms of animal and plant life. The more impure the 
 water, the more abundant these forms. The solid particles 
 are probably either silica or the carbonate of lime. Run a 
 little strong sulphuric acid under the cover-glass. Car¬ 
 bonate of lime is dissolved with effervescence of carbon 
 dioxide, which will form numerous minute bubbles. Silica 
 is unaffected by the sulphuric acid, and vegetable sub¬ 
 stances are more or less blackened. 1 
 
 Striped Muscular Fibre. — The tissues of the larger ani¬ 
 mals may be prepared for examination by teasing them 
 to pieces on the glass slip in a drop of water, glycerine, or 
 some staining solution. The striated fibre of the voluntary 
 muscles is thus easily prepared. Borax-carmine or hse- 
 matoxylin is a good medium to employ. The fibrillse, the 
 nuclei, the transverse striations, and the sarcolemma, or 
 investing membrane, are readily made out. The study 
 should be begun with a low power, and continued with 
 a high power. 
 
 A half per cent solution of common salt in water is also 
 a good medium for this study. A preparation may be 
 made by teasing up a bit of any of the active muscles 
 of the legs of an animal in the salt solution. After the 
 transverse striations have been studied, remove the salt 
 solution and run acetic acid under the cover-glass. This 
 causes the striations to show less distinctly, but the nuclei 
 
 i For more complete directions for tlie microscopical examination of 
 water, see the author’s “ Water Analysis for Sanitary Purposes: Chemical 
 and Biological.” Boston, 1892. Willard Small, Publisher, 24 Frank¬ 
 lin Street. 
 
78 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 and the sarcolemma are brought more clearly into view. 
 If now the acetic acid be washed out by applying succes¬ 
 sive drops of water to one side of the cover-glass and draw¬ 
 ing them under by means of a piece of filter paper at the 
 opposite side, and then a little borax-carmine stain be run 
 in, the nuclei take a deep color and are rendered very 
 conspicuous. They are somewhat elongated or fusiform, 
 and seem to be at the very surface of the fibres. 
 
 Unstriped Muscular Fibre. — In a similar way a study 
 may be made of the unstriped fibre of the involuntary 
 muscles by teasing out a piece of the wall of the stomach 
 or intestine. The elements of this muscle will be found to 
 be elongated cells, each containing a nucleus situated near 
 the centre, which stains more deeply than other portions. 
 
 Sections of Animal Tissues. — To obtain sections of soft 
 animal tissues it is necessary- to harden the specimen. 
 This is most conveniently done by placing small pieces 
 in alcohol for a few days. Better results are obtained if 
 the specimen is gradually hardened by remaining a few 
 hours in weak alcohol, say 50 per cent, then in 70 per 
 cent and 95 per cent alcohol. The pieces should be not 
 more than half an inch square and one-fourth of an inch 
 thick, and a large volume of alcohol should be used. 
 When sufficiently hard to give clean sections, the object 
 may be held in the hand or between pieces of carrot or of 
 pith moistened with alcohol, and sections cut by the free¬ 
 hand method already described. Or the object may be 
 freed from alcohol by soaking in turpentine a few hours, 
 and imbedded in paraffin by the method described in 
 Chapter IX. Both unstained and stained sections should 
 be examined. The staining is best accomplished by allow¬ 
 ing the sections to lie in a watch glass with a few drops 
 of the staining fluid until the right intensity of color is 
 
METHODS OF STUDYING FJRESH OBJECTS. 
 
 79 
 
 obtained. The best effects are secured when the specimen 
 is treated, before and after staining, according to the direc¬ 
 tions given in the Appendix in connection with the for¬ 
 mulas for the different staining solutions. 
 
 Blood, — Place on a cover-glass a small drop of fresh 
 blood obtained by pricking a fold of the skin on the back 
 of the wrist with a needle previously sterilized by pass¬ 
 ing it through the gas flame, and invert the cover-glass on 
 a glass slip. Observe the numerous red corpuscles float¬ 
 ing about in a colorless plasma. The corpuscles seem to 
 attract one another, and are seen to move towards one 
 another, forming rolls. The tendency to do this is in¬ 
 creased if the cover-glass is slightly moved on the slip. 
 The red corpuscles are seen to be round and very thin, 
 regular in shape, disk-shaped, hollowing in the centre, or 
 bi-concave. Among them the smaller and irregular white 
 corpuscles may possibly be distinguished, but it is not 
 always easy to do this, as in health the white corpuscles 
 are very few in number compared with the red corpus¬ 
 cles. In some diseases the relative proportion of the 
 white corpuscles is much increased. 
 
 Fresh blood from a frog is more favorable for the study 
 of the white corpuscles. The white corpuscles are rela¬ 
 tively much more numerous in the blood of the frog than 
 in human blood. The form of the white corpuscle will be 
 seen to be very irregular, the appearance being very much 
 like the amoeba. If the slide is slightly warmed, a true 
 amoeboid movement may be seen in these corpuscles, as 
 the irregular projections change in form and size, moving 
 the corpuscle about. The structure of the corpuscle is 
 granular in the central portion; clear and transparent in 
 the outer portions. If the specimen is treated with acetic 
 acid and then with borax-carmine, the nucleus will stain, 
 
80 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 showing one or more nucleoli; the outer portions remain 
 unstained. 
 
 The red corpuscles of frog’s blood are also very inter¬ 
 esting. They are much larger than the corpuscles of 
 human blood, oval in shape, and contain a distinct nu¬ 
 cleus which is slightly convex, being thicker than the 
 peripheral portions. If the specimen is treated with alco¬ 
 hol and then with hsematoxylin or with borax-carmine, 
 the nucleus takes a deep stain, the other portions remain¬ 
 ing uncolored. Iodine is also a useful staining agent for 
 blood corpuscles. 
 
 Very interesting preparations of frog’s blood may be 
 made as follows: Place a minute drop of the blood on a 
 cover-glass and lay a second cover-glass over it. Iiub the 
 cover-glasses together to obtain a very thin evenly dis¬ 
 tributed film of the blood on each. Place the cover- 
 glasses, prepared side up, on a sheet of paper and let them 
 dry thoroughly in the air or exposed to direct sunlight. 
 When dry, place on each a large drop of Kleinenberg’s 
 or Grenadier’s hsematoxylin and let it remain twenty 
 minutes. Do not let it dry up. If there is a tendency 
 to do so, add more of the stain. Wash thoroughly in 
 water by letting a gentle stream fall upon the cover-glass, 
 or, holding the cover-glass with the forceps, dip it in a con¬ 
 siderable volume of Avater. When the excess of hsema¬ 
 toxylin has been removed, place a weak solution of eosin 
 on the cover-glasses and allow it to remain five minutes. 
 Wash thoroughly again, and dry the cover-glasses as before. 
 The nuclei are stained purplish blue; the peripheral por¬ 
 tions red. Place a drop of Canada balsam on a clean glass 
 slip and place one of the thoroughly dried cover-glsisses on 
 it, prepared side down. Press it down gently, and a very 
 attractive and permanent object is made. 
 
METHODS OF STUDYING FRESH OBJECTS. 
 
 81 
 
 The double staining may also be accomplished by using 
 successively different aniline stains; fuchsin and methylene 
 blue, fuchsin and Bismarck brown, fuchsin and iodine 
 green are recommended. In making the preparations, the 
 cover-glass may be dried by passing it through the heated 
 air several inches above a gas flame, holding the cover- 
 glass with the forceps. Much care must be exercised, or 
 the corpuscles will be shrivelled. It is better ordinarily 
 to let the film dry at the ordinary temperature of the room. 
 
 The Circulation of the Blood in a Frog’s Foot. — The in¬ 
 teresting appearance of the blood circulating in the web of 
 a living frog’s foot may be seen at the cost of very little 
 trouble. No special piece of apparatus is required, but a 
 thin strip of wood five inches long and one and a half 
 inches wide, with a hole three-fourths of an inch in diam¬ 
 eter bored through it an inch from one end, makes an 
 excellent frog plate. In the centre of a piece of cloth 
 about eight inches square cut a small hole, and pass the 
 frog’s foot through it. Wrap the cloth around the frog 
 and saturate it with water. With a soft string bind the 
 frog loosely upon the strip of wood in such position that 
 its foot comes over the opening. Tie threads to its toes, 
 and so fasten them that a portion of the web is spread 
 horizontally over the opening, and all is ready for exami¬ 
 nation. A little care must be exercised that the strings 
 are not drawn too tight, or the circulation may be stopped. 
 The frog does not appear to be hurt, and after the first 
 fright is over usually lies very quietly. 
 
 Cartilage. — Cut as thin a section of cartilage as possible, 
 and mount in a half per cent salt solution. Cartilage 
 from the sternum of a frog gives particularly favorable 
 specimens. Slowly run under the cover-glass some iodine 
 solution. The cells of the cartilage will be brought into 
 
82 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 clear view as they take a deep brown stain from the 
 iodine. The cells occur singly, or two, three, or four 
 together. When there are three, one is larger than the 
 other two. The growth by cell division is evident from 
 this arrangement of the cells. Each cell contains a nu¬ 
 cleus ; some contain two nuclei. The nuclei sometimes 
 contain several nucleoli. Between the cells is a lifeless 
 matrix which does not take the stain. 
 
 Bone. — Bone may be decalcified by treating it for 
 several hours with very dilute hydrochloric acid. As the 
 hydrochloric acid destroys some of the soft tissues, better 
 results will be obtained if chromic acid is used, beginning 
 with a one-sixth per cent solution. After twelve hours 
 pour off this solution, and replace it with a one-third per 
 cent solution, and after another twelve hours with a one- 
 half per cent solution. Do not use a stronger solution 
 than this, but use a large volume and allow the specimen 
 to remain until softened. Wash thoroughly in water to 
 remove the acid. 
 
 Sections may now be easily cut with a razor. Wash 
 the sections in water to make sure that the acid is removed, 
 and stain in borax-carmine, which imparts an intense color. 
 Avoid overstaining, removing excess, if necessary, with 
 acid alcohol. 
 
 It is not difficult to prepare sections of bone by grinding. 
 A suitable piece of dry bone, a cross-section, for instance, 
 cut as thin as possible with a surgeon’s saw, is ground 
 smooth on one side by rubbing it over a fine-grained whet¬ 
 stone or hie. During the grinding, press gently but firmly 
 upon it with the fingers. Now place a large drop of 
 Canada balsam on a piece of glass about an inch square, 
 and hold the glass with the forceps, or better with a spring 
 clothes-pin, over the gas flame until the volatile portions 
 
METHODS OF STUDYING FRESH OBJECTS. 83 
 
 are driven off. Allow it to cool, but before it is quite 
 ready to set, place the smooth side of the bone on it, and 
 press it gently down until the balsam is cold. Using the 
 glass as a holder, continue to grind the bone on the whet¬ 
 stone or file until it is thin enough. Wash the surface 
 with alcohol, and examine the progress of the work from 
 time to time under the microscope, placing a drop of water 
 on the specimen and covering with a cover-glass. The 
 section may be thus studied on the bit of glass, or it may 
 be very easily made into a permanent mount. Immerse it 
 in chloroform or benzole until the balsam dissolves and the 
 specimen slips off from the piece of glass. Use no force to 
 remove it, or it will be injured. Place a drop of balsam on 
 a glass slip, and heat to drive off the volatile portions so 
 that, upon cooling, the balsam becomes quite hard. Treat 
 a cover-glass in the same way. Remove the section of 
 bone from the chloroform or benzole, using a camel’s-hair 
 brush as a lifter, and drain off the fluid by touching .the sec¬ 
 tion with a piece of filter paper. Place the dried section 
 on the centre of the slip, invert the cover-glass over it, and 
 hold some distance above the flame until the balsam just 
 melts. Set aside to harden. The object of this treatment 
 is to prevent the balsam from penetrating the specimen 
 and rendering the structure too transparent. 
 
 Epithelium. — Epithelium is to the internal cavities of 
 the body what the skin is to the outside. It does not, 
 however, form a firm and continuous covering to the under¬ 
 lying tissues. It exists in various forms of loosely joined 
 cells. These forms run into one another insensibly, but 
 are usually distinguished as pavement and columnar 
 epithelium. The cells of the former are thin and flat. 
 Their arrangement somewhat resembles the arrangement 
 of the stones of a pavement. The cells of columnar 
 
84 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 epithelium are thickly crowded together, arranged like 
 aggregates of columns. The columnar epithelium may 
 have cilia or hairlike appendages. 
 
 To obtain specimens for examination, scrape with a dull 
 knife-blade the inside of the cheek, the roof of the mouth, 
 or the mucous membrane of the intestine of a recently 
 killed animal, and mount the scrapings in water, or a half 
 per cent salt solution. The cells are nucleated. Scrapings 
 from the tongue or the roof of the mouth of a recently 
 killed frog are especially good for the study of ciliated 
 epithelium. The movements of the cilia are best studied 
 by watching the specimen under a high power for some 
 time, while the cells die, the movements of the cilia in full 
 life being too rapid to be observed well. 
 
 The methods of studying vegetable and animal speci¬ 
 mens may, of course, he indefinitely extended. If sufficient 
 has been said to give the beginner a start in the right 
 direction and awaken interest in these fascinating processes 
 of investigation, the object of this chapter is attained. 
 The use of so many different stains and reagents is wont 
 to seem to the beginner an almost insurmountable obstacle 
 to the work. The expense involved is very slight, and 
 a little experience shows that the trouble-of the manipula¬ 
 tive processes is small. As nearly all the reagents and 
 stains keep well, and some are even improved by age, they 
 should be prepared in leisure hours and kept ready for use 
 in suitable bottles. When the trouble of using is thus 
 reduced to a minimum, there soon comes to be a fascina¬ 
 tion in studying the effects produced by different reagents 
 and combinations of reagents. A previous knowledge of 
 chemistry is, of course, an advantage in this work, but is 
 by no means a prerequisite. 
 
CHAPTER VI. 
 
 MOUNTING OBJECTS FOR THE MICROSCOPE. 
 
 The process of mounting objects for the microscope for 
 permanent preservation is very simple. Many different 
 media are employed, but it is well for the beginner to 
 become thoroughly familiar with the working of one before 
 attempting to use others. One who has mastered the 
 methods given in this chapter will find no difficulty in 
 using any medium. 
 
 Mounting in Canada Balsam. — The medium most uni¬ 
 versally employed is Canada balsam. This medium pre¬ 
 sents the fewest difficulties. It is therefore well suited to 
 the needs of the beginner. Canada balsam may be obtained 
 of dealers in microscopical supplies in the pure form, or 
 combined with various solvents, as benzole, xylol, or 
 chloroform. Ihese solutions of balsam, when properly 
 prepared, harden much more quickly than the pure balsam. 
 In their preparation the pure balsam is heated at a moder¬ 
 ate temperature until the volatile portions are driven off, 
 so that when cold the balsam is hard and chips easily. 
 This hardened balsam is then dissolved in benzole, xylol, 
 or chloroform. When an object is mounted in this solu¬ 
 tion, the solvent quickly volatilizes, and leaves the cover- 
 glass securely cemented to the glass slip by the hard balsam. 
 Ignorant or unscrupulous dealers sometimes simply mix 
 fresh balsam with the solvent, and sell the mixture under 
 the name of benzole, xylol, or chloroform balsam. Such 
 a mixture is as slow in drying as the pure balsam. 
 
 85 
 
86 PRACTICAL METHODS IN MICROSCOPY. 
 
 If the object to be mounted contains moisture, as is 
 ordinarily the case, it must first be thoroughly dehydrated, 
 or a cloudiness will result around the object from the fact 
 that the water does not mix with the balsam. The dehy¬ 
 dration may, in most cases, be sufficiently accomplished by 
 soaking for some minutes in strong commercial alcohol, 
 commonly spoken of as 95 per cent alcohol. The use of 
 absolute alcohol will insure perfect dehydration, but there 
 are comparatively few cases where it is a necessity. 
 
 After dehydration the alcohol is removed, and the speci¬ 
 men cleared, by placing it for a few minutes in oil of 
 cloves, turpentine, benzole, or some other substance which 
 drives out the alcohol, and which is itself miscible with 
 balsam. Perfect clearing is important, and it is often well 
 to pass the specimen through two changes of the clearing 
 agent. Both dehydration and clearing may be effected 
 after the object is placed on the slide. The specimen is 
 covered with a large drop of alcohol or of the clearing 
 agent. After a few minutes this is drained off, and another 
 portion of the fluid applied. Repeat the process still again 
 for especial thoroughness. 
 
 A glass slip is thoroughly cleaned and warmed over a gas 
 flame to drive off all adhering moisture. The slip is placed 
 on the turn-table, the object is exactly centred, and a small 
 drop of Canada balsam is dropped upon it. The cover- 
 glass, thoroughly cleaned and warmed, is allowed to fall 
 gently upon this. The cover-glass should be handled with 
 the forceps to prevent leaving finger marks or stains upon 
 it. One edge should be brought in contact with the 
 balsam at one side of the object. It should then be slowly 
 and gradually lowered to position to prevent displacement 
 of the object by a too violent movement of the balsam. 
 The cover-glass should then be pressed down and exactly 
 
MOUNTING OBJECTS FOR THE MICROSCOPE. 
 
 87 
 
 centred. Gentle pressure may be applied, if desired, by 
 means of spring clips; or by means of lead bullets slightly 
 flattened by a blow from a hammer so that they will not 
 roll; or by large-headed screws, which are excellent for 
 this purpose. 
 
 The balsam may be allowed to harden at the ordinary 
 temperature. If pure balsam is used, the hardening will 
 require some weeks, so that it is often desirable to hasten 
 the process. The slides may be placed in the oven of a 
 water-bath, and a temperature of between 180° and 140° P. 
 maintained for several hours. If a higher temperature is 
 used, or if the heating is unduly prolonged, the -balsam 
 will turn yellow. Another way is to put the slides in the 
 kitchen oven at night just after the fire is dumped. In 
 the morning the balsam will ordinarily be well set. A 
 steam radiator or the mantle-piece above a stove serves 
 a similar purpose. The slides should of course be pro¬ 
 tected from dust. Each should bear a temporary label to 
 prevent mistakes in identification. 
 
 This is all that is really necessary for a permanent 
 mount. The object will be preserved indefinitely without 
 further treatment. With practice it is not difficult to 
 gauge the amount of balsam so that the space between 
 the cover-glass and the glass slip will be exactly filled 
 without overflow. If this has not been accomplished, and 
 there is an overflow of balsam, the slide may be placed on 
 the turn-table, and the thoroughly hardened balsam cut off 
 with a chisel-shaped instrument. A little water placed on 
 the slide will prevent the balsam chips from flying about, 
 and will help in cleaning the slide. When the superfluous 
 balsam has been dislodged by means of the chisel, the slide 
 is placed under a gentle stream of water from a faucet and 
 carefully rubbed with a small brush. The final portions 
 
88 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 may be removed with a piece of cloth wet with alcohol, 
 after which the slide is carefully wiped with a dry 
 cloth. 
 
 Mounting in Glycerine-Jelly. — To mount with glycerine- 
 jelly requires rather more skill than to mount with Canada 
 balsam, on account of the difficulty of getting rid of air 
 bubbles, which must be removed before the mount is sealed. 
 There are, however, no serious obstacles to be overcome. 
 For many objects, noticeably delicate plant sections, 
 glycerine-jelly is superior to balsam. Delicate tissues are 
 shrivelled by dehydration. Glycerine-jelly does not require 
 that the water should be removed from the specimen, as 
 water and the jelly are readily miscible. 
 
 The bottle of glycerine-jelly is placed in a dish of water, 
 and the water heated until the jelly melts. Carefully 
 clean a slide, place it on the turn-table, put the object on 
 its centre, and breathe upon it. Working quickly, place a 
 drop of the melted jelly on the object, removing any air 
 bubbles by pricking them with a needle. Breathe upon a 
 clean cover-glass, put a little of the hot jelly upon it, invert 
 it over the specimen, and centre exactly. If air bubbles 
 are formed under the cover-glass, they may sometimes 
 be removed by introducing a fine wire. 
 
 Another method of procedure is to place a small piece of 
 the solid glycerine-jelly on the centre of the glass slip, and 
 carefully warm until it melts. The object is placed in this, 
 and the air bubbles are removed, if any are formed. A 
 piece of the glycerine-jelly is placed on a cover-glass held 
 in the forceps, and melted. The cover-glass is then in¬ 
 verted over the object and centred. 
 
 Set the slide away with a light weight upon it for 
 twenty-four hours for the jelly to harden thoroughly. 
 Remove the superfluous jelly with a knife and by washing 
 
MOUNTING OBJECTS FOR THE MICROSCOPE. 89 
 
 in cold water, using a cloth wet with water to remove the 
 final portions. 
 
 Glycerine-jelly mounts must be sealed with some tough 
 cement to prevent the entrance of air and the parting of 
 the cover-glass from the slide. Preliminary to doing this 
 the glycerine-jelly around the edge of the cover-glass may 
 be firmly set and rendered insoluble by slightly moistening 
 it with a strong solution of potassium dichromate and 
 exposing to the sunlight. The slide is placed upon the 
 turn-table, and the potassium dichromate applied with 
 a brush. When the slide is ready for the cement, a sable- 
 liair brush with long hair is lightly charged with some 
 transparent cement, such as shellac cement, or Berry s 
 hard oil finish (see Appendix). The turn-table is rapidly 
 whirled and a light ring of cement formed about the edge 
 of the cover-glass. The brush is charged a second time, 
 and the ring is reinforced by a second coat, ihe slide is 
 now set aside for the cement to harden. Several additional 
 coats should be applied at intervals until a firm ring has 
 been formed to hold the cover-glass securely in place. 
 
 Mounting in Thin Fluids. — It is sometimes desirable to 
 mount in clear glycerine, or in some fluid of even less 
 consistency. The principal difficulty arises from the ease 
 with which the cover-glass is displaced when it is attempted 
 to apply the cement ring, lo overcome this difficulty, 
 after the cover-glass is in position over the object, and the 
 superfluous fluid which runs out from under the cover has 
 been removed with a piece of filter or blotting paper, place 
 minute drops of melted paraffin or sealing wax at three 
 equidistant points around the cover-glass. Upon harden¬ 
 ing, these drops will hold the cover-glass in position while 
 the rings of colorless cement are being applied. In many 
 cases a shallow cell, such as is described in the next para- 
 
90 
 
 PEACTICAL METHODS IjST MICEOSCOPY. 
 
 graph, is required to hold the fluid medium, especially 
 when the object is of appreciable thickness. 
 
 Dry Mounting. — Some objects are rendered too trans¬ 
 parent when mounted in any of the above-mentioned 
 media. I he markings on starches, scales of moths, dia¬ 
 toms, and many similar objects are much less distinct 
 when imbedded in these media than when they are viewed 
 dry. Ihe principal objection to dry mounting is the fact 
 that after some time moisture is liable to collect in the 
 mount and obscure the view of the object. For dry 
 mounts glass slips should be prepared with shallow cells, 
 formed of rings of transparent cement, of a size to suit the 
 cover-glass to be used. These cells should be well seasoned 
 before they are used. Many of the slips may be prepared 
 in leisure hours and kept on hand ready for use. 
 
 When ready to mount an object, run a very thin fresh 
 coat of transparent cement on the outer edge of the hard¬ 
 ened ring. Place the object in position on the slip, or, 
 if light like starch or the scales of a moth, dust a little 
 of it on a cover-glass. Lay the cover-glass, prepared side 
 down, in position over the cement ring. The ring of 
 fresh cement should be of such size that the edge of the 
 cover-glass will extend into it slightly all around. Place 
 a light weight on the cover-glass and set the slide away 
 until the cement is hard. I hen place it upon the turn¬ 
 table again and apply a light coat of cement over the 
 juncture of the cover-glass with the cement ring. If too 
 much cement is used, it will surely run under the cover- 
 glass. When the first coat is dry, add another light one, 
 and repeat until the mount is thoroughly sealed. 
 
 The sweating of dry mounts may in a measure be pre¬ 
 vented, when the object will not be injured by exposure 
 to the air, by making small openings through the hardened 
 
MOUNTING OBJECTS FOR THE MICROSCOPE. 
 
 91 
 
 cement with a fine needle to give free circulation of the 
 air through the cell. Much care must be exercised in 
 making the opening, or the cover-glass will be broken. 
 
 Cells of paraffin are also much used for dry mounts. 
 They are very easily prepared. The paraffin is heated 
 quite hot and applied with a brush in the usual way of 
 forming rings on the turn-table. The ring hardens in a 
 few minutes and is then neatly trimmed down with a 
 chisel-shaped instrument, using the turn-table as a lathe. 
 A coat of transparent cement is applied to the ring, and 
 the object is mounted and sealed as already described. 
 
 Mounting in Cells with Fluid Media. — It is often desired 
 to preserve objects that are too thick to mount without 
 some means of raising the cover-glass from the slide. 
 Many delicate objects — whole insects, for example would 
 be crushed and much injured if mounted in the ordinary 
 way, unless especiady prepared for that style of mounting. 
 All such objects may be mounted in cells of depths suited 
 to each case. When the objects are quite thin, rings of 
 transparent cement of sufficient thickness may be built up. 
 These should be prepared beforehand and kept ready for 
 use. A well-seasoned cell is much more reliable than one 
 freshly made. In making these cells apply light coats of 
 the cement at intervals, giving time for each to dry before 
 the next application is made. For thicker objects, rings 
 of glass, hard rubber, or metal should be purchased of 
 some dealer in microscopical supplies. These are fastened 
 to glass slips by smearing one side with transparent cement, 
 placing them in exact central position on the slip on the 
 turn-table, and setting aside with a light weight on them 
 until the cement is hard. A coat of the cement is then 
 run around the outside of the cell and allowed to harden. 
 
 In mounting, the cell is filled even full with Canada 
 
PRACTICAL METHODS IN MICROSCOPY. 
 
 balsam, glycerine-jelly, glycerine, or other mounting 
 medium to be used, taking care not to let the medium 
 touch the upper surface of the ring. Place the slide on 
 t e turn-table and run a ring of transparent cement on the 
 upper surface of the ring. Lower the cover-glass to posi¬ 
 tion and see that there are no air bubbles. The edge of 
 the cover-glass should be slightly imbedded in the ring 
 of fresh cement. If skilfully done, the cement and 
 atmospheric pressure will hold the cover-glass in position. 
 Any overflow of the mounting medium should now be 
 removed unless the medium is Canada balsam. It is gen¬ 
 erally better to wait for the balsam to harden before at¬ 
 tempting to remove it. Aqueous media may be washed 
 away, the cover-glass being held in position with spring 
 clips, if necessary. After this cleaning, the cover-glass is 
 secured in position by successive light rings of transparent 
 cement. 
 
 It is important for success in cell-mounting that the 
 upper surface of the cell be perfectly even and flat. 
 Cement cells may be brought to this condition, when 
 necessary, by means of a fine file, or by heating to soften 
 the cement, and pressing squarely against the top of the 
 cell with a glass slip. No attempt should be made to use 
 curtain rings and other cheap substitutes for cells, as no 
 amount of skill will enable one to succeed in closing the 
 cell if its upper surface does not make perfect contact 
 with the cover-glass all around. 
 
 Finishing Slides. For practical purposes, slides made 
 by the processes above described may be regarded as fin¬ 
 ished. The appearance and the durability of the slide 
 will, however, be much improved by applying neat rings of 
 colored cements. Before applying these colored cements, 
 balsam as well as other mounts should be thoroughly 
 
MOUNTING OBJECTS FOB THE MICROSCOPE. 
 
 sealed by the application of several light coats of trans¬ 
 parent cement. When this cement has thoroughly hard¬ 
 ened, the edges should be turned true with a chisel-shaped 
 instrument, using the turn-table as a lathe. A somewhat 
 broad ring of some colored cement may now be applied, 
 entirely covering the transparent cement and extending 
 slightly on to the cover-glass on the inner side and out on 
 to the slip on the outer side. Give this cement time to 
 harden and turn both edges true. _ A narrower ring of 
 some contrasting color is now applied in such a manner 
 as to leave a narrow ring of the first color on either side. 
 When dry, a ring of a third color may similarly be applied 
 so as to leave a ring of the second color on either side of 
 it. With a little practice, rings of as many different colors 
 as desired may be very quickly and neatly applied, and the 
 appearance of the slide much improved. 
 
 There is in some quarters a professed contempt for this 
 ornamentation of slides. Carried to excess it is unques¬ 
 tionably a serious waste of time. The contempt usually 
 comes from those who have never acquired the skill to do 
 a neat piece of work. One who has once acquired this 
 skill knows that he possesses the ability to do neat work 
 more rapidly than he could before do slovenly work. 
 There is, too, a real pleasure in a piece of neatly exe¬ 
 cuted mechanical work, and skill attained in this woik is a 
 training for the many other mechanical operations of the 
 microscopist. 
 
 In applying cement rings, a long-haired sable brush should 
 be used. The brush known among artists as the Rigger 
 brush is excellent. The brush should first be lightly 
 charged and the turn-table rapidly whirled. This first 
 application forms a track which the cement will follow in 
 the subsequent applications. The brush is now more 
 
94 PRACTICAL METHODS IN MICKOSCOPY. 
 
 heavily charged so that a small drop forms at the end. 
 The turn-table is rapidly whirled, the brush held nearly 
 vertically or slanted in a line tangent to the ring to be 
 formed, and the drop of cement approached until it just 
 touches the track formed for it. The judgment must be 
 used as to the amount of cement to be now applied. As a 
 rule it is better to apply the cement in light coats, giving 
 time for each to harden. In this way the spreading of the 
 cement is avoided. 
 
 It now remains to place neat permanent labels on the 
 slide, and it is finished. These labels should state the 
 common and scientific name of the object, call attention 
 to any particular points of structure or preparation, name 
 the mounting medium and stains employed, the thickness 
 of the cover-glass, and any other facts which will be of 
 value in future examinations of the slide. 
 
 Cleaning Glass Slips and Cover-Glasses. — It often happens 
 even with experienced microscopists that imperfect mounts 
 are made, and an easy way of cleaning the slip and cover- 
 glass is desirable. A wide-mouthed bottle should be pro¬ 
 vided for this purpose, a pint fruit jar, for instance. In 
 this place a strong solution of borax, washing soda, or 
 caustic potash. Into this solution drop the spoiled slides, 
 here to remain until a number have been so deposited, 
 and until a convenient season presents itself to attend 
 to them. The solution dissolves not only balsam, but 
 all resinous media and cements. After a few days the 
 cover readily slips off from even seasoned mounts, and the 
 balsam and cements are easily removed by scraping with 
 the thumb nail. Sharp instruments should not be employed 
 to scrape a slide that is to be used again, as scratches are 
 almost sure to result. 
 
 The writer uses ordinarily borax water, as being most 
 
MOUNTING OBJECTS FOR THE MICROSCOPE. 
 
 95 
 
 agreeable to handle, and finds the glass clean enough for 
 most practical purposes. To make sure that the slides and 
 covers are optically clean, soak them for some days in the 
 following mixture : dissolve 10 grammes of potassium di¬ 
 chromate in 50 c.c. of water by the aid of heat. When 
 the solution is cool, immerse the containing vessel in cold 
 water, and pour into it 50 c.c. of sulphuric acid slowly and 
 carefully with constant stirring. 
 
 Upon removing the slides and cover-glasses from this 
 solution, wash them thoroughly in clean water. 
 
CHAPTER VII. 
 
 PRACTICE MOUNTING. 
 
 The object of this chapter is to furnish illustrations of 
 the methods of the preceding chapter, and to afford means 
 of introducing the beginner to some of the manipulations 
 necessary in the making of microscope slides. An object 
 prepared for the microscope is not unfrequently an object 
 of supreme mystery to one who is wholly inexperienced in 
 the processes. Minute and explicit directions will be here 
 given for a few simple, easily accessible, and easdy prepared 
 objects. If the beginner will follow these directions with 
 patience and care, the mystery will be solved and be 
 proved to be no mystery at all. 
 
 For this practice work the necessary equipment consists 
 of a few glass slips and cover-glasses; a small bottle of 
 benzole or xylol balsam; a bottle of sodium or calcium 
 hypochlorite; a bottle of borax-carmine staining fluid; one 
 each of iodine green, alcohol, turpentine, benzole, and a weak 
 solution of caustic potash; a pair of improvised dissecting- 
 needles; and plain forceps. With this equipment, not 
 only the work of this chapter, but quite a wide range 
 of investigation may be accomplished, and a considerable 
 variety of work in the preparation and mounting of objects 
 may be done. It is better, perhaps, to purchase the borax- 
 carmine staining solution ready for use, though it is not 
 difficult to prepare it according to the directions given in 
 the Appendix. However it is obtained, it is of importance 
 to know its alcoholic strength, and to soak the object to be 
 
 96 
 
PRACTICE MOUNTING. 
 
 97 
 
 stained in alcohol of the same strength before it is placed 
 in the stain, and after staining to rinse off the the super¬ 
 fluous staining fluid in alcohol of the same strength. If 
 these precautions are neglected, solid particles of carmine 
 will be deposited on the specimen. The iodine green 
 should be purchased in the solid form. A strong solution 
 of this in alcohol should be made and filtered. This 
 solution may be diluted with alcohol to any desired degree 
 for use. 
 
 Insect Scales, Starch, and Similar Light Objects. — These 
 objects afford interesting material for slides, and are 
 especially easy of preparation. The wing of a moth is 
 touched to a cover-glass, or a little starch is dusted on a 
 cover-glass, and the cover-glass is inverted over a small drop 
 of balsam on a glass slip. Hold the clean cover-glass in 
 the forceps by one edge, bring the opposite edge into light 
 contact with the balsam, and allow the cover to fall by its 
 own weight gradually upon the balsam. Carefully press 
 down and exactly centre the cover-glass. Pay no attention 
 to air bubbles unless they are quite large, as they will 
 disappear as the balsam hardens. The centring of the 
 object and of the cover-glass may be accomplished on 
 the turn-table, or by using the common expedient of 
 drawing a figure one by three inches in dimensions on a 
 slip of paper, finding its centre by drawing diagonals, and 
 describing a circle the size of the cover-glass about this 
 centre. The balsam is now hardened, and the slide cleaned 
 and finished according to the directions given in the pre¬ 
 ceding chapter. 
 
 Hairs. — Hairs should be soaked in turpentine for some 
 days to render them more transparent. They should then 
 be cut in lengths suited to the size of cover-glass used, 
 placed on a glass slip, a small drop of Canada balsam 
 
98 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 allowed to fall on them, and a clean cover-glass placed 
 over them. Many hairs polarize finely. Among easily 
 accessible and interesting hairs may be mentioned human 
 hairs (especially those of the beard), caterpillar, rat, mouse, 
 raccoon, and bat hairs, and the bristles of the hog. The 
 hog’s bristles are especially attractive objects under the 
 polariscope. 
 
 Insects. — When the beginner has acquired a little ex¬ 
 perience in mounting the above-mentioned objects, he may 
 advance to parts of insects, as the wing or foot of a fly, and 
 to the mounting of small insects whole. Soak the object for 
 twelve hours in a weak solution of caustic potash to cleanse 
 it from any adhering dirt or oily particles, and to soften 
 the harder portions of chitinous skeletons. If a whole 
 insect is being treated, it should now be placed between 
 two pieces of glass, and the soft visceral portions removed 
 by gently and carefully pressing the pieces of glass to¬ 
 gether. The soft portions are then carefully brushed off, 
 placing the piece of glass bearing the insect under water 
 in a shallow dish and using two camel’s-hair brushes, one 
 to hold the insect, the other to remove the adhering 
 matter. Renew the water until the caustic potash has 
 been wholly washed out. Place the object in 95 per 
 cent alcohol for twenty-four hours to remove the water. 
 Then treat with turpentine. The longer the object remains 
 in the turpentine, the clearer and more transparent will 
 it become; but as the balsam has a similar effect, it may 
 remain in the turpentine only long enough to become 
 permeated. It is well to allow it to remain at least twenty- 
 four hours. Warm a glass slip by passing it to and fro 
 above a gas or lamp flame to drive off all moisture. 
 Transfer the object to the centre of the slip and drain off 
 the superfluous turpentine. Place a drop of Canada 
 
PRACTICE MOUNTING. 
 
 99 
 
 balsam on the object and cover with a previously warmed 
 and cleaned cover-glass. 
 
 The insect world offers such variety that one might 
 spend his life in studying and mounting insect preparations. 
 A dissecting-microscope is of great use in separating and 
 dissecting out by means of dissecting-needles the parts 
 to be mounted. Parts which are too large to be mounted 
 as above must, of course, be mounted in a cell prepared 
 as described in the preceding chapter. The process of 
 mounting is similar and but little more difficult than when 
 no cell is used, as long as balsam is the medium employed. 
 
 Vegetable Sections. — Place some transverse sections of 
 a plant stem, — a geranium stem is good for the first trial, 
 — cut‘by the free-hand method described in Chapter V., 
 in a considerable volume of sodium hypochlorite for sev¬ 
 eral hours to bleach them. Wash thoroughly in several 
 changes of water and transfer to 35 per cent alcohol. In 
 a few minutes transfer to borax-carmine stain. Let the 
 sections remain in the stain until upon the examination of 
 one under the microscope it is found to be sufficiently 
 colored. This operation will require a length of time 
 varying from a few minutes to several hours, depending on 
 the strength of the fluid. Transfer to slightly acid 35 per 
 cent alcohol. In a few minutes transfer to a weak solu¬ 
 tion of iodine green in alcohol, where the sections should 
 remain at least half an hour. They will probably now 
 appear of a uniform green color. Place in 95 per cent 
 alcohol until the green is removed from the parenchymal 
 portions, but take care that they do not remain too long, 
 or the green stain will be wholly discharged. Transfer to 
 oil of cedar or to benzole to remove the alcohol and clear. 
 After a few minutes mount in balsam. 
 
 Sections which will bear repeated transferals are best 
 
100 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 treated in Avatch glasses or individual butter dishes of 
 white porcelain. When the sections must be left for 
 some time in a fluid, two-drachm vials, to be obtained at 
 the drug store, are very convenient, as they may be set 
 away for hours tightly stoppered, and there is no loss by 
 evaporation, as would be the case if Avatch glasses are used. 
 
 Preparations made by teasing, and any delicate tissues, 
 may be passed through these processes on the glass slip. In 
 each operation cover the specimen Avell with a large drop 
 of the fluid. It is well to repeat the washing in alcohol 
 and the clearing in oil of cedar or benzole to make sure of 
 thorough Avork. 
 
 Animal Sections. — Place some sections of animal tissues, 
 cut as described in Chapter V., in 70 per cent alcohol for 
 a feAv minutes, transfer to Kleinenberg’s hsematoxylin, 
 letting them remain until, upon the examination of one 
 under the microscope, they are seen to be sufficiently 
 colored. Remove to 70 per cent alcohol, and in a few 
 minutes transfer to 95 per cent alcohol, then to absolute 
 alcohol, and to oil of cloves. Mount in Canada balsam. 
 
 If it is desired to use other stains, folloAv the directions 
 given in the Appendix in connection Avith the formula for 
 each stain for the treatment before and after staining. 
 Use a camel’s-hair brush to handle the sections. Both 
 animal and vegetable sections may be secured on the 
 centre of the glass slip by means of white of egg or 
 Schallibaum’s collodion fixative, used as described in the 
 Appendix. 
 
 Diatoms. — It is not the purpose here to go far into the 
 very extensive subject of diatoms. The attention of 
 the beginner is more particularly called to recent diatoms. 
 At some pond, stream, or spring, gather some of the 
 broAvn or yellowish brown slime adhering to any of the 
 
PRACTICE MOUNTING. 
 
 101 
 
 water plants. Place some of this slime in a test-tube with 
 enough nitric acid to cover it, and boil for some minutes. 
 Add a small quantity of potassium dichromate and boil 
 again. The diatoms will usually be well cleaned by this 
 process, if it is continued sufficiently long. Pour the 
 contents of the tube into a large volume of water in a tall 
 glass vessel and allow the vessel to stand undisturbed for 
 several hours until the diatoms have settled. Without 
 disturbing the diatoms at the bottom, decant off the liquid 
 above them. Fill the vessel with clean water and allow 
 it to stand again. Repeat this process until the acid has 
 been entirely removed. 
 
 If, now, it is desired to separate diatoms of different 
 densities, fill the vessel again with water, and let it stand 
 until the heavier diatoms have settled. Decant off the 
 water containing the lightest diatoms. Fill the original 
 vessel again with water, and again let the heavier forms 
 settle. Decant the lighter forms into another vessel. By 
 a continuance of this process the diatoms of different 
 weights are quite easily separated. The sediments from 
 the different vessels used are finally collected in small 
 bottles with a little water or alcohol. 
 
 To mount the diatoms, shake the bottle, place by means 
 of a pipette a drop of the water containing the diatoms on 
 a cover-glass, evaporate to dryness b} r holding the cover- 
 glass above a flame, moisten with turpentine, place a small 
 drop of balsam on the cover-glass, heat until the balsam 
 boils, invert over a glass slip, centre, harden, and finish, 
 as already described. 
 
 If one has the desire and patience to do the, as it seems 
 to the writer, unprofitable work of arranging diatoms in 
 figures, coat a cover-glass with gelatine fixative and let it 
 dry. Under a low power arrange the diatoms in the 
 
102 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 desired pattern, using a mechanical finger, or simply a 
 hog’s bristle, or cat’s whisker, attached to a wooden 
 handle by means of wax. When the pattern is formed, 
 or from time to time during its formation, breathe gently 
 on the cover-glass to soften the gelatine. Upon drying, 
 the diatoms will be held fast in the gelatine. Mount in 
 balsam. 
 
 Some of the fossil diatoms may be cleaned by the 
 process described above. Others it is more difficult to 
 clean. Much of the foreign matter may in some cases 
 be removed by heating the earth to a red heat, placing it 
 for this process on a piece of platinum foil or of porcelain. 
 It is frequently necessary to break down the mass of 
 diatomaceous earth by boiling with a solution of caustic 
 potash or caustic soda. These solutions attack the dia¬ 
 toms, forming silicates of potassium and sodium. If they 
 are used, the action should not be allowed to continue 
 long, but as portions of the mass are broken down, they 
 should be poured into a large volume of water to wash out 
 the reagent. It is, perhaps, better to break down the mass 
 of earth with ammonia of moderate strength, as ammonia 
 does not act upon the diatoms to any perceptible extent. 
 When the mass has been broken down, pour the diatoms 
 into a considerable volume of water, and allow them to 
 settle. This requires several hours. Decant off the water, 
 and place the residue in a dish in which it may be heated. 
 If the foreign matter cannot now be removed by the use 
 of nitric acid with potassium dichromate, use hydrochloric 
 acid, aqua regia, and sulphuric acid successively. 
 
 Certain earths are best treated, after the breaking down 
 with ammonia, by dropping in powdered potassium dichro¬ 
 mate while the residue is boiling, and then pouring’ in 
 strong sulphuric acid slowly and with constant stirring. 
 
PRACTICE MOUNTING. 
 
 103 
 
 Some workers recommend boiling the diatoms in strong 
 sulphuric acid, at the same time sifting in pulverized 
 potassium chlorate. This process is dangerous, as violent 
 explosions are liable to occur. It should be a last resort. 
 Flocculent matter is often very troublesome. It may 
 sometimes be removed by boiling the diatoms in a strong 
 solution of soap. The processes are long and tedious. 
 Fine sand can be removed only by the process of decanta¬ 
 tion already described for separating the diatoms of dif¬ 
 ferent weights. Gatherings of the thin coating of black 
 mud on the bottoms of springs, ponds, and streams may be 
 treated in this way and yield rich results in fresh diatoms. 
 
CHAPTER VIII. 
 
 BOTANICAL SECTIONS. 
 
 In some of the preceding pages a few simple directions 
 have been given for the preparation and mounting of 
 vegetable specimens. If a considerable study of structural 
 botany is to be made, more elaborate methods leading to 
 more perfect results are desirable. Even if one is not 
 especially a student of botany, the making of plant sections 
 is attractive; from no other material can showy and 
 interesting specimens be so easily prepared as from plant 
 life. 
 
 For purposes of sectioning, vegetable specimens may be 
 divided into two classes. 
 
 1. Hard specimens, or those that cannot be cut without 
 
 softening. 
 
 2. Soft specimens, including annuals, young shoots, buds, 
 
 seeds, cones, and similar products of plant life, many 
 of which must be hardened or otherwise prepared by 
 special processes. 
 
 Hard Specimens. — Most solid wood specimens may be 
 sufficiently softened for cutting by soaking in water for 
 some hours, changing the water occasionally, or by boiling 
 in water for a few minutes. Three pieces of each specimen 
 should be prepared to give sections in three different direc¬ 
 tions, — transverse, radial, and tangential. A transverse 
 section is made across the stem in a direction transverse to 
 the length. A radial section is made lengthwise the stem, 
 
 105 
 
106 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 along a radius of the circles of annual growth, or from the 
 bark towards the centre of the tree. A tangential section 
 is made lengthwise the stem, tangent to the circles of 
 annual growth. 
 
 If no microtome is available, the pieces should be two 
 or three inches long, that they may be firmly held in the 
 hand, and about one-fourth inch in cross-section. Use a 
 razor ground flat on one side, and follow the directions for 
 cutting free-hand given in Chapter Y. 
 
 For the microtome, specimens should be cut about one- 
 fourth inch square and one inch long. If a microtome 
 having a clamp is used, the softened piece of wood is fixed 
 in the clamp and cut without further preparation. The 
 nearer to the supporting clamp the section is cut, the more 
 perfect it will be, as the springing of the specimen is 
 reduced to the minimum. If a well-microtome is used, 
 the specimen is fixed in the well by wedges of pine or 
 other very soft wood which are sunk in the well, and do 
 not come within one-fourth of an inch of the cutting plate. 
 These wedges hold the specimen firmly, but leave one- 
 fourth of an inch of it exposed for cutting. The specimen 
 is placed at one side, close to the wall of the well, so that 
 the wall may give it support and prevent the specimen 
 from springing. In cutting, keep the knife and the 
 specimen wet with water, dilute glycerine, or a mixture of 
 glycerine and alcohol, using a camel’s-hair brush to apply 
 the liquid. Transfer the sections from the knife to a 
 glass of water with a brush, or by dipping the blade of the 
 knife in the water to float the section off. 
 
 Soft Specimens. — All specimens of a consistency suit¬ 
 able to be cut without special preparation may, of course, 
 be cut free-hand, or may be placed directly in the clamp 
 of the microtome and be cut at once. In many cases, how- 
 
BOTANICAL SECTIONS. 
 
 107 
 
 ever, immersion in alcohol for a few days is an advantage, 
 when the changes resulting from the alcohol are of no 
 account, as the alcohol removes all resinous substances, 
 which stain and clog the knife. If the specimen is made 
 hard and brittle by the alcohol, immerse it for a short 
 time in a mixture of alcohol and glycerine, half and half, 
 which will restore it to a soft condition. 
 
 Specimens which are too soft to be cut directly may be 
 held between two pieces of cork, the specimen projecting 
 about one-fourth of an inch above the cork. The whole 
 should be clamped low down in the jaws of the micro¬ 
 tome clamp to prevent springing. Do not cut through 
 the cork, as it dulls the knife. Some specimens are advan¬ 
 tageously held between pieces of pith, carrot, potato, or 
 similar substance which will not injure the knife. Others 
 are so soft that they must be imbedded in paraffin or 
 celloiclin. Some even require to be hardened before they 
 are imbedded. The hardening is accomplished by gradual 
 dehydration to prevent shrinkage. The specimen is placed 
 in 50 per cent, 70 per cent, and 90 per cent alcohol, remain¬ 
 ing in each several hours. Soft tissues thus hardened may 
 be kept indefinitely in 90 per cent alcohol. So also tis¬ 
 sues which do not require hardening may be preserved. 
 Specimens collected in July or August may be thus kept 
 for winter use. They are very conveniently kept in two- 
 drachm vials. The specimen should be completely cov¬ 
 ered with the alcohol, and each vial should be labelled. 
 The more woody specimens, which become hard and brittle 
 in the alcohol, must be soaked twenty-four hours in the 
 mixture of alcohol and glycerine before they are cut. In 
 cutting alcoholic material, keep the specimen and knife 
 wet with dilute alcohol. Handle the sections with a brush. 
 
 Imbedding in Paraffin. — Three grades of paraffin may 
 
108 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 be obtained of dealers in microscopical supplies: hard, 
 melting at 130° F.; medium, melting at 125° F.; soft, 
 melting at 120° F. They may be mixed in any propor¬ 
 tions necessary to give the right consistency for the tem¬ 
 perature of the room in which the sectioning is to be 
 done. Paraffin that will melt at about 125° F. is suitable 
 in most cases. For paraffin moulds, obtain at the gas- 
 fitter’s pieces of the brass tubing used as the casing for gas 
 fixtures. These pieces should be about one inch long and 
 from one to two inches in diameter. 
 
 Prepare the object by dehydrating in alcohol of gradu¬ 
 ally increasing strength, up to absolute alcohol. Soak in 
 chloroform twenty-four hours, then twenty-four hours in 
 chloroform in which has been dissolved as much paraffin as 
 will dissolve. As chloroform is somewhat expensive, the 
 spirits or oil of turpentine is often substituted for it. 
 
 Melt the paraffin over a water-bath, but be sure that 
 it is no warmer than is necessary to keep it fluid, or the 
 specimens may be shrivelled. Pour on to a piece of paper 
 enough of the melted paraffin to make a circle a little 
 larger than the mould to be used. As the paraffin begins 
 to stiffen fix the specimen upright in it, the end to be cut 
 being down. Place a mould over the specimen, sinking it 
 down into the circle of paraffin, and fill it with melted 
 paraffin. Set aside, for the paraffin to become thoroughly 
 hard. The hardening may be hastened, if so desired, by 
 placing the whole in cold water as soon as the paraffin has 
 stiffened sufficiently to bear this treatment. When the 
 paraffin is hard, pass the mould two or three times 
 through a gas flame, and the paraffin cast may then be 
 easily pushed out. Trim off the opposite sides so that it 
 may be more easily held in the jaws of the microtome, and 
 cut away the paraffin to within an eighth of an inch of 
 
BOTANICAL SECTIONS. 
 
 109 
 
 the object on the end to be cut, and it is ready for section¬ 
 ing. The cast may be held directly in the clamp of the 
 microtome, or it may be fastened to the roughened end of 
 a cork by means of a little melted paraffin, and the coik 
 held in the jaws of the clamp. If a well-microtome is 
 used, the object may be imbedded in paraffin in the well. 
 Imbedded specimens may, of course, be cut free-hand in 
 the manner already described. Keep the specimen and 
 the knife well wet with alcohol, applying it with a brush. 
 Transfer the sections with a small brush to chlorofoim, 
 turpentine, benzole, or oil of cloves, any one of which will 
 dissolve the paraffin. Remove this solvent by transfer¬ 
 ring to two changes of alcohol before staining. 
 
 Imbedding in Celloidin.—Celloidin may be obtained of 
 dealers in microscopical supplies. It is a form of nitro¬ 
 cellulose. It dissolves in a mixture of equal parts of 
 ether and absolute alcohol. Prepare two solutions, one 
 quite thin; the other, thick. The object, which for this 
 method should be not more than one-third of an inch in 
 length, is thoroughly dehydrated in absolute alcohol and 
 soaked in the thin solution of celloidin for twenty-four 
 hours or longer. It is then transferred to the thick solu¬ 
 tion, where it is to remain some hours. It may then be im¬ 
 bedded as follows: Place a little of the thick solution of 
 celloidin on a cork and let it dry for a few minutes. Add 
 successive layers of celloidin, letting each dry, until a 
 considerable mass is built up. Transfer the object from 
 the thick solution of celloidin to this mass and build the 
 celloidin up over it. Weight the cork and immerse the 
 whole in 82 per cent alcohol, and in twenty-four to forty- 
 eight hours the celloidin will be sufficiently hardened to 
 cut. Stronger alcohol dissolves the celloidin and must not 
 be used. Objects imbedded in celloidin may be kept 
 
110 PRACTICAL METHODS IN MICROSCOPY. 
 
 indefinitely in 82 per cent alcohol. In cutting, keep the 
 object and the knife wet with 50 to 70 per cent alcohol. 
 Plant sections should be cut to the thickness of one row of 
 cells, which varies in different specimens from to 
 T 2 V 0 an inch. 
 
 It is to be understood that the somewhat complicated 
 processes of imbedding in paraffin and celloidin are not 
 recommended for general use. The simple processes 
 already described of cutting the sections without imbed¬ 
 ding the specimen, or after imbedding it in pith or carrot, 
 are amply sufficient for the greater number of objects that 
 are to be cut, and will naturally be used, on account of 
 their greater simplicity, when good sections can be ob¬ 
 tained by them. There are, however, many delicate 
 tissues that cannot be cut into thin sections unless their 
 parts are supported by some substance that will infiltrate 
 into the specimen. Resort is then had to the more trouble¬ 
 some methods. Another method highly recommended for 
 this class of objects by many experienced workers is 
 
 Imbedding in Transparent Soap. — The soap mass is pre¬ 
 pared as follows: Any good white soap is cut into thin 
 slices and thoroughly dried by exposure to the sun. The 
 soap is then crumbled to a fine powder and mixed with 
 90 per cent alcohol to form a stiff mass. Glycerine and 
 90 per cent alcohol are then added till the proportions by 
 weight are: soap, 10 parts; glycerine, 22 parts; alcohol, 
 35 parts. 
 
 Place the object in 90 per cent alcohol, imbed in the 
 soap liquefied by heat, cut sections dry or wet with alcohol, 
 and remove the soap with warm water or alcohol. 
 
 Staining. — The staining of vegetable sections is a very 
 simple matter. Many sections take the stain more readily 
 if they are first bleached. Sections cut without imbed- 
 
BOTANICAL SECTIONS. 
 
 Ill 
 
 ding, or after being freed from paraifin, are placed in 
 water and then in an aqueous solution of calcium 01 
 sodium hypochlorite in a stoppered bottle. Ihe time 
 required for bleaching varies with different specimens 
 from one or two hours to one or two days. Ihe speci¬ 
 mens must be watched to guard against maceration. If 
 the action is slow, decant the calcium hypochlorite and 
 add a fresh portion from time to time. The bleaching 
 will be more complete if the sections are transferred from 
 the calcium hypochlorite to very dilute hydrochloric acid 
 for a few minutes. After the acid wash thoroughly in 
 water to remove the acid. The sections may now be 
 stained in carmine, hsematoxylin, or in aniline dyes. The 
 Appendix gives formulas for preparing the staining solu¬ 
 tions, but the beginner is advised to obtain the stains 
 ready prepared by some dealer in microscopical supplies. 
 After some experience has been acquired with the ready- 
 prepared stains, trials may be made in preparing one s 
 own stains. It is best to confine one’s self to a very few 
 kinds until some experience has been gained. As a lule 
 alcoholic stains are more satisfactory than the aqueous 
 stains, if the object is to be mounted in balsam. In using 
 the carmine stains it is to be remembered that it is very 
 important that the object be immersed in alcohol of the 
 same strength as the stain before staining, and transfeired 
 from the stain to alcohol of the same strength. If this 
 precaution is not observed, solid particles of carmine will 
 be precipitated in the cells of the tissue. Ihe object is 
 then passed through the higher grades of alcohol to com¬ 
 plete dehydration. 
 
 Objects cut in celloidin may be transferred to water or 
 dilute alcohol and stained in carmine or lisematoxylin. 
 The celloidin is not colored by these stains, and the sec- 
 
112 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 tions are mounted without removing the celloidin. Ani¬ 
 line dyes stain the celloidin, and must not be used for 
 these sections. After staining transfer to alcohol of the 
 same strength as the stain, then to water, then to glycerine, 
 in which the sections should soak some minutes before 
 mounting. Mount in glycerine or glycerine-jelly. If 
 Canada balsam is to be the mounting medium, transfer the 
 section from the stain to alcohol of the same strength as 
 
 O 
 
 the stain, dehydrate in 95 per cent alcohol very quickly, 
 clear with chloroform, oil of cedar, oil of bergamot, or oil 
 of origanum, but not with oil of cloves, which dissolves 
 celloidin. 
 
 It is to be remembered that the object of staining is to 
 differentiate structure, and that that staining is most suc¬ 
 cessful which brings out most perfectly the different parts 
 of the specimen. Overstaining obscures structure and 
 defeats the very purpose for which the staining is under¬ 
 taken. Delicate staining also, as a rule, gives results that 
 are in better taste than deeply stained objects. The 
 thicker the section, the more careful must one be not to 
 overstain. It is also to be remembered that many speci¬ 
 mens are not suitable for double staining. The more 
 homogeneous the structure, the less likely is the specimen 
 to take two different stains. 
 
 The same dye prepared by different formulas acts dif¬ 
 ferently on the same tissues. Experience alone can tell 
 which stain, or which combinations of stains, will give the 
 most satisfactory results. The same staining fluid also 
 acts quite differently on sections of different plants. The 
 acidity, alkalinity, or other chemical properties of the 
 plant tissue modify the effect of the stains, so that each 
 plant is, as it were, a law to itself in its behavior towards 
 the staining fluid. 
 
BOTANICAL SECTIONS. 
 
 118 
 
 Order of Procedure with Vegetable Specimens to be cut in Paraffin. 
 
 1. a. If the material is fresh, dehydrate in 50 per cent, 70 per cent, 
 
 95 per cent, and absolute alcohol. 
 
 b. If the material is alcoholic and hard, soak in alcohol and 
 glycerine, equal parts, until soft; if the material is alcoholic 
 and soft, omit this step. 
 
 2. a. Remove alcohol by soaking in benzole or oil of cloves. Soak 
 
 24 hours in benzole containing all the paraffin it will dis¬ 
 solve. Or, 
 
 b. Soak in chloroform 24 hours, then 24 hours in chloroform in 
 which has been dissolved as much paraffin as will dissolve. 
 
 3. Imbed in paraffin. 
 
 4. Cut sections. 
 
 5. Remove paraffin by soaking in chloroform, turpentine, benzole, 
 
 or oil of cloves. 
 
 6. Remove chloroform, turpentine, benzole, or oil of cloves by 
 
 soaking in tw T o changes of 95 per cent alcohol. Bleach 
 the section, if it seems desirable. 
 
 7. a. Place in 35 per cent alcohol. 
 
 b. Stain in Grenadier's Borax-Carmine. 
 
 c. Place in 35 per cent alcohol, to which has been added a drop 
 
 or two of hydrochloric acid. 
 
 d. Pass through 50 per cent, 70 per cent, and 95 per cent alcohol. 
 S. a. Stain in an alcoholic solution of iodine green or methyl 
 
 green. 
 
 b. Wash out excess of stain in 95 per cent alcohol, and place for 
 a few minutes in absolute alcohol. 
 
 9. Clear in oil of cedar or benzole. 
 
 10. Mount in pure Canada balsam. 
 
 Number 8 may be omitted. In this case, place the sec¬ 
 tions in absolute alcohol, clear in oil of cedar, oil of cloves, 
 oil of marjoram, oil of turpentine, or benzole. Mount in 
 pure balsam or in balsam dissolved in xylol, benzole, or 
 chloroform. Sections stained in the aniline dyes are liable 
 to fade in oil of cloves and in xylol, chloroform, or benzole 
 balsam, or in glycerine, or glycerine-jelly. They should, 
 
114 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 therefore, be cleared in benzole or oil of cedar and mounted 
 in pure balsam. Even then the aniline dyes are not very 
 reliable for permanent mounts. Sections stained with these 
 dyes should be kept in the dark, as exposure to strong light 
 fades the colors. 
 
 If the sections are to be mounted in glycerine or glyce¬ 
 rine-jelly, the above order may be adhered to through the 
 staining. The sections pass from the stain into alcohol 
 of the same strength as the stain, then into water. They 
 are then soaked some time in glycerine, and mounted in 
 glycerine or glycerine-jelly. 
 
 In the place of numbers 7 and 8 may be substituted any 
 of the stains mentioned in the Appendix, taking care to 
 treat the specimen before and after the staining accord¬ 
 ing to the directions given with the formula for each 
 stain. 
 
 Order of Procedure with Vegetable Specimens to be cut in Celloidin. 
 
 1. a. If the material is fresh, dehydrate in alcohol of gradually 
 
 increasing strength. 
 
 b. If the material is alcoholic and hard, soak in alcohol and glyce¬ 
 rine, equal parts, until soft; if the material is alcoholic and 
 soft, omit this step. 
 
 2. Soak 24 hours in thin celloidin. 
 
 3. Soak 24 hours in thick celloidin. 
 
 4. Imbed in celloidin and immerse in 82 per cent alcohol for 24 to 
 
 48 hours. 
 
 5. Cut sections, keeping the knife and specimen wet with 50 to 70 per 
 
 cent alcohol. Bleach the sections, if necessary. 
 
 6. a. Place in 35 per cent alcohol with the celloidin on the sections. 
 
 b. Stain in Grenacher’s Borax-Carmine. 
 
 c. Place in 35 per cent alcohol to which a few drops of hydro¬ 
 
 chloric acid have been added. 
 
 d. Place in 82 per cent alcohol. 
 
BOTANICAL SECTIONS. 
 
 115 
 
 7. a. Place in 95 per cent alcohol for not more than 30 seconds. 
 
 b. Place on a glass slip and flood with chloroform, to clear the 
 
 section. Apply the chloroform at least twice to insure per¬ 
 fect clearing. 
 
 c. Working quickly, before the chloroform can evaporate, drop 
 
 on pure or chloroform balsam, and cover with a cover-glass. 
 
 In place of number 6 may be substituted hsematoxylin 
 or one of the other carmine solutions, remembering the 
 precautions as to treatment before and after staining. To 
 secure success with the celloidin method, the dehydration 
 must be rapidly accomplished, and chloroform must be 
 used as the clearing agent. The essential oils, such as oil 
 of cloves, oil of cedar, etc., either dissolve the celloidin 
 or cause the section to shrivel, and should not be used by 
 preference, although it is true that they are so used by 
 some workers. 
 
CHAPTER IX. 
 
 SECTIONS OF ANIMAL TISSUES. 
 
 Hardening. — Most animal tissues require to be hardened 
 before they can be cut into thin sections. For most 
 tissues ordinary alcohol is the best as well as the most 
 convenient reagent to accomplish this. Care must be 
 taken, however, with delicate tissues, or they will be 
 distorted and contracted because of the shrinkage which 
 results from the fact that the alcohol removes the water 
 from the tissue faster than the alcohol can take its place. 
 With most tissues it is sufficient to begin with 60 per cent 
 and pass through 70 per cent and 95 per cent alcohol. 
 With the more delicate tissues it is best to begin with 40 
 per cent and pass through 50 per cent, 60 per cent, 70 per 
 cent, and 95 per cent alcohol. The time should be short 
 in the lower grades, as there is danger of maceration if 
 the object is left too long in them. 
 
 The specimen to be hardened should be cut into small 
 pieces. Persons not accustomed to the process ordinarily 
 use pieces that are far too large. A piece one-third of an 
 inch square and one-fourth of an inch thick will give 
 more sections than can be used, and it is only a waste of 
 time and of alcohol to use larger pieces. The larger 
 pieces not only do not harden so quickly, but they do not 
 harden so uniformly. Using a piece of one of the softer 
 tissues — a piece of liver, for instance — of the above 
 dimensions, wash it in cold water, and place it in 40 per 
 cent alcohol for one hour; then in 50 per cent alcohol, two 
 
 117 
 
118 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 hours; 60 per cent, six hours; 70 per cent, twenty-four 
 hours; 95 per cent, twenty-four hours; and it will ordi¬ 
 narily be sufficiently hardened. The volume of the alcohol 
 should in each case exceed the volume of the specimen 
 many times. Pint bottles with wide mouths are conven¬ 
 ient vessels to contain the alcohol. Only one or two 
 pieces of the tissue should be immersed at a time in each 
 of these bottles, and the alcohol should be changed after 
 having been used a few times, the weakest being thrown 
 away and that in each of the other bottles being consid¬ 
 ered as of the next lower grade, the strongest alone being 
 renewed. 
 
 Staining. — Many animal tissues in pieces of the size 
 above mentioned may be stained in toto and better results 
 obtained than when the staining is done after the sections 
 are cut. Tissues which are found by experience not to 
 stain well as a whole may be prepared for imbedding and 
 cutting, and be stained after the sections are cut. Car¬ 
 mine and hsematoxylin stains are most commonly used for 
 animal preparations. The pieces are immersed for twenty- 
 four hours in alcohol of the same strength as the stain to 
 be used. If Kleinenberg’s hsematoxylin is to be used, 
 immerse the pieces in 70 per cent alcohol; if Grenadier’s 
 borax-carmine is to be used, immerse in 35 per cent 
 alcohol. It is impossible to give definite directions as to 
 the time required for staining. The pieces must be 
 watched, and when they appear to be deeply stained they 
 are removed to alcohol of the same grade as the stain, 
 made slightly acid with hydrochloric acid. They remain 
 in the acid alcohol twenty-four hours, and are trans¬ 
 ferred to alcohol of the same strength, and pass through 
 the higher grades to complete dehydration in absolute 
 alcohol. 
 
SECTIONS OF ANIMAL TISSUES. 
 
 119 
 
 Imbedding in Paraffin. — The alcohol must now be 
 removed from the specimen to prepare it for the pene¬ 
 tration of the paraffin. Place in chloroform for twenty- 
 four hours, adding a little sulphuric ether or oil of cloves 
 to the chloroform; then place for another twenty-four 
 hours in a solution of paraffin in chloroform, at the ordi¬ 
 nary temperature; then in some of the same solution on 
 the water-bath, maintaining a temperature of about 130 F. 
 for several hours, or until bubbles cease to rise from the 
 specimen; then in clear paraffin on the water-bath for 
 several hours, at the same temperature. It is then ready 
 for imbedding in the manner described under botanical 
 
 specimens. 
 
 On account of the cost of chloroform, turpentine is 
 most commonly used to remove the alcohol and piepaie 
 the specimen for the paraffin, though it is open to the 
 objection that it has a tendency to render objects brittle. 
 The above process need be modified only by substituting 
 the turpentine for the chloroform. The process is, how¬ 
 ever, sometimes abridged. The object is placed in turpen¬ 
 tine, at the ordinary temperature, for some hours, or until 
 it appears clear. It is then placed directly in melted 
 paraffin on the water-bath for several hours, and then 
 imbedded. The temperature of the paraffin should not 
 in any case be allowed to rise much above the melting- 
 point of the paraffin. 
 
 Oil of cloves may also be used in connection with tur¬ 
 pentine. It drives out the alcohol more thoroughly, and 
 tends to better results. The object is transferred from 
 alcohol to a mixture of alcohol and oil of cloves, half and 
 half, then to clear oil of cloves, then to turpentine, and 
 finally to melted paraffin on the water-bath. 
 
 If the casts are spongy, it is an indication of a lack 
 
120 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 of thoroughness in the processes. Such casts should be 
 thrown back into the melted paraffin for further treatment. 
 Casts made by the chloroform method will surely be spongy 
 if the chloroform has not been wholly expelled by heating 
 on the water-bath. When the details of this method are 
 carefully attended to, the results cannot be surpassed. 
 
 Imbedding in Celloidin. — The method of imbedding ani¬ 
 mal specimens in celloidin is precisely the same as that 
 detailed under botanical sections, and need not be repeated 
 here. Perfect dehydration before immersing the object 
 in the solution of celloidin is of the greatest importance. 
 The celloidin method gives very perfect results. 
 
 Cutting Sections. Ihe cork bearing the celloidin cast 
 is held in the jaws of the microtome clamp, and the sur¬ 
 face of the specimen and the knife are kept wet with 50 
 to 70 per cent alcohol. 
 
 Ihe paraffin cast is held directly in the jaws of the 
 miciotome, or is fastened to the roughened surface of a 
 cork by means of melted paraffin. It is an advantage of 
 this method that the sections may be cut dry. A section- 
 smoother should be placed on the knife to prevent the 
 sections curling. If there is a tendency to crumbling, 
 smear the surface of the specimen ivith collodion before 
 each section is cut and let it dry a minute or two. 
 
 Mounting. To mount a section from a celloidin cast, 
 transfer it to a glass slip, cover it with chloroform, drain, 
 and cover again with chloroform, add pure or chloroform 
 balsam before the chloroform evaporates, and cover with 
 a cover-glass, do mount a section from a paraffin cast, 
 diy a clean slide, place it on the turn-table, and with a 
 camel s-hair brush apply to its centre a very thin coat of 
 collodion fixative, transfer a section from the microtome 
 knife to the centre of the slide with a camel’s-hair brush, 
 
SECTIONS OF ANIMAL TISSUES. 
 
 121 
 
 and smooth it out by pressing gently on it with a piece of 
 smooth oiled paper. Place the slide in the oven of the 
 water-bath, the temperature being maintained at about 
 130° F., to evaporate the clove oil from the fixative. The 
 section will then be firmly secured to the centre of the 
 glass slip. Remove the paraffin from the section by pour¬ 
 ing upon it a large drop of turpentine. In a few minutes 
 drain off the turpentine and add a fresh drop. It may be 
 necessary to repeat this operation three or four times to 
 remove all traces of the paraffin. Another way is to 
 immerse the slip bearing the section in a bottle of turpen¬ 
 tine for fifteen or twenty minutes. Wipe the slip free of 
 turpentine and mount at once in Canada balsam. 
 
 If it is desired to mount in glycerine or glycerine-jelly, 
 remove the turpentine by immersing the slip in two 
 changes of 95 per cent alcohol, remove the alcohol by 
 immersing in water, pour a large drop of glycerine on the 
 object, drain it off, and then mount in glycerine or glyce¬ 
 rine-jelly. 
 
 Order of Procedure with Animal Tissues when the Object is to be stained 
 in toto and cut in Paraffin. 
 
 1. Harden in alcohol of gradually increasing strength. 
 
 2. a. Place in 70 per cent alcohol. 
 
 b. Stain in Kleinenberg’s hsematoxylin. 
 
 c. Place in 70 per cent alcohol, slightly acidulated with hydro¬ 
 
 chloric acid, if overstained; otherwise omit this step. 
 
 d. Wash thoroughly in 70 per cent alcohol, and dehydrate with 
 
 95 per cent and absolute alcohol. 
 
 3. Soak in chloroform or turpentine for 24 hours. 
 
 4. Soak for 24 hours in chloroform or turpentine containing all the 
 
 paraffin that will dissolve. 
 
 5. Place in the same solution on the water-bath, temperature about 
 
 130° F., for several hours. 
 
122 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 6. Place in clear paraffin on the water-bath at the same temperature 
 
 for several hours. 
 
 7. Imbed in paraffin. 
 
 8. Cut sections dry. 
 
 9. Fix on glass slip with collodion fixative. 
 
 10. Place the glass slip in the oven of the water-bath, temperature 
 
 about 130° F., until the clove oil is evaporated from the fixative. 
 
 11. Remove paraffin with turpentine. 
 
 12. Mount in Canada balsam. Or remove the turpentine with alco¬ 
 
 hol, place in water, then in glycerine, and mount in glycerine or 
 glycerine-jelly. 
 
 In place of number 2 may be substituted Grenadier’s 
 hsematoxylin or any of the carmine stains, observing the 
 precautions as to treatment before and after staining. 
 
 Order of Procedure with Animal Tissues to he cut in Paraffin and stained 
 after they are cut. 
 
 1. Harden in alcohol of gradually increasing strength. 
 
 2. Clear by soaking in chloroform or turpentine 24 hours. 
 
 3. Place for 24 hours in chloroform or turpentine containing all the 
 
 paraffin that will dissolve. 
 
 4. Place for several hours iit*melted paraffin on the water-bath, tem¬ 
 
 perature about 130° F. 
 
 5. Imbed in paraffin. 
 
 6. Cut sections dry. 
 
 7. Fix sections on glass slip with collodion fixative. 
 
 8. Place slip in the oven of the water-bath, temperature about 
 
 130° F., to evaporate the clove oil from the fixative. 
 
 9. Immerse the glass slip bearing the section in a bottle of turpen¬ 
 
 tine for 15 or 20 minutes. 
 
 10. Remove the turpentine from the section by immersing the slip in 
 
 two different bottles of 95 per cent alcohol. 
 
 11. a. Cover the specimen with 70 per cent alcohol. 
 
 h. Immerse the slip in a bottle of Kleinenberg’s hmmatoxvlin, 
 or put a drop of the stain on the specimen, letting it remain 
 until the section is sufficiently colored. 
 
SECTIONS OF ANIMAL TISSUES. 
 
 123 
 
 c. Immerse in 70 per cent alcohol, slightly acidulated with hydro¬ 
 
 chloric acid, if overstained ; otherwise omit. 
 
 d. Dehydrate in 70 per cent, 95 per cent, and absolute alcohol. 
 
 12. Remove the alcohol with benzole or turpentine (do not use oil of 
 
 cloves, as it will dissolve the collodion fixative), and mount in 
 Canada balsam. Or, omitting the dehydration in number 11, 
 place in water, then in glycerine, and mount in glycerine or 
 glycerine-jelly. 
 
 Order of Procedure with A nimal Tissues to be stained in toto and then 
 imbedded in Celloidin. 
 
 1. Harden in alcohol of gradually increasing strength. 
 
 2. Stain in hsematoxylin or carmine with the proper treatment 
 
 before and after staining. 
 
 3. Dehydrate in 95 per cent and absolute alcohol. 
 
 4. Soak in thin celloidin for 24 hours. 
 
 5. Soak in thick celloidin for 24 hours. 
 
 6. Imbed in thick celloidin on a cork in the manner already de¬ 
 
 scribed. 
 
 7. Immerse in 82 per cent alcohol for 24 to 48 hours. 
 
 8. Cut sections, keeping the knife and specimen wet with 50 to 
 
 70 per cent alcohol. 
 
 9. Place a section on the centre of a glass slip and cover it with 95 
 
 per cent alcohol. Immediately drain off the alcohol and covei 
 with chloroform. Repeat the chloroform twice, and before the 
 chloroform can evaporate mount in chloroform balsam or pure 
 balsam. 
 
 Order of Procedure with A nimal Tissues to be imbedded in Celloidin, the 
 Sections to be stained after cutting and fixed in Position on the Glass 
 Slip. 
 
 1. Harden in alcohol of gradually increasing strength to perfect 
 
 dehydration in absolute alcohol. 
 
 2. Soak in thin celloidin for 24 hours. 
 
 3. Soak in thick celloidin for 24 hours. 
 
 4. Imbed in celloidin. 
 
 5. Immerse in 82 per cent alcohol for 24 to 48 hours. 
 
124 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 6. Cut sections, keeping the knife and specimen wet with 50 to 70 
 
 per cent alcohol. 
 
 7. Stain in hsematoxylin or carmine. 
 
 8. Place in 95 per cent alcohol not more than 30 seconds. 
 
 9. Place on the centre of a glass slip, and fix in position by placing- 
 
 on the section just enough of a thin solution of bleached shellac 
 in alcohol to cover it. 
 
 10. Place in the oven of a water-bath, temperature about 130° F., to 
 
 evaporate the alcohol. 
 
 11. Clear in chloroform and mount in Canada balsam. 
 
CHAPTER X. 
 
 CHEMICAL CRYSTALS. 
 
 The preparation of chemical crystals, eithei foi tem¬ 
 porary examination or for permanent mounting, is both 
 easy and attractive. A very large number of chemical 
 substances crystallize in most beautiful forms from solu¬ 
 tions. The same chemical often gives crystals of very 
 different forms when treated in different ways. For many 
 substances it is necessary only to make a solution in 
 water, place a drop of the solution on a cover-glass or 
 glass slip, and allow it to evaporate at the 01 dinary tem¬ 
 perature of the room. The solution may be made in a 
 small test-tube or in a watch glass. Very small quantities 
 only of the chemicals and of the solvent should be used, 
 as but a small drop of the solution is needed. Another 
 method of making the solution is to place a drop of water 
 or other solvent on a cover-glass or glass slip, add a few 
 particles of the chemical, and apply gentle heat. 
 
 Different forms of crystals are obtained from solutions 
 of different strengths. If saturated solutions do not give 
 good crystals, especially if the results are massive and 
 coarse, try weaker solutions. Some of the most delicately 
 beautiful crystals form from very weak solutions. Othei 
 solvents than water may be used, as alcohol, ethei, chloio- 
 form; and the greatest variety of interesting forms of 
 crystals results. The forms also vary according as the 
 evaporation is rapid or slow. The best results are obtained 
 from some substances by retarding the evaporation, by 
 
 125 
 
126 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 placing the cover-glass with the drop of solution on it in 
 a damp, cool cellar, until the crystals are formed; sugar is 
 an example of such substances. Crystallization may also 
 be retarded by adding to the solution gelatine in varying 
 quantities. A few strips of gelatine are placed in a test- 
 tube with a considerable volume of water and, after soak¬ 
 ing some minutes, are dissolved by the application of heat. 
 A little of this solution is poured into another test-tube or 
 into a watch glass, and the chemical under treatment is 
 dissolved in it. The crystallization is allowed to take 
 place at the ordinary temperature of the room. Many of 
 the common chemical salts give excellent results when 
 treated by this method. 
 
 Some solutions need to be evaporated by the application 
 of artificial heat or by exposure to the direct rays of the 
 sun. Potassium chlorate, potassium chromate, potassium 
 dichromate, potassium oxalate, potassium permanganate, 
 potassium iodide, sodium chloride, cobalt chloride, ammo¬ 
 nium nitrate, copper nitrate, oxalic acid, cadmium sulphate, 
 iron sulphate, barium chloride, nickel nitrate, and many 
 other common chemical salts may be dissolved in water 
 and treated by this method. 
 
 In other cases no solution is made, but a small amount 
 of the solid substance is placed on a cover-glass and held 
 a few inches above a flame until it melts. The cover-glass 
 is then withdrawn from over the flame and, upon cooling, 
 the substance recrystallizes in its own water of crystalliza¬ 
 tion. Salicine, santonine, and benzoic acid are substances 
 which give especially fine results by this method. 
 
 For temporary examination a solution of the chemical 
 salt is made by one of the methods described, and a few 
 drops placed on a glass slip previously somewhat warmed. 
 The slip is placed on the microscope stage, slightly inclined, 
 
CHEMICAL CRYSTALS. 
 
 127 
 
 and the crystallization watched with a low power, — two 
 inch, one inch, or three-fourths inch objective, — under polar¬ 
 ized light. If crystallization does not begin within a few 
 minutes, the slip is again slightly warmed. A cover-glass 
 should not be placed over the crystals in this examination. 
 
 Interesting and instructive crystals are also foimed 
 by filling shallow cells, made by cementing rings to glass 
 slips, as described in Chapter VI., with a saturated solution 
 of the chemical under examination and setting aside, pro¬ 
 tected from the dust, until the crystals are formed. A 
 thread laid diametrically across the cell, just touching the 
 liquid, will often cause the crystallization to start along 
 this diameter and so be in a convenient part of the cell 
 for observation. The crystals formed in a considerable 
 quantity of liquid are much more characteristic of the 
 substance than the tabular crystals, which result when a 
 thin layer of liquid crystallizes on a flat surface. For 
 this reason no study of the crystallization of a substance 
 is complete until an examination has been made of the 
 crystals formed in the “mother liquor.” The crystals 
 may be permanently preserved in these cells. After the 
 crystals have been formed, fill the cell even full of the 
 saturated solution of the substance used, place the slip on 
 the turn-table, and run a fresh ring of shellac cement 
 on the outer edge of the cell, breathe on a cover-glass, 
 place it over the cell, press it gently down, and see that 
 there are no air bubbles. When dry, finish as already 
 described for cell mounts. Slides thus prepared should be 
 handled carefully, to prevent displacement of the crystals 
 by sudden jars. 
 
 For permanent mounting it is better, as a rule, to form 
 the crystals on the cover-glass rather than on the slip. A 
 cover-glass is cleaned, slightly warmed, a drop of the solu- 
 
128 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 tion of the chemical in water, gelatine-water, alcohol, 
 ether, chloroform, or other solvent, is placed on the centre, 
 and the crystals are allowed to form. It is not to be ex¬ 
 pected that every trial will give results worthy of preser¬ 
 vation, but a little patience will meet with sure reward. 
 I he crystals may be mounted in various ways. Carpenter 
 recommends castor oil as the best preservative. Balsam 
 mounting is much easier, and is most excellent for those 
 substances that will stand the balsam without dissolving. 
 The following substances, crystallized by this method, are 
 permanent in pure balsam: potassium chlorate, potassium 
 ferrocyanide, potassium ferricyanide, potassium oxalate, 
 potassium chromate, potassium dichromate, copper sul¬ 
 phate, iron sulphate, tartaric acid, ammonium oxalate, 
 boiax, salicine, cane sugar, and doubtless many others. 
 
 In mounting, a drop of pure Canada balsam is placed on 
 the centre of a glass slip, the cover-glass on which the 
 crystals have formed is inverted over it, centred, and gen¬ 
 tly pressed down, care being taken to disturb the crystals 
 as little as possible. The balsam must be allowed to 
 harden at the ordinary temperature. 
 
 A method applicable with a few substances, and giving 
 most beautiful results, is to place a small drop of balsam 
 on a slide, heat until the volatile matters are driven off, 
 then add a little of the solid chemical, and heat again until 
 solution of the chemical in the balsam is effected, put on 
 a warm cover-glass properly centred, and set it aside to 
 cool. This method possesses the advantage of effecting 
 the mounting with a minimum of trouble, and the added 
 advantage that the slide may, immediately upon cooling, 
 be freed from the superfluous balsam and receive the fin¬ 
 ishing rings of cement. Snlphonal and salicine give excel¬ 
 lent results by this method. 
 
CHEMICAL CRYSTALS. 
 
 129 
 
 Most chemical crystals may be preserved in dry mounts. 
 A shallow cell is necessary, best formed of shellac cement, 
 though paraffin cells are popular for this purpose. It is 
 best to have on hand a quantity of cells ready prepared, 
 of various depths, and to use in each case one that is 
 but little deeper than the thickness of the crystals to be 
 mounted. Crystals of deliquescent and efflorescent sub¬ 
 stances, if mounted dry, must be mounted and thoroughly 
 sealed immediately after they are formed and before the 
 air has had time to act upon them. 
 
 In the examination of crystals polarized light should be 
 employed. Even crystals belonging to the cubical system 
 are brought out more clearly in some positions of the 
 Nicols than they are in ordinary light. These crystals do 
 not polarize, of course, unless, as sometimes happens, the 
 film is subjected to a strain while the crystals are forming. 
 Crystals of other systems polarize with endless variety of 
 colors. The selenite plate accompanying the polarizing 
 apparatus, or in lack of this a selected film of mica, placed 
 on the stage under the slide, gives a colored field which 
 aids in bringing out the forms and colors for crystals which 
 do not happen to be of the right thickness to polarize well 
 of themselves; but the use of the selenite detracts rather 
 than adds to the interest of other crystals which are of 
 suitable form and thickness to give brilliantly beautiful 
 colors without adventitious aid. In the course of the 
 examination, the rotating Nicol should, of course, be turned 
 to obtain different color effects, and to enable the observer 
 to watch the change of each color to its complementary. 
 
 It is hardly necessary to give an extended list of chemi¬ 
 cal substances from which crystals may be formed. Many 
 nitrates, some sulphates, most chlorates, oxalates, citrates, 
 and tartrates form polarizing crystals. 
 
CHAPTER XI. 
 
 ROCK SECTIONS BY SIMPLE MEANS. 
 
 Within recent years the study of the microscopic 
 structure of rocks has come to be recognized as an impor¬ 
 tant feature of geological investigation. The microscope 
 helps in the determination of the soundness and of the 
 purity or impurity of minerals, and aids in deciding upon 
 the nature and character of the impurities. Before the 
 application of the microscope to rock sections, chemical 
 analysis was the only means of determining the composi¬ 
 tion of rocks. In many instances where chemical analysis 
 fails, the microscope decides with certainty on the mineral 
 composition; and in the case of certain minerals, nearly 
 all that we know of their origin has been learned from the 
 microscopical study of their sections. It is true that the 
 microscope cannot settle all questions, hut it has already 
 largely superseded chemical analysis where the end in 
 view is simply the determination of the mineral ingredi¬ 
 ents ; and in most cases where a quantitative determination 
 of the minerals is to be made by chemical processes, the 
 microscope lends important aid in making the preliminary 
 qualitative determination. The value of the microscopical 
 study of rocks is being more and more recognized. 
 
 The practical use of the microscope for these purposes 
 involves a vast amount of stud} r — study of great interest, 
 and which, on account of the newness of this line of re¬ 
 search, offers peculiar attractions. It is not the presump¬ 
 tion of this chapter to give an exhaustive treatment of 
 
 131 
 
132 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 petrology, but to state simple methods for the preparation 
 of thin sections of rocks for microscopical study, with a 
 few hints at the methods of studying these sections. 
 
 Specialists have devised special apparatus for the prepa¬ 
 ration and examination of rock sections, which is quite' 
 out of the reach of most private workers on account of 
 the expense involved. A great deal may be done with 
 the usual microscopical outfit. Many sections of rocks 
 yield very interesting results when examined by ordinary 
 light; for the examination of most rocks polarized light is, 
 however, essential. A revolving stage gives an additional 
 means for their study. A plate of selenite or mica gives 
 a colored field which, in many cases, is a help in bringing 
 out the characteristic features of minerals. This outfit of 
 apparatus is all that is really necessary for a large amount 
 of work in this interesting line of microscopical investi¬ 
 gation. 
 
 If one has the use of a lathe, slices of rocks may be 
 sawed by means of a thin disk of iron or copper charged 
 at its edge with diamond dust, and these slices may then 
 be ground down on emery wheels. This method of work¬ 
 ing, however, involves too much expense for most private 
 workers. 
 
 Very simple means suffice for much interesting and 
 instructive work in grinding rock sections. All that is 
 needed is a smooth piece of iron — an old stove cover or a 
 piece of smooth sheet iron will do — some coarse emery, 
 say, No. 120, or even coarser for many rocks, some flour 
 of emery, bits of window glass, and some Canada balsam. 
 
 For a specimen to begin with, granite is good. Select 
 a granite that is firm and sound. With a hammer chip 
 off a piece about half an inch square and as thin as pos¬ 
 sible. Place a little of the coarse emery on the iron plate, 
 
ROCK SECTIONS BY SIMPLE MEANS. 
 
 133 
 
 together with a little water. Holding the specimen firmly 
 with the fingers, grind one side flat. Wash off the coarse 
 emery, and continue the grinding with flour of emery in 
 water on a plate of window glass until the surface is quite 
 smooth. Wash the specimen again and dry it. Now place 
 a few drops of Canada balsam on a piece of window glass 
 about an inch square, and heat to drive off the volatile 
 portions of the balsam. A spring clothes-pin makes an 
 excellent holder for this operation. The balsam, after 
 cooling, should be quite hard and firm, but not brittle. 
 Care should be taken not to let the balsam take fire. 
 Place the specimen, smooth side down, in the warm bal¬ 
 sam, and press down upon it gently until the balsam has 
 cooled, seeing that no air bubbles are enclosed between 
 the specimen and the piece of glass. When the balsam is 
 cold, the specimen will be firmly cemented to the glass. 
 Using the glass as a holder, continue to grind until the 
 specimen is reduced in thickness to such a degree that 
 light is readily transmitted through it. 
 
 The specimen should be examined at intervals under 
 the microscope to watch the progress of the work. Wash 
 it free of the emery, place a drop of water on it, and cover 
 with a cover-glass. As the specimen becomes thin, care 
 should be taken that the balsam is not worn away up to 
 its edge. If this happens, the specimen may be rapidly 
 broken away. The remedy is to clean and dry the speci¬ 
 men, place a little fresh balsam on it, drive off the volatile 
 matter, and let the balsam set around the specimen again. 
 The latter portion of the grinding should be done in the 
 flour of emery on a plate of glass, as before. 
 
 When the specimen has been reduced to the desired 
 degree of thinness, place on it a large drop of Canada 
 balsam, and heat until the object is released from the piece 
 
134 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 of glass. Then with a needle carefully slide the specimen 
 into a watch glass of alcohol, and let it remain until the 
 balsam and contained dirt have been removed from it, 
 draining off the alcohol and adding a fresh portion once 
 or twice, if necessary. The specimen may now be lifted 
 by means of a camel’s-hair brush, and placed on the centre 
 of a microscope slide, dried carefully in gentle heat, and 
 mounted in Canada balsam. 
 
 Very brittle specimens are liable to be broken in the 
 process of removal from the glass holder to the alcohol. 
 This may in a measure be guarded against by placing a 
 cover-glass over the specimen after the fresh balsam has 
 been added. Heat and remove specimen and cover-glass 
 together to the alcohol. The cover-glass will also serve 
 to support the specimen in removing it from the alcohol to 
 the slide. See that the section is on the centre of the 
 cover-glass, remove, drain, and dry. Have ready a slide 
 with a drop of balsam on its centre, invert the cover-glass 
 and specimen over the balsam, and centre. In the case of 
 excessively brittle specimens, it is best to omit the wash¬ 
 ing in alcohol. Remove the specimen, supported by a 
 cover-glass, directly to a glass slip on which a drop of 
 balsam has been previously placed. 
 
 The amount of material available for the microscopical 
 study of rocks is unlimited. The dark rocks are, in gen¬ 
 eral, so opaque that they must be ground to extreme thin¬ 
 ness unless the sections are to be examined by reflected 
 light; but any of the lighter colored stones yield good 
 results. A cabinet of minerals affords numerous speci¬ 
 mens for instructive study. Fossils often yield most beau¬ 
 tiful specimens. Silicified wood, for instance, though very 
 hard and requiring much patience in grinding, amply com¬ 
 pensates for the labor by revealing the minute plant struct- 
 
ROCK SECTIONS BY SIMPLE MEANS. 
 
 135 
 
 ure as plainly as a piece of a growing tree. Pieces of 
 silicified wood are sometimes found which, by a blow from 
 a hammer, cleave into thin films, ready to mount for the 
 microscope. The study of the microscopic structure of 
 fossil plants, though until recently but little attention 
 was paid to it, is now recognized as a valuable adjunct to 
 the study of growing plants. Indeed, it is only by the 
 study of fossil specimens that the historical development 
 of modern plants can be investigated. 
 
 All rocks of crystalline structure afford much interest¬ 
 ing material. When a section of such a rock is viewed by 
 means of polarized light, the variegated coloring is very 
 beautiful. As the analyzer is turned, each color changes 
 to its complementary. The labor bestowed in grinding 
 rock sections is amply repaid if no further object is sought 
 than to obtain beautiful and interesting objects for a 
 cabinet. 
 
 In the study of the sections of a crystal in a rock sec¬ 
 tion, it is to be remembered that the form of the section 
 will depend upon the direction in which the section was 
 cut. A cubical crystal might, for instance, give any of 
 the following shapes: an equilateral, an isosceles, or a 
 scalene triangle, a square, an irregular quadrilateral, or 
 a parallelogram. The same rock section will often give 
 several different sections of crystals of the same mineral. 
 At the most, the study of a small number of sections of 
 the same rock will usually give quite a definite idea of the 
 shapes of the crystals that it contains. In some cases, 
 however, the crystals are much changed in shape by com¬ 
 pression in the rock mass, so that the study of the crystal¬ 
 line structure is made complicated. 
 
 If the revolving stage of the microscope has a gradu¬ 
 ated edge, and the tube of the instrument has centring 
 
136 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 screws, so that its axis may be made to pass accurately 
 through the centre of the stage, the angles of the crystals 
 may be measured. An eye-piece with crossed hairs is 
 used. The point of intersection of the hairs is made to 
 coincide with the vertex of the angle to be measured, 
 and one of the hairs is made to coincide with one side of 
 the angle. The stage is then revolved until the same hair 
 coincides with the other side of the angle, and the number 
 of degrees through which it has been turned is read from 
 the graduated edge of the stage. The measurement of 
 the angles affords valuable means for the identification of 
 crystals. 
 
 The presence or absence of cleavage is another means 
 of identifying minerals. The cleavage lines come out 
 very plainly under the microscope, even when they cannot 
 be made out in the massive structure with the unaided 
 eye. The angle formed between the cleavage planes 
 determines at once several different minerals. It is to be 
 remembered that the direction in which the section is cut 
 relatively to the face of the crystal modifies the size of the 
 angle, so that it is important to determine in what direc¬ 
 tion the section is made with reference to the face of the 
 crystal before relying upon the angle to decide what the 
 mineral is. 
 
 In studying sections of crystals under the microscope, 
 the lack of purity is very noticeable. Minerals which to 
 the unaided eye seem to be perfectly pure are often found 
 to contain large amounts of other substances, either crys¬ 
 talline or amorphous. Crystals enclosed within crystals 
 are very common; these crystals are sometimes large 
 enough to be determined; sometimes so small that identi¬ 
 fication is impossible. Cavities are also frequently found 
 which contain a small bubble of water, or of liquid car- 
 
ROCK SECTIONS BY SIMPLE MEANS. 
 
 137 
 
 \ 
 
 bonic acid, or a small cube of common salt, which has 
 crystallized from the supersaturated salt water that was 
 enclosed in the rock at the time of its formation. 
 
 As we have seen in Chapter III., polarized light fur¬ 
 nishes the means for determining whether a substance is 
 singly refractive or doubly refractive. If some portions 
 of a rock section, viewed in polarized light, do not restore 
 the light when the Nicols are crossed, and do not give 
 color effects when the analyzer is turned, or when the 
 stage is rotated, it is at once known that they are singly 
 refractive or isotropic. If, under these conditions, the light 
 is restored or color effects are produced, these portions of 
 the section are doubly refractive or anisotropic. 
 
 The singly refractive portions of the section are either 
 amorphous, or are crystals of the isometric system, or are 
 sections of crystals of other systems cut perpendicularly to 
 an optic axis. Amorphous substances are readily distin¬ 
 guished from crystalline substances, from the fact that the 
 latter almost invariably show either a definite polygonal 
 outline or cleavage lines. Isometric crystals may be dis¬ 
 tinguished from crystals of other systems cut perpen¬ 
 dicularly to an optic axis by means of convergent polarized 
 light. To obtain the convergent polarized light, petro- 
 graphical microscopes are provided with a convex lens, to 
 be mounted just above the polarizer, or with some other 
 means of converging the light. If a section of an isometric 
 crystal, cut in any direction, is viewed in this light, nothing 
 more is noticed than when ordinary light or parallel polar¬ 
 ized light is used. Sections of crystals belonging to the 
 tetragonal and hexagonal systems cut perpendicularly to 
 the vertical axis, which, as we have seen, act like isometric 
 crystals in parallel polarized light, when viewed in conver¬ 
 gent polarized light, show rings of prismatic colors and 
 
138 PRACTICAL METHODS IN MICROSCOPY. 
 
 a distinct black cross when the Nicols are crossed. This 
 cross remains stationary when the crystal is revolved. It 
 is changed to a white cross as the analyzer is revolved, 
 while at the same time the colors of the rings change to 
 their complementaries. These basal sections of hexagonal 
 and tetragonal crystals are further distinguished by the 
 outline of the section, which is a polygon of some multiple 
 of three sides for the hexagonal crystals, and of four or 
 eight sides for tetragonal crystals. There is but one direc¬ 
 tion in hexagonal and tetragonal crystals, viz. that parallel 
 with the vertical axis, along which parallel polarized light 
 can pass without modification, and it is only when con¬ 
 vergent polarized light passes in this direction that the 
 rings of prismatic colors and the black cross result. 
 Parallel polarized light passing in any other direction 
 through crystals of these systems is again polarized. The 
 field is dark when the vertical axis of the crystal corre¬ 
 sponds to the planes of vibration of either Nicol, light in 
 other positions of the crystal, the Nicols being crossed. 
 Color effects are produced as the crystal is rotated or the 
 analyzer turned. These crystals are uniaxial. 
 
 In the case of crystals of the orthorhombic, monoclinic, 
 and triclinic systems, there are two directions in which 
 parallel polarized light passes unmodified. In all other 
 directions it is doubly refracted. These crystals are 
 biaxial. A section perpendicular to either of the optic 
 axes behaves like an isotropic substance in parallel polar¬ 
 ized light. In convergent polarized light, more or less 
 elliptical rings of prismatic colors are seen; but instead 
 of a black cross, as in basal sections of crystals of the 
 tetragonal and hexagonal systems, when the Nicols are 
 crossed, a single bar of black will be seen. A further 
 distinguishing characteristic is the fact that when the 
 
ROCK SECTIONS BY SIMPLE MEANS. 139 
 
 crystal is revolved the black bar revolves in the opposite 
 direction. 
 
 If a section of a biaxial crystal, cut perpendicularly to 
 the line which bisects the acute angle formed by the optic 
 axes, is viewed in convergent polarized light with the 
 Nicols crossed, a series of elliptical rings of prismatic 
 colors with two black hyperbolas will be observed in one 
 position of the crystal. If the crystal is rotated, the 
 hyperbolas give place to a black cross with unsymmetrical 
 arms. If the analyzer is turned, the black hyperbolas 
 change to white, and the prismatic colors to their com- 
 plementaries. 
 
 Any section of an orthorhombic crystal, placed so that 
 any one of the three crystallographic axes is parallel with 
 the planes of vibration of the light, is dark between 
 crossed Nicols. Some sections of monoclinic crystals are 
 dark between crossed Nicols when a crystallographic axis 
 is parallel with the planes of vibration of the light, while 
 others restore the light. In the latter case, if the section 
 is revolved through a certain number of degrees, differing 
 in different crystals, the field becomes dark. Sections of 
 triclinic crystals are never dark between crossed Nicols 
 when a crystallographic axis is parallel with the planes of 
 vibration of the light. On turning the crystal through 
 some degrees, the field becomes dark. 1 he numbei of 
 degrees through which the crystal must be turned in 
 these two cases is a valuable aid towards determining 
 the crystal. The position of the crystallographic axis can 
 usually be determined by the outlines of the crystal oi by 
 cleavage lines. 
 
 The determination of the system to which a crystal 
 belongs is, of course, an important step towards its identi¬ 
 fication. In some cases this determination is quite easy; 
 
140 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 in other cases it is hardly necessary to say that it is most 
 difficult. Systematic study of the crystals in rocks pre¬ 
 supposes extensive training in crystallography, calls for 
 the keenest powers of observation, and the highest exer¬ 
 cise of the reasoning faculties. What has been here said 
 is intended merely to suggest to the interested beginner 
 some of the methods employed in the microscopical exam¬ 
 ination of rock sections. 
 
 Cleavage lines, angles of crystals, outlines of crystals, 
 determination of the system to which crystals belong-, en¬ 
 closure of crystals and amorphous substances in crystals, 
 cavities containing air, water, or liquid carbonic acid, 
 effect on the light, color of the crystals, association of 
 minerals, are, then, some of the characteristics relied upon 
 in the identification of the components of a rock. 
 
 Rolling Sand. Although not strictly belonging under 
 the heading of this chapter, a slide of rolling sand is 
 properly classed with rock specimens. Such a slide is 
 not very difficult of preparation, and is a most interesting 
 addition to one’s collection. Clean quartz sand from the 
 seashore or the beach of a pond or stream is required. A 
 cell of such depth that the sand is free to move after the 
 cover-glass is in position is used. The mounting fluid is 
 glycerine slightly diluted with water. 
 
 Place a small quantity of the sand in a cell, fill the cell 
 full of the fluid, and see that there are no air bubbles 
 clinging to the sand or to the edges of the cell. Run a 
 ring of shellac cement around the outside of the top of 
 the cell, and put the cover-glass in position. See that no 
 air bubbles are enclosed. Clean and finish the slide in the 
 manner already described for glycerine mounts. 
 
 For examination, slightly incline the stage of the micro¬ 
 scope, having the Nicols crossed. Hold the slide edge- 
 
ROCK SECTIONS BY SIMPLE MEANS. 
 
 141 
 
 wise for a moment until the sand runs down to one side. 
 Place the slide in position on the stage with the sand 
 uppermost. As the sand now rolls slowly down (it 
 appears, of course, to roll up hill), a most magnificent 
 display of color changes is seen as the different posi¬ 
 tions of the grains of sand bring different thicknesses of 
 the quartz between the Nicols. The specimen is very 
 showy. 
 
CHAPTER XII. 
 
 THE STUDY OF BACTERIA. 
 
 In the study of bacteria the microscope plays only a 
 subordinate part, yet without it this study would be im¬ 
 possible. The numerous methods of preparation, culture, 
 and isolation of bacteria lead up to an examination under 
 the microscope. Some account of such methods in their 
 simpler forms would seem then to have a place in these 
 pages. Much may be done with powers of the microscope 
 not higher than four or five hundred diameters. The proc¬ 
 esses employed may be learned, and very definite ideas of 
 what bacteria are, and the means used by specialists in 
 their investigations, may thus be acquired. For the more 
 satisfactory study of such preparations, however, a sub¬ 
 stage condenser and the very highest powers of the micro¬ 
 scope are necessary, used with the highest degree of , 
 manipulative skill. It is not to be expected that the 
 finer points of detail will be seen except by these means. 
 
 The beginner is cautioned to remember that bacteria are 
 the minutest organisms in nature, and that they can be 
 seen under moderate powers of magnification only by the 
 closest observation. In looking for them, focus sharply 
 with the fine adjustment and expect to see exceedingly 
 minute rods, spheres, and curves, or, in the case of fila¬ 
 mentous forms, very fine segmented threads. These are 
 all highly transparent, unless they have been submitted 
 to the action of some staining fluid. They may easily be 
 
 143 
 
144 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 overlooked, but a little persevering observation will culti¬ 
 vate the eye to detect them. 
 
 The name bacteria is applied to countless numbers of 
 forms of vegetable organisms. They exist everywhere. 
 They are the active cause of putrefaction and of fermen¬ 
 tation. The decay of all animal and vegetable substances 
 is occasioned directly by the rapid multiplication of dif¬ 
 ferent kinds of these organisms. Many chemical changes 
 in organic substances are due to their agency. The sour¬ 
 ing of milk, for instance, is caused by the action of bacte¬ 
 ria, which convert the sugar of the milk into lactic acid, 
 and this into butyric acid and other products. The devel¬ 
 opment of flavors in the ripening of cheese is brought 
 about through the action of bacteria. It is believed that 
 bacteria play an important part in the stomach and intes¬ 
 tines in the digestion of food. 
 
 Under favorable conditions the multiplication of bacteria 
 is enormously rapid. As long as they can obtain an 
 abundance of food material of the right nature, they mul¬ 
 tiply by fission ; but if food material fails, or the conditions 
 for this kind of development are otherwise not right, a 
 clear round or oval spot appears in each individual organ¬ 
 ism of many of the species. This is the resting-spore. It 
 continues to live without nourishment after the rest of 
 the organism has disappeared; it resists drought and cold. 
 These spores, in the dry condition, are taken up by the air, 
 and are afterwards deposited on all exposed surfaces. 
 When they are brought in contact with suitable nourish¬ 
 ing material they spring into life with great vigor. 
 
 The reproduction is effected by the splitting of the 
 cells. Hence the name, Schizomycetes, or fission-fungi. 
 There are, however, good reasons for believing that they 
 do not belong with the fungi. Various forms occur. All 
 
THE STUDY OF BACTERIA. 
 
 145 
 
 are enclosed in a structureless gelatinous membrane. One 
 general division includes minute spherical cells which 
 divide by a wall running across in one direction, or by 
 walls crossing one another; these receive the general name 
 of Micrococcus. Another division, including straight, 
 rod-like forms, receives the name Bacillus. If the rods 
 are very short and thick, with somewhat rounded ends, the 
 name Bacterium is employed. Slender, long rods are 
 called Leptothrix; thick, long rods, attached at one end 
 of the filament to some supporting object, are called Beg- 
 giatoa. Crenothrix is a name used for a form of large 
 rods having about them an especially distinct gelatinous 
 envelope. Branching forms are called Cladothrix. The 
 cells of the rod-like forms multiply by first increasing in 
 length, and then throwing a partition transversely through 
 the middle of the cell, the new cells formed repeating the 
 process indefinitely, giving rise to chains of cells con¬ 
 nected by a highly transparent membrane. The indi¬ 
 vidual cells are so small that the minute structure is very 
 hard to study. So far as is known, each cell is filled with 
 protoplasm of uniform composition, but differing some¬ 
 what in different species. The belief that protoplasm is 
 present is based upon the fact that only the more opaque 
 portions are stained when iodine is run under the cover- 
 glass. Of the coiled forms, those that are short, stout, and 
 have but few coils are called Spirillum; those that are 
 long, slender, and have many coils are called Spirochseta. 
 Vibrio is a name applied to loosely curved and segmented 
 forms. In each of these general divisions are innumerable 
 individual forms, many of which have been studied and 
 given particular names. It is to be remarked, however, 
 that there is more or less of confusion in the use of these 
 names in the literature of bacteriology. 
 
146 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 The prevailing colors of bacteria are gray, yellowish 
 white, and white. Some species are bright, and are 
 readily recognized in masses as red, blue, green, or brown. 
 In some cases the coloring matter exists in the protoplasm ; 
 more frequently it is found in the envelope of cellulose¬ 
 like material which surrounds the protoplasm. The colora¬ 
 tion arises from the power which bacteria possess of 
 chemically separating the substances in which they thrive, 
 and of forming new compounds, or upon their power of 
 absorbing ingredients from the surrounding substances. 
 Crenothrix and Cladothrix are of a distinct brown color, 
 due to the oxide of iron thus absorbed from the water in 
 which they thrive. The yellow color of Beggiatoa is due 
 to sulphur, which has in a similar way been separated from 
 the sulphur water in which these bacteria grow. The 
 blood-like drops which form on bread and some other 
 substances when left in a moist atmosphere under suitable 
 conditions of temperature are simply masses of bacteria, 
 the Bacillus prodigiosus , which have developed from 
 germs deposited from the atmosphere. The blue color 
 which sometimes under favorable conditions develops in 
 milk is explained in a similar way. The green color 
 which develops on cheese is another example. It is 
 characteristic of bacteria as an order of plants that they 
 are devoid of chlorophyll. Yet there are exceptions to 
 the rule, — at least some forms give the characteristic blue 
 color when treated with iodine solution, showing that 
 starch, the product of chlorophyll, is present. The color 
 is in some cases an important aid towards the identification 
 of the bacteria. 
 
 In connection with color may be mentioned the phos¬ 
 phorescence seen at times on the ocean and on decaying 
 wood. It is caused, in some cases, by different kinds of 
 
THE STUDY OF BACTERIA. 
 
 147 
 
 bacteria, which have the power of separating phosphorus 
 from some of its compounds. 
 
 Some forms of bacteria are endowed with the power of 
 motion, or at least have this power at certain stages of 
 their existence; other forms are incapable of moving. It 
 is probable that in the case of some of the moving forms 
 the motion is effected by the contractions of the proto¬ 
 plasm of the cell. In other forms there are hair-like 
 appendages, either from the ends or sides of the cell. 
 These cilia, or flagella, are believed to consist in some 
 cases of protoplasm, and to be organs of motion; in other 
 cases it is believed that the cilia are merely prolongations 
 of the membrane enveloping the cell, and that they have 
 nothing to do with the production of motion. These 
 appendages are naturally difficult to make out, but the 
 number of forms possessing them is now known to be very 
 large. 
 
 The observer is cautioned to remember that almost all 
 finely divided solid matters suspended in a fluid exhibit 
 a peculiar oscillatory motion called the Brownian move¬ 
 ment. The study of the Brownian movement made in 
 Chapter IV. should be recalled, as many kinds of bacteria 
 exhibit this movement. Careful observation will readily 
 enable one to distinguish between the progressive move¬ 
 ment from place to place of the really motile bacteria and 
 this purely mechanical vibratory movement. The hay 
 bacillus, described later, affords good material for the 
 study of both these movements. Another movement about 
 which one might be mistaken is the motion of Vibriones. 
 A filament of Vibriones is shaped like a corkscrew, and 
 the movement is believed to be a revolution on the axis of 
 the filament. Under the microscope it appears by an opti¬ 
 cal illusion to be a snake-like, undulatory motion. In the 
 
148 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 case of Spirocliseta and Spirillum, the serpentine motions 
 are believed to be real. 
 
 Many forms of bacteria are perfectly harmless. In fact, 
 they are essential to life either as a direct agent in digestion, 
 as already noted, or by their action in the decomposition 
 of animal and vegetable substances, and the preparation 
 of the soil for the production of plant life. With the air, 
 the water, and with our food, we receive countless numbers 
 of bacteria into our systems, where they are either inert or 
 perform the functions for which they were designed in the 
 economy of nature for our well-being. The investiga¬ 
 tions of recent years have equally well established the fact 
 that other forms are pathogenic. It now scarcely admits 
 of question that cholera, typhoid fever, diphtheria, con¬ 
 sumption, and man}^ other diseases, are caused and propa¬ 
 gated by particular bacteria. 
 
 With these introductory remarks, which are intended to 
 call the attention of the beginner to the nature and impor¬ 
 tance of the subject, Ave will turn our attention to the 
 methods employed in the study of these exceedingly minute 
 organisms. As already intimated, culture methods must 
 be employed in the satisfactory study of most forms of 
 bacteria. The remainder of this chapter will haA^e to do 
 Avith forms which can be studied without special cultiva¬ 
 tion, and Avith those which can be cultivated by very 
 simple means. The folloAving chapter Avill describe the 
 methods now employed in the cultivation, isolation, and 
 examination of forms Avhich cannot be so directly studied. 
 
 Bacteria of the Fur of the Teeth. — Scrape a little of the 
 fur from the teeth, and place it on a cover-glass in a thin, 
 even layer Avith a small drop of Avater. Invert the cover- 
 glass on a slide, and examine with the highest power at 
 command. Several forms of bacteria Avill undoubtedly be 
 
THE STUDY OF BACTERIA. 
 
 149 
 
 found. Minute spheres of Micrococci are clustered to¬ 
 gether; a spiral form, Spirilla dentium, or, as it is some¬ 
 times called, Spirochceta denticula , darting back and forth 
 \vith characteristic movements, may be present; possibly 
 the comma bacillus of the teeth, a form curved like a 
 comma (,) and closely resembling the comma bacillus of 
 Asiatic cholera, will be seen in active motion; larger than 
 any of these, straight, rod-like forms will be found, a vari¬ 
 ety of Leptothrix. If a little iodine solution be run under 
 the cover-glass, the protoplasm in the largest rods will 
 contract sufficiently to show that the rods are segmented. 
 Possibly a blue color, due to the presence of starch gran¬ 
 ules, will be seen. With the exception of comma bacillus, 
 the bacteria of the mouth cannot be cultivated by the ordi¬ 
 nary means. 
 
 Putrefaction Bacteria. — Expose to the air of a living- 
 room at the ordinary temperature pieces of moist bread, 
 boiled egg, boiled potato or turnip, for twenty-four to 
 forty-eight hours. Small spots, either translucent or 
 colored, will form. These are colonies of bacteria that 
 have developed from germs deposited from the air upon 
 the substance used. 
 
 Transfer a little from one of these spots to a cover-glass 
 with a small drop of water, and examine as already de¬ 
 scribed. Numerous rod-like and spherical bacteria will be 
 seen, many of the rods in active motion. 
 
 For further study, boil some green leaves in a flask. 
 Almost any kind of leaves will do. Cabbage or lettuce 
 leaves are good. In another flask boil some ground peas. 
 Filter each solution after an hour, and set aside open at 
 the ordinary temperature. In about forty-eight hours 
 skin-like formations will be seen on the surfaces of the 
 liquids in the flasks. This formation is called a pellicle. 
 
150 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 A little of this is examined under the microscope in the 
 manner already described. 
 
 Again, soak some hay in water at the ordinary temper¬ 
 ature for four or five hours. Filter the infusion into a flask, 
 and boil gently for a few minutes. Plug the mouth of 
 the flask with a wad of cotton-wool, and place it aside at 
 the ordinary temperature for twenty-four or forty-eight 
 hours. A pellicle forms. This is formed by the hay 
 bacillus, Bacillus subtilis. The germs of this bacillus are 
 able to withstand boiling for a few minutes. Other bac¬ 
 teria present have probably been killed by this treatment. 
 We have then a pure culture, as it is called, of this 
 variety. The cotton-wool plug is an efficient filter for the 
 air that enters the flask, no germs being able to pass 
 through it. If the boiling was not continued long enough, 
 other forms of bacteria will also be present. 
 
 If a bit of the pellicle be examined at the right stage 
 of development, filamentous forms will be seen imbedded 
 in a transparent, gelatine-like substance. In the active, 
 swarming state, the Bacillus subtilis divides into filaments 
 of twos, fours, and eights, with flagella at the ends of the 
 filaments. These bacteria will not stain by the ordinary 
 methods. 
 
 For still another study, place some Spirogyra, a water 
 plant described in Chapter V., in a flask of water, and 
 allow it to stand until decomposition begins. From time 
 to time examine a drop of the water. Very lively motile 
 coiled bacteria, Spirochceta plicatalis , are likely to be 
 found. These bacteria are common in any stagnant water 
 containing decaying organic matter. In the same water 
 the straight, comparatively thick rods of Beggiatoa alba 
 may be found. These are filamentous. The protoplasm 
 is granular in appearance, due to the particles of sulphur. 
 
THE STUDY OF BACTEMA. 
 
 151 
 
 That these granules are sulphur may be proved by allow¬ 
 ing the preparation to dry and then running a little carbon 
 bisulphide under the cover-glass. This will dissolve the 
 sulphur, and the granulation will become less distinct. 
 
 In all of these cases, the masses taken from the pellicle 
 show the bacteria in the zoogloea stage. The pellicle is 
 composed of motionless bacteria surrounded by a gelatine¬ 
 like, transparent substance which is a product of their 
 growth. If we take our specimen at the right time, we 
 have the bacteria in the active, swarming stage, when they 
 are in active motion. At the proper stage of development, 
 we may see the resting-spores. In order, then, to form 
 correct ideas of bacteria, one should make frequent exami¬ 
 nation, to see them in the different stages of their develop¬ 
 ment. 
 
 Cover-Glass Preparations. — In the study of the above, as 
 well as of all other bacteria, it is of advantage to make a 
 so-called cover-glass preparation. A very thin layer of 
 the medium containing the bacteria is placed evenly on a 
 cover-glass. This is best effected by placing a minute 
 drop on a cover-glass and laying another cover-glass on 
 this. The two cover-glasses are now gently rubbed to¬ 
 gether, and then separated by sliding them apart. They 
 are then dried at the temperature of the room, or by hold¬ 
 ing them in the forceps some distance above a gas flame, 
 being careful not to scorch the film. The bacteria are 
 now stained by placing on each cover-glass a solution of 
 fuchsin, Bismarck brown, methyl violet, or other staining 
 solution. This is allowed to act from ten to twenty min¬ 
 utes, or longer. The dye is now washed off with distilled 
 water; and, if there has been considerable diffuse staining 
 of the ground tissue, the cover-glass is washed in alcohol. 
 The cover-glass is now dried as before, and a small drop 
 
152 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 of oil of cedar, turpentine, or xylol is placed upon it. It is 
 then inverted on a glass slip, and is ready for examination. 
 
 In some cases double staining is of advantage. After 
 the surplus of the first stain has been washed away, as 
 above, the cover-glass is not dried, but is treated with a 
 contrast stain from ten to twenty minutes, washed, treated 
 with alcohol if necessary, dried, and mounted in oil of 
 cedar, turpentine, or xylol for examination. Fucjisin 
 and Bismarck brown, Bismarck brown and methyl violet 
 or methyl blue, give good results. These stains may be 
 used in water solution or in alcoholic solution, with the 
 exception of Bismarck brown, the alcoholic solution of 
 which deteriorates on standing. A nearly saturated solu¬ 
 tion in alcohol of the other dyes may be kept on hand and 
 diluted with about twice their bulk of water, and filtered, 
 at the time of using. As a rule, the bacteria take only 
 one of the colors. The object of the second color is to 
 stain the ground tissue, and so set forth the bacteria by 
 contrast. 
 
 A cover-glass preparation made as above may be perma¬ 
 nently preserved by simply draining off the oil of cedar, 
 turpentine, or xylol, and mounting in xylol or benzole 
 balsam. Do not use chloroform balsam, as the chloroform 
 dissolves the aniline dye. The bacteria are very much 
 more distinctly seen when mounted in balsam than when 
 mounted temporarily in oil of cedar, etc. 
 
 The Bacillus Tuberculosis. — The Bacillus tuberculosis is 
 a slightly curved non-motile rod, containing frequently 
 from two to six spores. These spores are very difficult to 
 make out. The rods are from 1.5 /a to 3 p long and from 
 .2 p to .3 p thick. Numerous methods have been devised 
 for the demonstration of the Tubercle bacilli. Two reliable 
 methods are here given. 
 
THE STUDY OF BACTERIA. 
 
 153 
 
 Ehrlich's Method. 
 
 Mix 1 c.c. of aniline oil in 20 c.c. of distilled water, and 
 filter. To this add an equal volume of a saturated solu¬ 
 tion of fuchsin in alcohol. A cover-glass preparation of 
 sputum is made with this as a first stain. The stain is 
 allowed to act for half an hour or longer. The cover-glass 
 is washed first in 33 per cent nitric acid, and then in dis¬ 
 tilled water. Nitric acid is used because it discharges the 
 color from all bacilli likely to be present except the Tubercle 
 bacilli. The bacilli are stained red. As a contrast stain, 
 either methyl blue or Bismarck brown is now applied in 
 saturated alcoholic solution. The ground tissue will be 
 stained blue or brown, the bacilli remaining red. Wash 
 in distilled water and in alcohol, clear in xylol, and mount 
 in xylol balsam. 
 
 « 
 
 The Ziehl-Neelsen Carbol-Fuchsin Method. 
 
 A cover-glass preparation of sputum is treated with a 
 staining solution prepared by dissolving one part of 
 fuchsin in ten parts of absolute alcohol and then adding 
 100 parts of a 5 per cent solution of carbolic acid in 
 water. This solution may he heated in a watch glass 
 until it steams, and the prepared cover-glass floated on it, 
 prepared side down, from three to five minutes; or the 
 solution may be used cold, in which case it should be 
 allowed to act for twenty-four hours. The cover-glass is 
 then washed in alcohol and in 25 per cent sulphuric, nitric, 
 or hydrochloric acid. The acid removes the color from 
 everything except the bacilli. Wash again in alcohol, 
 and then in a saturated water solution of lithium carbon¬ 
 ate to fix and intensify the stain. As a contrast stain, use 
 
154 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 a saturated solution of methyl blue in water, allowing it 
 to act ten or fifteen minutes. Wash and dry the cover- 
 glass, clear in aniline oil or xylol, and mount in xylol 
 balsam. 
 
 The Typhoid Bacillus. — Whether typhoid fever is caused 
 by a micrococcus or a bacillus is still disputed. The pre¬ 
 vailing weight of opinion among the foremost investiga¬ 
 tors is that it is caused by a short, somewhat thick bacillus 
 with slightly rounded ends, each rod being from 2 fi to 3 ^ 
 in length and about one-third as thick. The central por¬ 
 tion of the cell will not take aniline stains, and gives an 
 appearance closely resembling spores. These spots were 
 long believed to be true spores, and are so described in 
 works of high authority published within a very few years. 
 The most trustworthy authorities now believe that they 
 are not spores, but that the failure of these portions to 
 take the stains is due to the fact that degeneration of the 
 protoplasm is here going on. The typhoid bacilli are 
 found in the mesenteric glands, spleen, liver, occasionally 
 in the kidney, and in the dejecta of typhoid patients, in 
 water that has been contaminated by such dejecta, and in 
 the milk of cows that have drunk of the contaminated water. 
 Cultivation may be made by the methods described in the 
 following chapter, and cover-glass preparations made from 
 the cultures. Cover-glass preparations may also be made 
 directly from the water, milk, or dejecta. A drop is dried 
 on the cover-glass, stained for half an hour with a satu¬ 
 rated solution of methyl violet in water, washed, and 
 finished as usual. 
 
 The Cholera Bacillus. — The special interest felt at pres¬ 
 ent, and at all times when our country is threatened with 
 the introduction of cholera, warrants a few directions for 
 the study of the cholera bacillus, although the writer has 
 
THE STUDY OF BACTERIA. 
 
 155 
 
 never had an opportunity to study it personally, and must 
 rely wholly upon the observations of others. The cholera 
 bacillus, or comma bacillus, is a decidedly curved, motile 
 rod with rounded or thickened ends, about one-half or one- 
 third as long, but about twice as thick, as the Bucillus 
 tuberculosis. These rods sometimes join end to end, and 
 form a true Spirillum; they sometimes take the shape of 
 the letter O, and sometimes of S. They are found in the 
 dejecta of cholera patients during the early and middle 
 stages of the disease, and in water contaminated from 
 such dejecta. 
 
 The method most highly recommended for their direct 
 examination is very simple. A drop of the suspected 
 water, or a bit of the white mucous particles found in 
 the dejecta, is spread in a thin layer on the centre of 
 a glass slip, covered with a very weak, watery solution of 
 methyl violet, and a cover-glass immediately placed over 
 it, any overflow of the staining fluid being taken up 
 with blotting paper. The bacilli take the stain slightly, 
 but do not lose their power of motion, as would, of course, 
 be the case if an ordinary cover-glass preparation were 
 made. 
 
 As these bacilli are so minute, the very highest powers 
 of the microscope must be used in the examination. The 
 examination, though difficult, is an eminently satisfactory 
 one on account of the distinctly characteristic form of the 
 bacillus. 
 
Large Continental Microscope. 
 
 Made by Bausch and Lomb Optical Co., Rochester, N.Y. 
 Fig. 38. 
 
CHAPTER XIII. 
 
 THE STUDY OF BACTERIA: CULTURE METHODS. 
 
 Bacteria are such exceedingly minute organisms that 
 with the highest powers of the most perfect modern lenses 
 even the trained eye and brain cannot m many cases defi- 
 nitely distinguish between similar forms. Various methods 
 of cultivation have, therefore, been devised to afford means 
 of studying the habits, effects, and other physiological 
 functions as a means of distinguishing the different species 
 when the forms of the individual bacteria cannot be relied 
 upon. Several species of pathogenic bacteria may easily 
 be mistaken for non-pathogenic varieties, if the appearance 
 under the microscope is the only means employed for their 
 identification. The Bacillus tuberculosis and the typhoid 
 bacillus both closely resemble harmless forms. 
 
 Another very important end served in the cultivation 
 and isolation of bacteria is the study of the effects pro¬ 
 duced by the different isolated forms when injected into 
 the bodies of living animals. A simple reference to the 
 researches and discoveries of Pasteur and Koch is all that 
 need be said about the importance of these methods o± 
 
 investigation. . , 
 
 It is the purpose of this chapter to describe m simp e 
 form the methods of culture study that have been devel¬ 
 oped within the last ten or twelve years in some of the 
 leading laboratories of the world, and that are now m 
 common use wherever practical studies of these organisms 
 
 are being made. 
 
 157 
 
158 PRACTICAL METHODS IN MICROSCOPY. 
 
 It may be said by way of introduction, that the general 
 method of procedure is to provide proper food material 
 which has been deprived of germs of all other kinds of 
 bacteria, keeping it at such temperatures as are best suited 
 to the variety under consideration. The different species 
 are separated by means which will be described. The 
 food material is deprived of bacterial germs by sterilization. 
 
 Sterilization. — Sterilization of a substance means simply 
 heating it to sucli a temperature that the bacteria in it are 
 killed. No forms can live at a continued temperature of 
 boiling water, but many germs resist this temperature for 
 a considerable interval. All germs are killed by heating 
 for one or two hours at a temperature of 160° to 200° C. 
 Apparatus, cotton-wool, everything that cannot be effect¬ 
 ually protected from the air, should be so sterilized. It is 
 especially important to fix this fact, and not to use test- 
 tubes and cotton-wool that have not been sterilized for the 
 culture media hereafter described, as contamination is 
 almost sure to follow. 
 
 In steam sterilizing, the apparatus and media are sub¬ 
 mitted to a temperature of 100° C. for half an hour to an 
 hour on the first day. This kills the developed bacteria, 
 but the spores withstand this treatment. After twenty- 
 four hours the spores will have developed, at least par¬ 
 tially. They are steamed again for twenty minutes to half 
 an hour, and given another twenty-four hours to develop, 
 when they are again treated in the same way. It has 
 been found that this treatment kills all known germs. 
 Apparatus such as test-tubes and flasks may be plugged 
 with cotton-wool and given this treatment, though it is 
 better to use the higher temperature of dry-air sterilizing, 
 to make sure of killing the germs that fall on the cotton¬ 
 wool from the air. 
 
THE STUDY OF BACTERIA: CULTURE METHODS. 159 
 
 The apparatus used in bacteriological work may also be 
 sterilized by washing in various chemical solutions. The 
 substance most commonly used is corrosive sublimate 
 (mercuric chloride). One gramme of the solid dissolved in 
 one litre of water gives the usual 1:1000 solution. 
 
 The Sterilizers. — Complicated and costly sterilizers have 
 been devised and used by different investigators, but for¬ 
 tunately very simple and inexpensive forms of apparatus 
 are now in the market for other purposes, which are admi¬ 
 rably adapted for bacteriological studies. Both of the 
 articles here described are now in use in prominent labo¬ 
 ratories in this country for practical work. 
 
 For hot-air sterilizing, the gas stove called the “ Cone,” 
 manufactured by the Central Oil Gas Stove Co., Boston, 
 does admirably. Glass plates, pipettes, 
 cotton-wool, and any other apparatus, 
 may be sterilized by heating from one to 
 two hours at a temperature of 160° to 
 200° C. These articles may, of course, 
 be sterilized in the oven of an ordinary 
 cooking-stove. All implements thus 
 
 sterilized should be carefully protected 
 from the dust until they are cool enough 
 to use. 
 
 For steam sterilizing, the Arnold Steam 
 Sterilizer, which may be purchased for 
 $2.50 of various dealers, or of the manu¬ 
 facturers, Wilmot Castle & Co., Roch¬ 
 ester, N.Y., serves the purpose excellently well. It is 
 verv simple in construction. It may be used over any 
 source of heat, as a Bunsen burner, oil stove, or an 01 di¬ 
 nary stove. Water placed in the pan finds its way through 
 small openings into a shallow closed vessel below, dhis 
 
160 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 vessel communicates directly with the sterilizing chamber 
 above. Steam is quickly generated. The sterilizing 
 
 chamber has an outer 
 jacket placed over it from 
 above. The steam, there¬ 
 fore, accumulates under 
 sufficient pressure to main¬ 
 tain a constant temperature 
 of 100° C. A thermometer 
 mounted in a cork may be 
 inserted through the covers, 
 and, by regulating the flame, 
 any desired temperature 
 below 100° C. may easily 
 be maintained. With the 
 Arnold Sterilizer as the only 
 means of sterilizing, a great 
 deal of thoroughly reliable 
 practical work may be done. 
 
 Food Materials. —Various 
 substances are used to fur¬ 
 nish the bacteria suitable food. Among those most 
 commonly employed are preparations of gelatine and of 
 agar-agar, milk, bouillon, slices of potato, carrot, turnip, 
 and other vegetable substances. 
 
 Gelatine Medium. — There are many different formulas 
 for the preparation of this medium. The following is a 
 good one. Half a pound of lean meat is soaked in 500 c.c. 
 of distilled water for twenty-four hours. The meat extract 
 thus obtained is decanted into a flask, and to it is added 
 
 I 
 
 —'I 
 
 sterilizin 
 
 J—H k 
 
 H 
 
 CD ^ 
 
 
 
 
 pt 
 
 V'ff 
 
 Fig. 40. 
 
 Gelatine .... 
 
 Salt. 
 
 Peptone (Merck’s) 
 
 50 grammes 
 2.5 grammes 
 5 grammes 
 
THE STUDY OF BACTERIA : CULTURE METHODS. 161 
 
 Allow this mixture to stand a short time for the gela¬ 
 tine to soften, then boil for half an hour. It is now tested 
 with litmus paper for acidity, and dry sodium carbonate 
 added cautiously until it has a faint alkaline reaction on 
 the litmus paper. It is now filtered through a piece of 
 cotton cloth or of flannel, and again through three or four 
 layers of absorbent cotton. As the gelatine hardens too 
 rapidly to allow the medium to be filtered in the open air, 
 the filtering apparatus is placed inside the steam sterilizer, 
 where the filtering is readily accomplished, the sterilizer 
 taking the place of a somewhat expensive hot-filtering 
 apparatus sometimes advised. After the filtering, the 
 medium should be quite clear. One-half of it is now 
 poured into another flask, and made faintly acid by adding 
 acetic acid cautiously until blue litmus paper just turns 
 red. The other half is kept in the alkaline condition. In 
 the subsequent work these two portions are, of course, 
 kept separate, and labelled alkaline and acid. 
 
 Test-tubes are now fdled about one-third full of the 
 medium, and the mouth of each is tightly plugged with 
 cotton-wool. These test-tubes are placed in the steam 
 sterilizer in wire stands, or simply in empty beakers, to 
 hold them upright, and thoroughly sterilized by heating 
 for half an hour on each of three successive days. They 
 are then ready for immediate use, or they may be kept 
 indefinitely, and used as occasion may require. It is, in 
 fact, a good plan to keep them several days before using, 
 as, if they have not been thoroughly sterilized, a change 
 in appearance will indicate the fact. Paper bags may be 
 drawn over the cotton-wool plugs to protect them from 
 the dust with its contaminating bacteria. If the medium 
 is found to melt in hot weather, the amount of gelatine 
 may be increased to 60 or 75 grammes. 
 
162 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 Agar-agar Medium. — Agar-agar is a kind of gelatine 
 made from a sea-weed growing in the Japan Sea. It has 
 recently come into the market for a number of commercial 
 uses, and is now imported into this country by the ton. 
 It is used by some packers of meat to give the meat a 
 richer appearance. It is inexpensive, and may be obtained 
 of dealers in biological supplies. It is used as an ingredi¬ 
 ent of a culture medium because it will stand a relatively 
 high temperature without liquefying. 
 
 The agar-agar medium is prepared by the formula given 
 for gelatine medium, substituting for the 50 grammes of 
 gelatine 5 grammes of agar-agar which has been soaked 
 in salt water for ten or twelve hours, and the salt water 
 drained off. 
 
 Agar-agar and ordinary gelatine are sometimes mixed 
 in different proportions, to give a medium combining the 
 qualities of both of these substances. 
 
 Test-Tube Cultures. — When it is desired to make a culti¬ 
 vation of bacteria from any source, some of the test-tubes 
 prepared as above are taken. The cotton-wool plug is 
 passed through a flame, ignited, and the flame quickly 
 blown out. The object of this treatment is to destroy 
 any bacteria which may have been deposited on the cotton¬ 
 wool with the dust that has settled on it from the air. A 
 sterilized pipette with a fine point is touched to the bac¬ 
 teria to be studied, the cotton-wool plug is quickly re¬ 
 moved just enough to allow the pipette to be admitted to 
 the tube, and the point of the pipette is touched to or is 
 drawn along the surface of the medium. The pipette is 
 withdrawn, and the cotton-wool plug is replaced. This 
 operation should consume as little time as possible, and 
 every precaution should be taken to prevent dust from the 
 air from entering. The tubes are set aside at the ordinary 
 
THE STUDY OF BACTERIA: CULTURE METHODS. 163 
 
 temperature, or are kept at some particular temperature, 
 as the conditions of the study may require. The tubes 
 are- examined from time to time to watch the development. 
 A turbidity of the gelatine, or translucent or colored spots, 
 after twenty-four to forty-eight hours, show that the bac¬ 
 teria are growing. Cover-glass preparations may at any 
 time be made from the growth. 
 
 Cultures at Elevated Temperatures. — Very many investi¬ 
 gations, in fact, the larger number, are made at the ordi¬ 
 nary temperature of the room. Cultures at temperatures 
 above the ordinary temperatures of the air are necessary 
 in some cases. Disease-producing bacteria, for instance, 
 must be cultivated at about the temperature of the human 
 body. Apparatus for these cultures may he improvised at 
 very little expense. Fill a large tin pan — the larger the 
 better — with water, and heat over a Bunsen burner or oil 
 stove. Provide as a cover a piece of board, through which 
 holes have been bored of a size suited to permit the test- 
 tubes to pass through. Test-tubes with flaring mouths 
 should be used. After the culture tubes are prepared and 
 inoculated, they are passed through the holes in the cover 
 and immersed in the water. A thermometer mounted in 
 a cork is inserted in one of the holes in the cover. 
 
 The water is heated with the full flame until the required 
 temperature is reached, then the flame is turned down. 
 With a little experience, it is very easy to maintain a tem¬ 
 perature varying not more than two or three degrees in 
 twenty-four hours. The greater the body of water, the 
 easier it is to maintain a constant temperature. 
 
 Plate Cultures. — Bell-glasses and plates of window glass 
 about four inches square are required. Ordinary tumblers 
 may be used as bell-glasses. For some purposes shallow 
 dishes, sold in pairs by the dealers under the name of 
 
164 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 bacteriological or Petri dishes, are convenient. A pair 
 of these dishes placed under a bell-glass is used in place of 
 the glass plate and tumbler. This apparatus is thoroughly 
 sterilized by dry heat, or by washing with a 1:1000 solu¬ 
 tion of corrosive sublimate. 
 
 A tube of gelatine or agar-agar medium is melted by 
 holding it in a beaker of warm water, and is then inocu¬ 
 lated with the bacteria to be studied, using the precautions 
 given under tube culture. The tube is thoroughly shaken, 
 the contents poured upon the glass plate, and the bell- 
 glass quickly placed over the plate. The plate is care¬ 
 fully levelled, and the joint between it and the bell-glass 
 is made air-tight by smearing with vaseline. In the course 
 of some hours, if the plate is kept at a suitable tempera¬ 
 ture, spots caused by the growth of the bacteria will 
 develop. Cover-glass preparations may be made from 
 these colonies, or a cover-glass may be laid over a colony, 
 and, the plate being placed on the stage of the microscope, 
 an examination made. 
 
 Method of Isolating Bacteria. — The method commonly 
 employed for the isolation of species is an application of 
 the plate culture. It is usually spoken of as the method 
 of dilution. Several test-tubes of gelatine, or agar-agar 
 medium, are melted at a low temperature by immersing 
 them in warm water. With the usual precautions to pre¬ 
 vent contamination from the dust in the air, one of the 
 tubes is inoculated with bacteria. The tube is well shaken, 
 thoroughly to incorporate the bacteria with the medium. 
 A drop or two from this tube is used to inoculate a second 
 tube. This tube is well shaken, and the third tube is 
 inoculated from it, and so on to any required degree of 
 dilution. The contents of these tubes are now poured 
 upon separate sterilized plates, covered with bell-glasses, 
 
THE STUDY OF BACTERIA: CULTURE METHODS. 165 
 
 and placed aside under suitable conditions of tempera¬ 
 ture. 
 
 After some hours, or in a few days at most, colonies 
 develop, but the plates of the most diluted preparation 
 will, of course, have the fewest. If the dilution was car¬ 
 ried far enough, the colonies will not run together, but 
 will be entirely separate. It is accepted that each separate 
 colony thus obtained is the growth from a single individ¬ 
 ual, or at least from a single chain of individuals of one 
 species. Cover-glass preparations may be made from these 
 colonies, or tubes of various media may be inoculated from 
 them, and what are known as pure cultures grown. 
 
 Cultures without Oxygen. — Bacteria are called aerobic 
 or anaerobic, according as to whether or not they can live 
 without oxygen. The simplest form of culture without 
 oxygen is made on a gelatine or agar-agar plate. The 
 plate may be prepared by pouring the inoculated gelatine 
 medium upon it, as already described, or the sterile gela¬ 
 tine may be poured upon the plate, and the inoculation 
 made in spots with the point of a pipette charged with 
 the bacteria. 
 
 Thin cover-glasses or thin films of mica are now laid 
 over some of the spots, and a ring of melted paraffin is 
 run around each film. Time is given for the growth of 
 the bacteria, and the observation is made whether they 
 grow equally well exposed to the air and under the mica 
 film, where the supply of oxygen is cut off. This culture 
 is a valuable means of distinguishing certain species. 
 
 Milk Cultures. — Advantage is taken of the fact that milk 
 is an excellent food for certain species of bacteria. The 
 milk is used as soon as possible after it is drawn from 
 the cow. Test-tubes are filled about one-third full of it, 
 plugged with cotton-wool, and sterilized by heating in the 
 
166 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 steam sterilizer for twenty minutes on three successive 
 clays. It may then be inoculated in the manner already 
 described. 
 
 Bouillon Cultures. — Bouillon, or clear soup, is used in 
 the same way that milk is used. It should be rendered 
 slightly alkaline with sodium carbonate. 
 
 Potato Cultures. — Potatoes, carrots, turnips, and other 
 vegetables may be used, either for test-tube cultures or for 
 plate cultures. For test-tube cultures, pieces of boiled 
 potato are cut with a sterilized apple-corer, and these 
 cylinders are cut obliquely from end to end. They are then 
 placed in test-tubes, large end down. This gives a large 
 sloping surface for the cultures. The tubes are plugged 
 with cotton-wool and thoroughly sterilized in the steam 
 sterilizer. They are inoculated in the manner described 
 for tube cultures. 
 
 For plate cultures, a boiled potato is peeled with a knife 
 sterilized by passing it back and forth through a flame, 
 slices are cut, placed on glass plates that have been steril¬ 
 ized by dry heat or by washing in the 1:1000 solution of 
 corrosive sublimate, and covered with sterilized bell-glasses. 
 Inoculation with the bacteria under examination is effected 
 by touching a sterilized pipette to the bacteria, and then 
 to the surface of the potato, either in spots or in lines. 
 
 Hanging-Drop Cultures. — This is a very convenient form 
 of culture for some investigations. Cells are made by 
 cementing rings of glass, rubber, or metal to microscope 
 slides, as described in Chapter VI. When the cultures 
 are to be made, sterilize the cells by washing in the 
 1:1000 solution of corrosive sublimate or in absolute alco¬ 
 hol. Smear the top surface of the cell thickly with vase¬ 
 line. Sterilize a cover-glass in the same way or by passing 
 it through the naked flame, place on its centre a drop of 
 
THE STUDY OP BACTERIA : CULTURE METHODS. 167 
 
 the sterilized agar-agar or gelatine medium, inoculate it 
 with the bacteria, and invert the cover-glass over the cell, 
 pressing it down gently. The vaseline makes an air-tight 
 joint. 
 
 Many of these slides may be prepared at the expense of 
 very little labor. They may be set aside at the required 
 temperature, and studied under the microscope whenever 
 one wishes. The drop of nutrient medium, being attached 
 to the cover-glass, is in a suitable position for examination. 
 If it is desired to keep the preparation moist, a very deep 
 cell may be used, and a drop or two of water that has been 
 sterilized by boiling may be placed in the bottom of the cell. 
 
 Another good cell for moist cultures is made by cutting 
 a hole a little smaller than the cover-glass in a piece of 
 thick pasteboard. Soak the pasteboard in boiling water 
 to sterilize it, place it while still wet and hot on the centre 
 of a microscope slide, and immediately place over it a 
 prepared cover-glass with its inoculated hanging drop. 
 To keep up the moisture, wet the pasteboard from time to 
 time in boiled water. If these cultures are to be prolonged 
 for some time, keep the slides under a bell-glass with a 
 dish of Avater. 
 
 A hanging drop of sterilized water, inoculated with the 
 bacteria, affords a most excellent means of studying the 
 movements of the motile forms. 
 
 Practical Applications of these Methods. — It is evident 
 from what has previously been said that material for study 
 may be obtained from any putrefying or decaying animal 
 or vegetable matter. In making practical studies of bac¬ 
 teria from any source, the bacteria are first isolated in the 
 manner abo\ r e described. Inoculations of the pure cultures 
 are then made in test-tubes of alkaline and of acid gela¬ 
 tine medium; of alkaline and acid agar-agar medium; in 
 
168 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 test-tubes of milk, bouillon, and potato, both at the ordi¬ 
 nary temperature and at different elevated temperatures ; 
 plate cultures are made with the different media; cultures 
 are made to determine whether the bacteria are aerobic or 
 anaerobic ; and hanging-drop cultures are started. 
 
 Observation is made as to whether the bacteria thrive in 
 the different media. The growth is usually easily seen 
 with the naked eye. The colonies are distinguished by 
 the changed appearance of the culture medium, a white, 
 gray, translucent, pearly, or colored spot indicating each 
 growth. Many bacteria in growing liquefy the gelatine 
 medium. The extent to which they do this, and the 
 manner in which they do it, whether in narrow lines 
 extending straight downwards or in diffusely spreading 
 masses, are carefully observed. At any stage of the prog¬ 
 ress, cover-glass preparations may be made and studied 
 under the microscope. In careful investigations several 
 sets of cultures are made, and the preparations of the same 
 kind are compared with one another to guard against error 
 arising from accidental contamination from the air. 
 
 A record of the results should give the source whence 
 the bacteria were taken; the shape and size as observed 
 and measured in cover-glass preparations ; the motility as 
 observed in hanging-drop cultures; the temperatures at 
 which the various test-tube and plate cultures thrive best; 
 a description of the appearance of the colonies on the 
 plate cultures; the effect in liquefying the gelatine 
 medium, both the alkaline and the acid; the effect on 
 milk, whether it is coagulated or not, how long it takes to 
 coagulate it, whether the milk is changed in color, and 
 whether it has an acid or an alkaline reaction at the close 
 of the investigation ; the effect on bouillon, whether it 
 changes in color or becomes turbid, the character of the 
 
THE STUDY OF BACTERIA: CULTURE METHODS. 169 
 
 precipitate, if one is formed, whether a skin is formed on 
 the surface; whether the organism can grow without 
 oxygen or not, as observed under the film of mica. 
 
 The results of these observations are now to be com¬ 
 pared with the observations recorded by other workers, so 
 far as their results can be obtained. It is to be remem¬ 
 bered that bacteriology is in its very infancy, and that it 
 is not yet possible to obtain perfectly reliable descriptions 
 of results for a very large number of bacteria. The descrip¬ 
 tions given by different workers often differ in important 
 particulars, even those of the very highest authoiities. 
 Bacteriology has, however, made sufficient progress to be 
 firmly established as a science. The newness and unde¬ 
 veloped condition of the science make it a peculiarly 
 attractive field for work, but the field is so vast that it 
 hardly seems proper to give in a book of this character 
 more than these general descriptions of the methods em¬ 
 ployed. 
 
CHAPTER XIV. 
 
 PHOTO-MICROGRAPHY. 
 
 The necessity for true representations of objects as seen 
 under the microscope, and the tediousness and difficulty of 
 making accurate free-hand or camera lucida drawings, 
 have naturally called the attention of microscopists to the 
 photographer’s camera as a means of securing, with a small 
 expenditure of time and labor, multiplied pictures of 
 microscopic objects. The rapid advances made in the art 
 of photography in very recent years have rendered the 
 accomplishment of this, to a certain extent, within the 
 reach of every microscopist. For certain classes of objects, 
 and for the purpose of fixing the details of structure in 
 the mind of the student, the free-hand or camera lucida 
 drawing must be made, but for many classes of objects, 
 and when it is desired to have numerous reproductions, 
 the photo-micrograph is largely superseding the laborious 
 processes of the draughtsman. 
 
 Complicated and costly apparatus has been devised for 
 the accomplishment of special purposes in photo-microg¬ 
 raphy. Such apparatus is a necessity if the highest 
 power objectives are to be used, as in photographing bac¬ 
 teria and the more delicate diatoms. Special apochro- 
 matic objectives, apparatus for using the sunlight, the cal¬ 
 cium light, or the electric light, and special orthochromatic 
 dry plates must be used to secure the best results with 
 these classes of objects. Very few private workers would 
 feel warranted in purchasing such outfits. Much may be 
 
 170 
 
PHOTO-MICROGRAPHY. 
 
 171 
 
 clone with very simple and inexpensive apparatus. We 
 shall deal here with simple apparatus and with low 
 powers of the microscope, such powers as, in fact, are 
 most serviceable in the general use of the microscope in 
 histological investigations, leaving to specialists the de¬ 
 scription of special apparatus and special processes. The 
 present chapter proposes to give in detail to those wholly 
 unacquainted with photography, all of the processes from 
 the setting up of the apparatus and the exposure of the 
 plate to the mounting of the finished photo-micrograph. 
 The experienced photographer will prefer processes and 
 solutions with which he is familiar, and numerous modifi¬ 
 cations may be made of the methods, but processes known 
 to produce good results are here given. 
 
 Apparatus. — Any microscope with a tube large in diam¬ 
 eter and with jointed pillar, and an ordinary amateur 
 photographic camera, omitting the lens, are all that is re¬ 
 quired. Such photographic outfits may be purchased at 
 varying prices, but an outfit costing ten or twelve dollars, 
 allowing the exposure of a five by eight inch plate, is 
 advised. 
 
 Arrangement of Apparatus. — The eye-piece, together with 
 the draw-tube, is removed from the microscope ; the mirror 
 and sub-stage fitting are removed or swung to one side. 
 The body of the tube is inclined at right angles to the 
 supporting pillar. The camera is used without its lens, 
 the microscopic objective being the only lens used. A 
 board four feet long by ten inches wide, placed on any 
 firm table, is used as a support for both camera and micro¬ 
 scope,— an arrangement which gives great steadiness. At 
 one end of the board, a rigid, upright support is placed, to 
 which the camera is firmly attached, at such a height that 
 the tube in which the photographic lens is usually inserted 
 
172 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 is exactly opposite the end of the inclined tube of the 
 microscope when it is placed in position on the board. 
 The tube of the camera should be of the same size as the 
 tube of the microscope, or larger. The junction of the 
 two tubes must be made light tight. This may be accom¬ 
 plished by winding a black cloth around the tubes, or an 
 adapting tube of tin or brass, painted dead black on the 
 inside, may be used (see Frontispiece). The light to be 
 used is an ordinary kerosene lamp, properly placed to 
 bring the flame in the optical axis of the microscope, no 
 mirror being used, and adjusted to give a clear circle of 
 light on the ground glass of the camera. One of the 
 attractions of photo-micrography is the fact that the ex¬ 
 posure can be made in the evening by lamplight. 
 
 This simple arrangement is advised for beginners. 
 When some experience has been obtained, a sub-stage con¬ 
 denser may be used; it will be required for the higher 
 powers of objectives. The eye-piece may also be used; 
 the only changes in the position of the microscope and 
 camera will be those required to adjust the focussing. The 
 time of exposure of the dry plate will, however, vary with 
 each different arrangement of the apparatus. 
 
 When the apparatus has been arranged as described, 
 light the kerosene lamp and put out all other lights in the 
 room. See that the circle of light on the ground glass is 
 perfect. Place a mounted microscopic object on the 
 stage of the microscope, and fasten it in position with the 
 spring clips. Adjust the objective to about its working 
 distance, and move the ground glass of the camera until 
 the image of the object is seen on it of such size as 
 desired. The image is much more distinctly seen if the 
 ground glass is well shaded from the light. By changing 
 the position of the ground glass and regulating the focus- 
 
PHOTO-MICROGRAPHY. 
 
 173 
 
 sing of the objective to correspond, the object may be pro¬ 
 jected on to the ground glass variously magnified from a 
 very few diameters to the full capacity of the ground glass. 
 
 When a circle of suitable size has been obtained, focus 
 sharply, first with the coarse adjustment, then with the 
 micrometer screw. In the final adjustment of the focus, 
 the image on the ground glass should be viewed with a 
 hand magnifying-glass. It is of advantage, too, to render 
 the ground glass more transparent by cementing with 
 Canada balsam one of the large rectangular pieces of thin 
 cover-glass used in serial mounts to the ground surface. 
 Smearing the ground surface of the glass with glycerine 
 is another means of accomplishing the same end. T he 
 image can then be much more sharply seen. Secuie the 
 sharpest image possible. 
 
 It is now to be remembered that light rays are focussed 
 a little nearer the lens than the chemical rays. Opticians 
 furnish objectives specially corrected for the chemical 
 rays. If an ordinary objective is used, the ground glass 
 is started back very slightly after the sharp image has 
 been obtained; or, what will effect the same result and is 
 usually easier of accomplishment, the objective is slightly 
 moved towards the object by means of the fine adjust¬ 
 ment. All is now ready for the dry plate. 
 
 Dry Plates.— Any of the reliable dry plates on the 
 market may be used. The beginner is advised to take 
 some one of the popular brands, as the Standard, Harvard, 
 Eagle, or Stanley, and follow the advice of the maker in 
 developing, using the formula for developer furnished with 
 the plates, continuing to use the same brand of plates and 
 the same developer until a considerable degree of skill in 
 their management has been acquired. After this, other 
 plates and other developers may be used, if it seems desir- 
 
174 PRACTICAL METHODS IN MICROSCOPY. 
 
 able. The beginner should not allow himself to be con¬ 
 fused by the many different brands of dry plates and by 
 the multitude of developers; nor should he experiment in 
 different kinds until he has gained sufficient experience to 
 qualify him to judge of the merits of the various kinds. 
 After a time he may make use of orthochromatic plates, 
 which are much superior to the ordinary plate for some 
 classes of objects. 
 
 Slow plates are better for the beginner than rapid ones, 
 as slight differences of exposure do not cause so great a 
 change in the plate, and, the development being less rapid, 
 the operator has more time to observe the changes. The 
 Carbutt “ B ” plate gives excellent results. Plates 31 by 
 4^ inches in size are well adapted for practice work. They 
 give a picture of very suitable size for many objects, and 
 are so inexpensive that one does not hesitate to discard a 
 negative that is not perfectly satisfactory and make another 
 trial. The small plate is placed in a kit, and the kit in 
 the plate-holder. Dry plates must, of course, be handled 
 in a dark room. 
 
 Exposure. — The plate-holder containing the dry plate 
 having been placed in position on the camera, a piece of 
 cardboard covered with black velveteen or other soft 
 cloth is carefully inserted between the object and the 
 objective in such manner as to shut off the light entirely 
 from the objective; it serves the purpose of the lens-cap 
 in ordinary photography. With the high-power objectives 
 the light must be cut off from below the stage, the object 
 and objective being shaded from diffused light by means of 
 a black cloth, or some mechanical means of cutting off the 
 light must be inserted in the camera or the tube of the 
 microscope. The slide of the plate-holder is now with¬ 
 drawn, and we are ready for the exposure. 
 
PHOTOMICROGRAPHY. 
 
 175 
 
 No fixed rules can be laid down for the time of exposure. 
 Experience in general photography is, of course, an advan¬ 
 tage here, but it is by no means a prerequisite. One 
 without experience is advised to make practice exposures 
 at first. The time will vary with different objectives and 
 with the different objects photographed. The average 
 time for the photo-micrographs reproduced in this book 
 was about eighteen seconds. They were photographed 
 through a |-inch objective, without condenser or eye-piece, 
 and are magnified about thirty diameters; the dry plates 
 were Carbutt “ B.” Using similar apparatus and a wood 
 section as an object, the beginner is advised to expose four 
 plates, giving them ten, fifteen, eighteen, and twenty 
 seconds, respectively. Develop, and take proofs to judge 
 of the results. Repeat this practice several times, until 
 the length of exposure is approximately learned. The 
 time, labor, and expense of doing this will be well repaid. 
 
 Measurement of Magnification. — After the exposure of 
 the plate, remove the object from the stage of the micro¬ 
 scope without disturbing any of the adjustments, and put 
 in its place a stage micrometer. With a pair of dividers 
 obtain the length of the image on the ground glass plate 
 of the camera of any number of divisions of the microme¬ 
 ter graduation. Apply the dividers to an accurate rule to 
 find the exact length of the image. Divide this length by 
 the known value of the divisions of the micrometer scale. 
 For example, suppose a stage micrometer with a scale 
 divided to hundredths of a millimeter is used, and the 
 image of 10 of these divisions, as measured by the dividers 
 and rule, is 7.5 mm. Then 7.5 mm., or 750 hundredths 
 of a millimetre, divided by 10 hundredths of a millimetre 
 gives 75 as the amount of the magnification. This method 
 gives a very accurate result. 
 
176 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 If a stage micrometer is not available, the magnification 
 may be roughly calculated, when the objective is the only 
 lens used, on the geometrical principle of similar triangles. 
 The length of the object and the length of its image are 
 the bases of two similar triangles of which the focal length 
 of the objective is the altitude of one, and the distance of 
 the ground glass from the optical centre of the objective 
 is the altitude of the other. The bases are to each other 
 as the altitudes. The focal length of the objective is 
 known, and the distance of the ground glass from the 
 optical centre of the objective can be measured more or 
 less accurately. We have then the ratio of the altitudes, 
 and this is the same as the ratio of the bases. Sup¬ 
 pose, for example, that the objective has a focal length 
 of one-fifth of an inch, and the measured distance of the 
 ground glass from the optical centre is thirty inches. 
 Then 30 -f- ^ = 150, the magnification of the image. The 
 results of this method are only approximate, owing to 
 the difficulty of making an accurate measurement of the 
 distance of the ground glass from the optical centre of 
 the objective. 
 
 Development. — The development must, of course, be 
 done in a dark room. Any room is a dark room at night, 
 and it is only necessary to provide a lamp giving ruby or 
 yellow light. The plate is taken from the plate-holder, 
 dusted lightly with a soft brush, laid film side up in a 
 shallow dish, and about four ounces of the developer, pre¬ 
 viously prepared according to the directions accompany¬ 
 ing the dry plates, quickly poured over it with a sweeping 
 motion to insure covering the whole surface of the plate 
 at the same instant. The dish is gently rocked to prevent 
 air bubbles or specks of dust from clinging to the plate 
 and interfering with the development. 
 
PHOTO-MICROGRAPHY. 
 
 177 
 
 If the plate has been properly exposed, the image will 
 begin to appear in from twenty to thirty seconds. The 
 development is to be continued until the image begins to 
 lose the bright yellow appearance, and until the exposed 
 portions of the plate where there is no image are quite 
 black when the plate is examined by transmitted light. 
 The development must not be continued too long, or the 
 plate will appear foggy and flat; on the other hand, the 
 development must not be too short, or there will be a lack 
 of detail and contrast. The directions accompanying the 
 formula for developer for producing more or less detail 
 and greater or less contrast should be followed. The 
 whole development should require two or three minutes. 
 Care should be taken that the temperature of the devel¬ 
 oper is not far from 68° to 70° F. 
 
 Some experience is necessary to produce good results. 
 Written directions more than the above are of little value. 
 Experience alone can teach. The pictures produced by 
 hosts of amateur photographers throughout the country 
 show that good work is within the reach of patient 
 workers. The development of a photo-micrographic nega¬ 
 tive differs in no respect from the development of an 
 ordinary negative, and the fascination in making repro¬ 
 ductions of the minute things of nature on an enlarged 
 scale is not at all inferior to the fascination experienced 
 in reproducing the large things on a reduced scale. 
 
 Fixing the Negative. — As soon as the negative is prop¬ 
 erly developed, it is washed thoroughly in clean water. It 
 is then placed in a shallow dish, film side up, and covered 
 with a solution of the hyposulphite of soda, strength one- 
 half pound to one quart of water. One-fourth ounce of 
 alum may be added to this, if desired, to render the film 
 harder and firmer. The negative is to remain in this fix- 
 
178 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 ing solution, with occasional rocking, until at least one 
 minute after the opaque yellowness seen on the back has 
 entirely disappeared. All the operations up to this point 
 must be performed in the dark room. The negative may 
 now be exposed to any light. It is thoroughly washed in 
 running water, if possible; otherwise, place it in a large 
 dish and change the water repeatedly. The washing will 
 occupy from one to two hours, and it is very important 
 that it be thoroughly done, as the hyposulphite of soda 
 will turn the negative yellow and ruin it, if any remains 
 in the film. After washing, the negative is set on edge 
 until it is thoroughly dried, when it is ready for printing. 
 
 Printing. — Silver prints are the most satisfactory. The 
 ready sensitized silver paper of the dealers is not very 
 reliable, and it is safer to obtain freshly sensitized paper 
 of a local photographer. This paper will keep good only 
 a few days, and so must be used at once. It must, of 
 course, be kept in the dark. It may be cut to the right 
 size with a sharp knife, placing the paper on a glass plate 
 and using a glass plate as a guide. This may be done by 
 lamplight or in subdued daylight. 
 
 Place a negative in a printing-frame, film side towards 
 the back. As it is seldom desirable that the whole ex¬ 
 posed portion of the plate should show in the photograph, 
 a piece of yellow paper has a circular or rectangular open¬ 
 ing cut from its central portion of the size desired for the 
 picture. This cut-out, as it is called, is laid on the nega¬ 
 tive, a piece of the sensitized paper placed on it, coated 
 side next to the negative, and the back of the printing 
 frame placed in position. The negative is now exposed to 
 the direct sunlight. At short intervals the progress of 
 the printing is examined by turning the spring and lifting 
 one part of the back of the printing-frame. The paper 
 
PHOTO-MICROGRAPHY. 
 
 179 
 
 is so lield by the other part of the frame that it will fall 
 back into exactly the same register after the examination. 
 It would not do, of course, to change the position of the 
 paper on the negative. This examination should be made 
 in subdued light. The print should be made somewhat 
 darker than the photograph is desired to be, as it will fade 
 somewhat in the subsequent processes. The print, on 
 being taken from the frame, is placed in a closed box to 
 protect it from the light. When a number of prints have 
 been made, they are ready for toning. 
 
 Toning. — The prints are washed in four changes of 
 water, remaining in each about ten minutes. The third 
 washing water should have about an ounce of sodium car¬ 
 bonate thoroughly dissolved in each twenty ounces of 
 water. The prints should be placed in each water face 
 down, one at a time, that they may not cling together. 
 
 Two solutions are needed: First, fifteen grains of chloride 
 of gold are dissolved in fifteen ounces of water. Each 
 ounce contains one grain of the chloride of gold, which is 
 sufficient to tone about fifteen three by four inch prints. 
 Second, one ounce of sodium acetate is dissolved in twenty 
 ounces of water. 
 
 The toning bath should be prepared at least twelve 
 hours before it is to be used, and is made by adding one 
 ounce of the chloride of gold solution and one ounce of 
 the sodium acetate solution to eight ounces of water. 
 The temperature of the bath when used should be from 
 75° to 80° F. 
 
 Transfer the prints, one by one, face down, from the 
 fourth washing water to the toning solution. Keep them 
 in motion, and be sure that they are not sticking together, 
 or they will tone unevenly. The prints should be exam¬ 
 ined from time to time by transmitted light. When the 
 
180 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 reddish brown color has given place to a rich purple, they 
 are sufficiently toned. They are then transferred to a dish 
 of clean water. All of these operations may be performed 
 in subdued daylight or by lamplight. The prints must 
 now be fixed by dissolving out the unchanged silver salt 
 by means of sodium hyposulphite. 
 
 Fixing the Prints. — The fixing solution consists of one 
 ounce of sodium hyposulphite dissolved in one quart of 
 water. It should be freshly prepared, and used but once. 
 Place the prints in the solution, face down, one by one, 
 and keep them in motion for fifteen minutes. This may 
 be done in any light. 
 
 Washing the Prints. — The prints are transferred from 
 the fixing solution to water in which a little salt has been 
 dissolved. The salt is used in this first wash water to 
 prevent the prints from blistering. After ten or fifteen 
 minutes the prints are transferred to clean water, which 
 must be repeatedly changed at intervals for several hours. 
 The best way is to place them in a vessel through which 
 water can be kept running in a gentle stream. It is of the 
 highest importance that the washing be thoroughly done, 
 as the prints will turn yellow and fade if even a trace of 
 the sodium hyposulphite is left in them. 
 
 Mounting. — After the washing the prints are ready for 
 mounting. They may be mounted at once; but as they 
 have become very tender from the continuous soakings, it 
 is better to allow them to dry first. They are then placed 
 in a dish of water until they are wet enough to be pliable. 
 Then they are laid, face down, in a pile on a sheet of glass. 
 With a small brush apply a thin coat of ordinary flour 
 paste, or of a paste made in the same way from starch, to 
 the back of the upper print. Place the print on a suitable 
 card, and over it a piece of blotting paper. With the hand, 
 
PHOTO-MICROGRAPHY. 
 
 181 
 
 or a small roller covered with velveteen, press firmly on 
 the blotting paper, rubbing from the centre to the outside, 
 until the print is in close contact with the card at all 
 points. Place the photograph between blotters and under 
 a weight until dry, or, if a burnisher is available, run it 
 through the burnisher while still damp. It only remains 
 to attach a suitable label, and the photo-micrograph is 
 finished. 
 
 Omega Prints. — A special sensitized paper, known as 
 Omega paper, may be obtained of the dealers. It keeps 
 fresh longer than the ready Sensitized silver paper, and 
 may be worked with less trouble. For these reasons, it is 
 a favorite paper with many amateur photographers. The 
 print closely resembles a silver print. It is printed upon 
 in the same way as silver paper, but is toned and fixed in 
 a single solution, which may be purchased ready prepared 
 with directions for use. The prints must be thoroughly 
 washed after toning. It is worthy of recommendation. 
 
 Bromide Prints. — Another special paper is known as 
 bromide paper. It is very sensitive, and may be printed 
 upon by lamplight. An exposure, varying with the den¬ 
 sity of the negative from ten to twenty seconds, to the 
 light of an ordinary kerosene lamp is sufficient. The 
 image must be developed and fixed in the same way as 
 the image on a dry plate, using ferrous-oxalate developer. 
 Explicit directions for using accompany each package of 
 the paper. The prints are in black and white, and are 
 very durable. For some classes of photo-micrographs they 
 are superior to silver prints. 
 
 Lantern Slides. — Lantern slides may be made by contact 
 from the negatives of photo-micrographs in the same way 
 as from ordinary negatives. The process is precisely the 
 same as the process of making a bromide print, and is very 
 
182 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 simple. Special, slow dry plates, or positive plates, are 
 made for the purpose. In the dark room place one of 
 these over a negative in the printing-frame so that the 
 films are in contact. The exposure is made by lamplight, 
 and must be learned by practice. It varies from five to 
 thirty seconds, according to the density of the negative. 
 The lantern slide is developed in the manner described 
 for bromide prints with ferrous-oxalate developer. The 
 formula for this developer accompanies the plates, and 
 may also be obtained from the catalogues of various deal¬ 
 ers in photographic supplies. After development the lan¬ 
 tern slide is washed free of the developer and fixed in 
 sodium hyposulphite. The sodium hyposulphite must be 
 thoroughly washed out, for the reasons which have already 
 been given in connection with the other kinds of plates. 
 When the plate is dry, it is covered with a piece of thin, 
 clear glass, an ornamental mat being placed between to 
 cut out the peripheral portions, and the edges of the two 
 pieces of glass are bound securely together by means of 
 strips of black gummed paper. 
 
N 0 T E. 
 
 The reproductions of photo-micrographs shown in the following 
 plates were made by the half-tone process, a process that gives a 
 decided dark tint to the white portions of a photograph. A good 
 photo-micrograph should show the structure of the object clearly, 
 and at the same time have an undarkened background. The photo¬ 
 micrographs that are reproduced here have clear and brilliant whites; 
 the dark background of the reproductions is due to the process used. 
 
 These photo-micrographs are introduced to give those who are 
 not familiar with the work some idea of the results that may be 
 obtained in low-power photo-micrography. Descriptions of the plates 
 will be found in the List of Illustrations, pages xii-xiv. 
 
/ 
 
 
 APPENDIX. 
 
 1. Grenacher’s Borax Carmine. 
 
 Water . 
 Borax . 
 Carmine 
 
 1 to 1J grammes 
 
 50 c.c. 
 
 2 grammes 
 
 Heat until the carmine dissolves. Add an equal volume of 70 per 
 cent alcohol, and let it stand 24 hours. Filter. 
 
 Objects to be soaked in 35 per cent alcohol before staining and 
 washed in same after staining. Excess of stain removed by acid 
 alcohol (alcohol, 35 per cent, 100 c.c.; hydrochloric acid, 4 or 5 drops). 
 A good fluid for staining objects in mass. 
 
 2. Grenacher’s Alum Carmine. 
 
 W ater 
 
 50 c.c. 
 
 Alum (ammonia or potash) . . 1 to 3 grammes 
 
 Carmine 
 
 J gramme 
 
 Boil 15 to 20 minutes, cool, and filter. Add a drop or two of car¬ 
 bolic acid to preserve it. 
 
 Objects soaked in water before staining, and washed in same after 
 staining. Excess of stain removed by water. 
 
 3. Beale’s Carmine. 
 
 Ammonia . 
 Water . . 
 
 Carmine 
 
 1 c.c. 
 20 c.c. 
 
 J gramme 
 
 201 
 
202 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 When the carmine is dissolved, add 28 c.c. water, and let it stand 
 open to the air, but protected from dust, until the odor of ammonia is 
 faint. Then add 48 c.c. glycerine and 12 c.c. alcohol. 
 
 In using, pour a little of the fluid into an open dish with an equal 
 volume of glycerine, and mix thoroughly. The action is slow, requir¬ 
 ing many hours. An old fluid is quicker in action than a fresh 
 one. 
 
 Objects soaked in water before staining and washed in same after 
 staining, then passed through very dilute hydrochloric acid and again 
 washed in water. 
 
 Excess of stain removed by passing object quickly through very 
 dilute ammonia (1 drop ammonia to 5 c.c. water), and then washing 
 thoroughly in water. 
 
 4. Gerlach’s Ammonia Carmine. 
 
 Water.30 c.c. 
 
 Carmine.2 to .4 grammes 
 
 Ammonia.A few drops 
 
 Mix and, after the most of the carmine has dissolved, filter. Allow 
 it to stand exposed to the air, but protected from dust, until the odor 
 of ammonia is faint. If the carmine separates from the solution, add 
 a small drop of ammonia. For use, add this fluid, drop by drop, to 
 water until the right shade is obtained. 
 
 Objects soaked in water before staining, and washed in water after 
 staining. Excess of stain removed by passing quickly through very 
 dilute ammonia. 
 
 5. Mayer’s Cochineal Tincture. 
 
 Alcohol, 70 per cent.10 c.c. 
 
 Powdered cochineal.1 gramme 
 
 Mix, let it stand some days, and filter. If the action is too intense, 
 dilute with 70 per cent alcohol. 
 
 Objects soaked in 70 per cent alcohol before staining, and washed 
 in same after staining. 
 
APPENDIX. 
 
 203 
 
 6. Kleinenberg's Haematoxylin. 
 
 a. To a saturated solution of calcium chloride in 70 per cent alcohol 
 add an excess of pure alum, and let it stand 24 hours; filter, and add 
 8 volumes of 70 per cent alcohol. If necessary, filter again. 
 
 b. Dissolve crystallized haematoxylin in absolute alcohol to satura¬ 
 tion. Add this to the first solution until the right intensity of color 
 is obtained. The fluid thus obtained may be diluted at any time by 
 further addition of the first solution. Object soaked in 70 per cent 
 alcohol before staining, and washed in same after staining. Object 
 must contain no acid, or the color will fade. 
 
 Excess of stain removed with acidulated alcohol (70 per cent alco¬ 
 hol, 100 c.c.; hydrochloric acid, \ c.c.). Wash thoroughly again in 
 70 per cent alcohol to remove acid. 
 
 An old solution is better than a fresh one. 
 
 7. Grenadier’s Haematoxylin. 
 
 Mix 4 c.c. of a saturated solution of haematoxylin crystals in abso¬ 
 lute alcohol with 150 c.c. of a saturated solution of ammonia alum in 
 water, and let the mixture stand exposed to the light for a week. 
 Filter, and add 25 c.c. of glycerine and 25 c.c. of methyl alcohol (wood 
 spirit). An old solution is better than a fresh one. 
 
 Object placed in water before and after staining. 
 
 8. Aniline Stains. 
 
 The aniline dyes give more satisfactory results when used in alco¬ 
 holic solution, though they may be used in watery solution. Make 
 saturated solutions in 95 per cent alcohol of fuchsin, Bismarck brown, 
 iodine green, methyl green, methyl violet, and eosin. Filter, and keep 
 as stock solutions to be diluted with 95 per cent alcohol or water to 
 any desired degree. These solutions do not keep very well. Small 
 quantities, therefore, should be prepared at a time. Excess of stain 
 should be discharged in 95 per cent alcohol. The specimen should be 
 cleared in benzole or oil of cedar. The aniline stains are not very 
 permanent. Objects cannot be stained in them in loto, with the excep¬ 
 tion of Bismarck brown. 
 
204 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 9. Fuchsin — Opal Blue. (Double Stain.) 
 
 a. Dissolve .1 gramme fuchsin in 44 c.c. 95 per cent alcohol. 
 
 b. Dissolve .1 gramme opal blue in 30 c.c. 95 per cent alcohol. 
 
 c. For use, mix equal parts of a and b. 
 
 10. Fuchsin — Iodine Green. (Double Stain.) 
 
 a. Dissolve fuchsin in 50 per cent alcohol. 
 
 b. Dissolve iodine green in 50 per cent alcohol. 
 
 c. Add a to b, drop by drop, until the color is violet. 
 
 11. Schallibaum’s Collodion-Fixative. 
 
 Ether-collodion. 1 P ai ’t 
 
 Oil of cloves.3 parts 
 
 If this is found not to work well, increase the proportion of collo¬ 
 dion. Place a thin coat of this on the centre of a dry slide, place the 
 section on this, and press it flat with a piece of oiled paper. Place 
 the slide in the water-bath until the oil of cloves has evaporated. If 
 these details have been properly attended to, the section will be firmly 
 fastened to the slide. The slide may be passed through successive 
 solutions, and the section will not be loosened. If too much of the 
 fixative is used, there will be a slight tendency to cloudiness. The 
 fixative does not take borax-carmine stain, but does take hsematoxylin 
 to a small extent. 
 
 12. Gelatine Fixative. 
 
 Dissolve 5 grammes of gelatine in 30 c.c. of 50 per cent acetic 
 acid. Then add 5 c.c. of 95 per cent alcohol and 1 c.c. of glycerine. 
 
 This solution is used to fix diatoms, scales of insects, and other 
 similar objects to the cover-glass or glass slip. Apply the fixative 
 with a brush and let it dry. After the objects are arranged, breathe 
 carefully on it. The moisture of the breath dissolves the gelatine suf¬ 
 ficiently to allow the objects to be stuck to it after it has again 
 hardened. 
 
 13. Transparent Cements. 
 
 The cement used in closing mounts should in every case be trans¬ 
 parent. When the mount has been securely closed, and the trans- 
 
APPENDIX. 
 
 205 
 
 parent cement seasoned, colored cement may be applied. Reliable 
 cements may be obtained ready prepared of the dealers in microscopi¬ 
 cal supplies, and it is perhaps better to obtain them thus. All the 
 cements prepared by Rev. J. D. King, Ph.D., Cottage City, Mass., 
 are very reliable. 
 
 Shellac cement may be made as follows: Make a saturated solution 
 of bleached shellac in 95 per cent alcohol. Let this solution stand 
 protected from the dust until, by evaporation of the alcohol, it is re¬ 
 duced to the consistency of a thick syrup. Add a little turpentine to 
 render the cement less brittle. Much of the bleached shellac on the 
 market is made artificially, and is worthless for this purpose. Only 
 real shellac should be used. Shellac dissolves very slowly in alcohol. 
 The bottle should be allowed to stand some days, with occasional 
 shaking. 
 
 In applying the cement, keep the brush clean by frequently washing 
 it in 95 per cent alcohol. If the cement becomes too thick, it may be 
 thinned with 95 per cent alcohol. This cement is especially useful 
 for mounts which are to be examined under immersion lenses, as the 
 cedar oil of the immersion fluid does not act upon it, as is the case 
 with most cements. If the mounts have already been ringed with 
 other cements, apply a light varnish of shellac cement. 
 
 Berry’s hard oil finish, used by painters, is highly recommended as 
 a transparent cement. 
 
 14. Colored Cements. 
 
 Colored cements may be made by adding to the transparent 
 shellac cement sufficient of a filtered saturated solution in 95 per cent 
 alcohol of fuchsin, iodine green, or opal blue, to give a strong color. 
 
 15. Hardening Fluids. 
 
 a. For most purposes alcohol, used as described in the preceding 
 pages, is the most satisfactory hardening fluid. As it is somewhat 
 expensive, other fluids may be desired. Other fluids are better than 
 alcohol for some special cases. Whatever fluid is used, the pieces of 
 tissue to be hardened should be small, a large volume of the fluid 
 should be used, and the fluid should be changed frequently, especially 
 during the early part of the process. 
 
 b. Muller’s fluid is much slower in action than alcohol, the time 
 
206 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 varying with different tissues from a few days to eight or ten weeks. 
 The colder the fluid, the better the results. The formula is : — 
 
 Distilled water. 1 litre 
 
 Potassium dichromate, K 2 Cr 2 0 7 . 25 grammes 
 
 Sodium sulphate, Na 2 S0 4 .10 grammes 
 
 c. Erlicki’s fluid is recommended for hardening brain tissues, which 
 require about two weeks. The formula is : — 
 
 Distilled water. 1 litre 
 
 Potassium dichromate, K 2 Cr 2 0 7 . 25 grammes 
 
 Copper sulphate, CuS0 4 . 5 grammes 
 
 16. Macerating Fluids. 
 
 a. Water. 
 
 b. Caustic potash, KOH, or caustic soda, NaOH 
 
 W ater. 
 
 c. Potassium hypochlorite, KCIO (Eau de Javelle) 
 
 Water . 
 
 cl. Alcohol of less than 30 per cent. 
 e. Schultze’s macerating mixture : — 
 
 Potassium chlorate, KCIO., . 
 
 Nitric acid, HN0 3 . 
 
 The last mixture may be used either hot or cold. 
 
 17. Mounting Media. 
 
 a. Canada balsam, pure or dissolved in benzole, xylol, or chloro¬ 
 form. Best purchased ready prepared. 
 
 b. Glycerine. 
 
 c. Glycerine-jelly. Best purchased ready prepared. One formula 
 
 is: — 
 
 Best gelatine.1 part 
 
 Water. 3 parts 
 
 Glycerine. 4 parts 
 
 30 grammes 
 70 c.c. 
 
 8 drops 
 100 c.c. 
 
 1 gramme 
 50 c.c. 
 
APPENDIX. 
 
 207 
 
 Soak the gelatine in the water until it is soft. Add the glycerine, 
 and heat over a water-bath. Then add two or three drops of carbolic 
 acid as a preservative. 
 
 d. Gum arabic dissolved in water. For vegetable sections. 
 
 e. Gum arabic dissolved in a saturated aqueous solution of potas¬ 
 sium acetate to the consistency of a thick syrup. (Hoyer’s Medium 
 for vegetable sections stained in aniline dyes.) 
 
 /. Gum arabic dissolved in a 10 per cent solution of chloral hydrate 
 in water, and one-tenth the volume of glycerine added. (Hoyer’s 
 Mounting Medium for vegetable objects stained in carmine solutions.) 
 
 g. Gum arabic dissolved in turpentine, or in equal parts of turpen¬ 
 tine and benzole. A subsitute for Canada balsam, but much inferior 
 to it. 
 
 h. Farrant’s Medium for either animal or vegetable objects. 
 
 Gum arabic.2 grammes 
 
 Water.4 c.c. 
 
 Glycerine.2 c.c. 
 
 Dissolve the gum arabic in the water and add the glycerine. Stir, 
 
 but do not shake, to effect solution. Strain through cloth, if necessary. 
 
 18. Solutions for Cleaning Glass Slips and Cover-Glasses. 
 
 a. Borax dissolved in water. 
 
 b. Washing soda (sodium carbonate, Na 2 C0 3 ) dissolved in water. 
 
 c. Caustic potash (potassium hydroxide, KOH) dissolved in water. 
 
 d. Potassium dichromate, K 2 Cr 2 0 7 ... 10 grammes 
 
 Water.50 c.c. 
 
 Sulphuric acid, H 2 S0 4 . 50 c.c. 
 
 19. Acids. 
 
 Sulphuric acid, H 2 S0 4 , hydrochloric acid, HC1, and nitric acid, 
 HN0 3 , should be used in full strength when the acid is simply men¬ 
 tioned. Special strengths of these acids have been mentioned for 
 particular purposes. For ordinary use, the common commercial acids 
 are sufficiently pure. If, however, the acids are purchased especially 
 for microscopical work, the chemically pure acids should be obtained. 
 
208 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 20. Alcohol of Percentage Strength. 
 
 In these pages, alcohol of a particular percentage strength has been 
 required for many purposes. It is not always convenient to stop 
 and compute the strength; the following table is therefore added. 
 The strength of ordinary commercial alcohol is assumed to be 95 per 
 cent. It should be this, but often falls short of it. The table is com¬ 
 puted not for mathematical accuracy, but for practical utility. It is 
 sufficiently accurate for all the purposes described in this book. 
 
 To Make 
 
 Use 
 
 95 % Alcohol. 
 
 Water. 
 
 95 % alcohol 
 
 100 parts 
 
 0 parts 
 
 90% “ 
 
 95 “ 
 
 5 “ 
 
 85% “ 
 
 89 “ 
 
 11 “ 
 
 82% “ 
 
 86 “ 
 
 14 « 
 
 80% “ 
 
 84 “ 
 
 16 “ 
 
 75% “ 
 
 79 “ 
 
 21 “ 
 
 70% “ 
 
 74 “ 
 
 26 “ 
 
 65% “ 
 
 68 “ 
 
 32 “ 
 
 60% “ 
 
 63 “ 
 
 37 “ 
 
 50% “ 
 
 53 “ 
 
 47 “ 
 
 40% “ 
 
 42 “ 
 
 58 “ 
 
 35% “ 
 
 37 “ 
 
 63 “ 
 
 30% “ 
 
 32 “ 
 
 68 “ 
 
 21. Iodine Solutions. (Test for Starch.) 
 
 a. Iodine dissolved in water. The solution takes place very slowly. 
 
 b. Iodine dissolved in alcohol. 
 
 c. Iodine dissolved in a water solution of potassium iodide, KI. 
 
 22. Chlorzinc Iodine. (Schultze’s Reagent. Test for Cellulose.) 
 
 Dissolve in hydrochloric acid all the metallic zinc that will dissolve, 
 and evaporate to the consistency of a thick syrup. Add to this solu¬ 
 tion all the potassium iodide, KI, that will dissolve. Then add all 
 the iodine that will dissolve. 
 
APPENDIX. 
 
 209 
 
 23. Ammoniacal Cupric Oxide. (Test for Cellulose.) 
 
 Dissolve in water all the copper sulphate, CuS0 4 , that will dissolve. 
 Add to this solution caustic soda, NaOH, or caustic potash, KOH, as 
 long as a precipitate is formed. Filter, and wash the precipitate in 
 water that has been boiled. Place this greenish precipitate in a 
 bottle and add just enough strong ammonia, NH 4 OH, to dissolve it. 
 To test it, see if fibres of cotton will dissolve in it. Keep in a dark 
 place. 
 
 24. Solutions used in Photo-micrography. 
 
 a. Developer for Dry Plates (Stanley’s). 
 
 No. 1. 
 
 Water.80 fluid ounces 
 
 Sulphuric acid.1 drachm 
 
 Pyrogallic acid.1 oz. Troy 
 
 No. 2. 
 
 Water.80 fluid ounces 
 
 Sodium carbonate.6 oz. Troy 
 
 Sodium sulphite (crystals) .... 6 oz. Troy 
 
 Mix equal parts of No. 1 and No. 2 for use. The same portion of 
 the developer may be used to develop three or four plates. It gives 
 greater intensity after it has been used once or twice than when fresh. 
 If the exposure is known to have been too long, use old developer; 
 if the exposure has been too short, use fresh developer. One part of 
 fresh developer and three parts of that which has been used are 
 recommended for ordinary use on plates that have been exposed 
 normally. Use the developer at a temperature of 68° to 70° F. 
 
 \ Ferrous-oxalate Developer (Carbutt’s). 
 
 No. 1. 
 
 Water.30 ounces 
 
 Potassium oxalate. 8 ounces 
 
 Citric acid.60 grains 
 
 Citrate of ammonia solution ... 2 ounces 
 
 < '■ " 
 
210 
 
 PRACTICAL METHODS IN MICROSCOPY. 
 
 The citrate of ammonia solution is prepared as follows: Dissolve 
 one ounce of citric acid in five ounces of water. Place a piece of 
 litmus paper in this solution and add aqua ammonia until the paper 
 loses its red color and is on the point of changing to blue. Add 
 enough water to make the solution up to eight ounces. 
 
 No. 2. 
 
 Water.32 ounces 
 
 Iron sulphate. 4 ounces 
 
 Sulphuric acid.16 drops 
 
 No. 3. 
 
 Water. 9 parts 
 
 Potassium bromide. 1 part 
 
 Mix for use two ounces of No. 1, one ounce of No. 2, one-half 
 ounce of water, and three to six drops of No. 3. This developer may 
 be used for lantern slides and for bromide prints. Temperature 
 68° to 70° F. 
 
 c. Fixing Solution. — For negatives, use one quart of water and 
 one-half pound of sodium hyposulphite. One-fourth ounce of alum 
 may be added to this to harden the film, if desired. The addition of 
 the alum gives rise to a precipitate. Allow this to settle and decant 
 the clear solution for use. The alum may be omitted from the fixing- 
 solution, and a separate solution of alum may be used to harden the 
 film after it has been fixed, or the use of alum may be entirely omitted. 
 In most cases, except in very warm weather, the film hardens suf¬ 
 ficiently without the treatment with alum. 
 
 Use for fixing silver prints one ounce of sodium hyposulphite dis¬ 
 solved in one quart of water. The prints remain in this solution fifteen 
 minutes, and are then washed in running water. It is of the highest 
 importance that the washing be done thoroughly, or the prints will 
 turn yellow and fade. 
 
 d. Toning solution for silver prints. 
 
 No. 1. 
 
 Gold chloride 
 Water . . . 
 
 15 grains 
 15 fluid ounces 
 
APPENDIX. 
 
 211 
 
 No. 2. 
 
 Sodium acetate 
 Water . . . 
 
 1 ounce 
 20 ounces 
 
 The toning bath is to be prepared at least twelve hours before it is 
 used. It consists of one ounce of No. 1, one ounce of No. 2, and eight 
 ounces of water. This quantity is sufficient for about fifteen three-by- 
 four-inch prints. When this number has been toned, add another 
 ounce of each of the solutions No. 1 and No. 2. This toning bath 
 will keep for some time, but is liable to spoil if kept too warm. Too 
 great economy should not be exercised in keeping it. Transfer the 
 prints to clean water. 
 
 25. Metric-English Equivalents. 
 
 1 gramme = 
 
 1 ounce avoirdupois = 
 1 ounce Ti’oy = 
 
 1 fluid ounce = 
 
 1 pint = 
 
 1 quart liquid = 
 
 1 quart dry = 
 
 15.4 grains 
 28.3 grammes 
 31.1 grammes 
 
 29.25 c.c. 
 
 16 fluid ounces 
 946 c.c. 
 
 = 1101 
 
 c.c. 
 
INDEX. 
 
 A. 
 
 Abbe sub-stage condenser, 30. 
 
 Abbe camera lucida, 31, 32. 
 
 Aberration, spherical, 10, 14. 
 
 Accessories, 24 et seq. 
 
 Acetic acid, 75, 77, 79, 204. 
 
 Achromatic lens, 11. 
 
 Acids, 207. 
 
 Acid, acetic, 75, 77, 79, 204. 
 carbolic, 72, 153, 201. 
 chromic, 71, 72, 82. 
 hydrochloric, 67, 70, 72, 75, 82, 102, 
 111, 113, 121, 153, 207. 
 nitric, 67, 101, 153, 206, 207. 
 sulphuric, 72, 77, 102, 103, 153, 207, 
 209. 
 
 Adjustable objectives, 26, 60. 
 
 Adjustment, coarse, 15. 
 collar, 24, 60. 
 fine, 15, 60. 
 
 Aerobic bacteria, 165. 
 
 Agar-agar, 160, 162. 
 
 Air bubbles, 58, 64. 
 
 Alcohol, 69, 73, 78, 86, 96, 110, 111, 
 113, 117, 123, 125, 205, 208. 
 absolute, 86. 
 acid, 82, 118, 201. 
 of percentage strengths, 208. 
 
 Alum carmine, 201. 
 
 Ammonia, 102. 
 carmine, 202. 
 
 Ammoniacal cupric oxide, 71, 209. 
 
 Anaerobic bacteria, 165. 
 
 Analyzer, 44. 
 
 Angles, of crystals, measurement of, 
 136. 
 
 Angular aperture, 28. 
 
 Aniline chloride, 72. 
 
 Aniline stains, 81, 111, 113, 203. 
 sulphate, 72. 
 
 Animal specimens, 76 et seq. 
 tissues, sections of, 78, 100. 
 
 Animalcules, 76. 
 
 Anisotropic substances, 53, 137. 
 
 Aperture, angular, 28. 
 numerical, 28. 
 
 Apochromatic objectives, 11, 170. 
 
 Arachnoidiscus Ehrenbergii, 54, 57. 
 
 B. 
 
 Bacillus, 145. 
 
 comma, of cholera, 155. 
 
 of the teeth, 148, 149. 
 prodigiosus, 146. 
 subtilis, 150. 
 
 tuberculosis, 152, 153, 157. 
 
 Ehrlich’s Method for, 153. 
 
 Ziehl - Neelsen Carbol - Fuchsin 
 Method for, 153. 
 typhoid, 154, 157. 
 
 Bacteria, 143 et seq. 
 aerobic, 165. 
 anaerobic, 165. 
 chlorophyll in, 146. 
 cilia of, 147. 
 colors of, 146. 
 described, 144. 
 isolation of, 164. 
 motion of, 147. 
 of the teeth, 148, 149. 
 pathogenic, 148. 
 protoplasm in, 145,146. 
 putrefaction, 149. 
 
 Balsam, Canada, 7, 18, 43, 80, 82, 85, 
 86, 92, 96, 112, 113, 121, 124, 132, 
 206. 
 
 213 
 
214 
 
 INDEX. 
 
 Balsam, Canada, mounting in, 85. 
 Banana, scalariform vessels in, 67. 
 Base, of microscope, 15. 
 
 Bath, water-, 39, 87, 119, 121, 122, 124. 
 Beale’s carmine, 201. 
 
 Beggiatoa, 146, 150. 
 
 Bell-glasses, 163. 
 
 Benzole, 85, 86, 96, 99,109, 113, 206. 
 
 balsam, 85, 96, 113. 
 
 Bergamot, oil of, 112. 
 
 Birefringent substances, 53. 
 
 Bismarck brown, 81,151, 152, 203. 
 Bleaching agents, 69. 
 
 Blood, 79. 
 
 circulation of, in frog’s foot, 81. 
 Bone, 82. 
 
 Borax, 94, 207. 
 
 carmine, 77, 79, 96, 99, 113, 201. 
 Grenadier’s, 113, 118, 201. 
 Botanical sections, 105 et seq. 
 
 Bouillon cultures, 166. 
 
 Bromide prints, 181. 
 
 Brownian movement, 58, 147. 
 
 Brush, camel’s-hair, 18, 68. 
 
 Rigger, 93. 
 
 Bubbles, air, 58, 64. 
 
 Bull’s eye condenser, 57. 
 
 C. 
 
 Calcium hypochlorite, 69, 96, 111. 
 Camel’s-hair brush, 18, 68. 
 
 Camera lucida, 31, 170. 
 lucida, Abbe, 31, 32. 
 photographic, 171. 
 
 Canada balsam, 7, 18, 43, 80,82, 85, 86, 
 92, 112, 113, 121, 122, 124, 128, 
 132, 133, 206. 
 mounting in, 85 et seq. 
 
 Carbolic acid, 72, 153, 201. 
 
 Care of microscope, 16. 
 
 Carmine, 111, 122, 123, 124. 
 alum, 201. 
 ammonia, 202. 
 
 Beale’s, 201. 
 
 borax, 77, 79, 96, 99,113, 201. 
 Gerlach’s, 202. 
 
 Grenadier’s, 113, 118, 201. 
 
 Carrot, 68, 78, 107, 160, 166. 
 
 Cartilage, 81. 
 
 Caustic potash, 70, 71, 94, 96, 98, 206, 
 207. 
 
 soda, 102, 206. 
 
 Cedar oil, 7, 99, 112,113, 114, 152. 
 Celloidin, imbedding in, 109, 114,120, 
 123. 
 
 Cells, cement, 90. 
 epidermal, 67. 
 glass, 91. 
 metal, 91. 
 
 mounting in, 90, 91, 166, 167. 
 paraffin, 91. 
 
 Cellular tissue, lignified, 72. 
 
 Cellulose, 71. 
 
 Cements, colored, 92, 93, 205. 
 
 transparent, 89, 92, 93, 204. 
 Chemical crystals, 125 et seq. 
 Chloroform, 85, 108, 109, 112, 113, 115, 
 119, 120,122,124,125, 206. 
 balsam, 85, 113,120, 206. 
 Chlorophyll, 65, 74. 
 
 in bacteria, 146. 
 
 Clilorzinc iodine, 71, 208. 
 
 Cholera bacillus, 154. 
 
 Ckromatophores, 73, 74. 
 
 Chromic acid, 71, 72, 82. 
 
 Cilia, of bacteria, 147. 
 
 of epithelium, 84. 
 
 Cladothrix, 145, 146. 
 
 Cleaning glass slips and cover-glasses, 
 94, 95, 207. 
 slides, 87. 
 
 Clearing, 86. 
 
 Cleavage, in minerals, 136, 140. 
 
 Clips, spring, 15, 87. 
 
 Cloves, oil of, 7, 86, 109, 112, 113, 115, 
 119, 121, 122. 
 
 Cochineal, Mayer’s, 202. 
 
 Collar adjustment, 24. 
 
 Collodion, 120. 
 
 fixative, 40, 100, 120, 122,123, 204. 
 Colors, complementary, 48, 49, 50, 135. 
 in thin plates, 47, 48. 
 of bacteria, 146. 
 primary, 5. 
 
 Comma bacillus, of cholera, 155. 
 
 of the teeth, 149. 
 
 Condenser, bull’s eye, 57. 
 immersion, 30. 
 sub-stage, 29, 30, 59. 
 
INDEX. 
 
 215 
 
 Conjugation, in Spirogyra, (55,66. 
 
 Corrosive sublimate, 159, 164, 166. 
 
 Cover-glass, 20. 
 
 cleaning of, 94, 95, 207. 
 gauge, 22, 23. 
 influence of, 20. 
 preparations, 80, 151, 165. 
 
 Crenothrix, 145, 146. 
 
 Cross, dark, 50, 51, 138. 
 white, 50,138. 
 
 Crystals, 41, 42. 
 angles of, 136. 
 biaxial, 41. 
 
 chemical, 51, 52, 53,125 et seq. 
 
 hexagonal, 138. 
 
 in rocks, 135. 
 
 isometric, 137. 
 
 monoclinic, 138. 
 
 orthorhombic, 138. 
 
 plant, 74, 75. 
 
 • tests for, 75. 
 tetragonal, 138. 
 triclinic, 138. 
 uniaxial, 41. 
 
 Cultures, at elevated temperatures, 
 163. 
 
 bouillon, 166. 
 hanging-drop, 166. 
 methods, 157 et seq. 
 milk, 165. 
 plate, 163,166. 
 potato, 166. 
 pure, 165. 
 test-tube, 162. 
 without oxygen, 165. 
 
 Cupric oxide, ammoniacal, 71, 209. 
 
 Currents, 58, 64. 
 
 Cutter, section, 36. 
 
 Cutting sections, 68,120. 
 
 D. 
 
 Dehydration, 86,108, 111. 
 
 Developer, ferrous-oxalate, 181, 209. 
 Stanley’s, 209. 
 
 Development, 176. 
 
 Diaphragm, 10, 15, 33,60. 
 
 Diatoms, 54,100, 101, 102, 103, 204. 
 
 Dispersion of light, 4,14. 
 in lenses, 10. 
 
 Dissecting microscope, 12, 35, 64, 99. 
 needles, 35, 64, 96. 
 
 Double-refracting objects, 53. 
 
 Draw-tube, 15, 22, 56. 
 
 Dry mounting, 90, 129. 
 plates, 173. 
 
 development of, 174,176. 
 orthochromatic, 171, 174. 
 
 Dust, removal of, from lenses, 16. 
 
 E. 
 
 Ehrlich’s Method for tubercle bacilli, 
 153. 
 
 Emery, 132. 
 
 Eosin, 203. 
 
 Epidermal cells, 67. 
 
 Epidermis of leaves, 67. 
 
 Epithelium, 83, 84. 
 cilia of, 84. 
 
 Ether, the, 1, 2, 41. 
 sulphuric, 119,125. 
 
 Exposure in photo-micrography, 174. 
 
 Eye-lens, 14. 
 
 Eye-piece, 13,14, 15. 
 
 Huyghenian, 14. 
 micrometer, 15, 33. 
 negative, 15. 
 
 Eyes, proper use of, 55, 56. 
 
 F. 
 
 Farrant’s mounting medium, 207. 
 
 Ferns, protliallia of, 74. 
 scalariform vessels in, 67. 
 spores of, 66. 
 
 Fibre, muscular, striped, 77. 
 muscular, unstriped, 78. 
 
 Field-lens, 14. 
 
 Filter paper, 69. 
 
 Finishing slides, 92, 93. 
 
 Fixative, gelatine, 101, 204. 
 
 Schallibaum’s, 100, 120, 122, 204. 
 shellac, 124. 
 
 Fixing negatives, 177. 
 silver prints, 180. 
 
 Fluid, Erlicki’s, 206. 
 
 homogeneous immersion, 7, 24. 
 Miiller’s, 205. 
 
 Fluids, hardening, 205. 
 
216 INDEX. 
 
 Fluids, macerating, 206. 
 
 thin, 89, 91. 
 
 Forceps, 67, 96. 
 
 Fossils, 134. 
 
 Fresh objects, 63 et seq. 
 
 Frog, blood of, 79, 80, 81. 
 
 Fuchsin, 81, 151, 152, 204. 
 
 Fungi, 67. 
 
 G. 
 
 Gauge, cover-glass, 22, 23. 
 
 Gelatine, 126, 160, 206. 
 fixative, 101, 204. 
 medium, 160. 
 
 Gerlach’s ammonia carmine, 202. 
 Glass, cover-, 20, 94, 95, 96, 207. 
 
 slip, 19, 94, 95, 96, 207. 
 
 Globules, oil, 58, 64. 
 
 Glycerine, 58, 69, 89, 92, 112, 113, 121, 
 122, 123, 140, 206. 
 
 jelly, 88, 92, 112, 113, 121, 122, 123, 
 206. 
 
 Gold chloride, 179, 210. 
 
 Granite, 132. 
 
 Green, iodine, 96. 
 
 Grenacher’s carmines, 113, 118, 201. 
 
 haematoxylin, 80, 122, 203. 
 
 Gum arabic, 207. 
 
 H. 
 
 Haematoxylin, 73, 77, 111, 123, 124. 
 Kleinenberg’s, 80, 100, 118, 121, 
 122, 203. 
 
 Grenacher’s, 80, 122, 203. 
 
 Hairs, 58, 67, 72, 97. 
 
 Hanging-drop culture, 166. 
 
 Hardening, 78, 107,117. 
 fluids, 205, 206. 
 of animal tissues, 78, 117. 
 of vegetable specimens, 107. 
 Homogeneous immersion objectives, 
 24, 26, 59. 
 fluid, 7, 24. 
 
 Hoyer’s mounting media, 207. 
 Huyghenian eye-piece, 14. 
 Hydrochloric acid, 67, 70, 72, 75, 82, 
 102, 111, 113, 121, 153, 207. 
 
 I. 
 
 Iceland spar, 7, 42, 43. 
 
 Imbedding, in celloidin, 109, 114, 120. 
 in paraffin, 107, 119. 
 in soap, 110. 
 
 Immersion objectives, 24, 26. 
 
 Insects, 97, 98. 
 
 Inulin, 75. 
 
 Iodine, 70, 71, 73, 74, 208. 
 
 green, 81, 96, 99, 204. 
 
 Isotropic substances, 53,137, 138. 
 
 K. 
 
 Kleinenberg’s haematoxylin, 80, 100, 
 118, 121, 122, 203. 
 
 L. 
 
 Labels, 94. 
 
 Lantern slides, 181. 
 
 Lenses, achromatic, 11. 
 apochromatic, 11. 
 dispersion in, 10. 
 double-convex, 8. 
 eye-, 14. 
 field-, 14. 
 
 Leptothrix, 145, 149. 
 
 Light, 1 et seq. 
 
 best kind to use, 55. 
 defined, 2. 
 dispersion of, 4. 
 path of, 3. 
 
 polarized, 41 et seq., 71, 75,127, 129, 
 132, 135, 137. 
 
 polarized, uses of, 51, 52. 
 refraction of, 3. 
 sources of, 2. 
 
 Undulatory Theory of, 1. 
 wave-lengths of, 1, 2. 
 
 Lignified cellular tissue, 72. 
 
 Litmus, 161. 
 
 M. 
 
 Macerating fluids, 206. 
 
 Maceration, 67. 
 
 Manipulation, 54 et seq. 
 
 Matter, Molecular Theory of, 1. 
 Mayer’s cochineal, 202. 
 
INDEX. 
 
 217 
 
 Methyl violet, 151, 152, 203. 
 
 Mica plates, 47, 48, 52, 53,129,132,165. 
 
 Micrococcus, 145. 
 
 Micrometer, eye-piece, 15, 33. 
 stage, 33, 175. 
 
 Micrometry, 33, 34,175. 
 
 Micromillimetre, 34. 
 
 Microscope, care of, 16. 
 compound, 13. 
 dissecting, 12, 35, 64, 99. 
 parts of, 15. 
 selection of, 15. 
 simple, 11, 12. 
 
 Microtome, 36, 37, 38, 106. 
 
 Minerals, cleavage in, 136, 140. 
 
 Mirror, 15, 55, 56, 57. 
 
 Molecules, 1, 2, 41. 
 
 Monorefringent substances, 53. 
 
 Moth, scales of, 54. 
 
 Mounting media, 206, 207. 
 objects, 85 et seq., 120 et seq. 
 practice, 96 et seq. 
 
 Mucor stolonifer, 67. 
 
 Muller’s fluid, 205. 
 
 Muscular fibre, striped, 77. 
 unstriped, 78. 
 
 N. 
 
 Needles, dissecting-, 35, 64, 96. 
 
 Negatives, 176, 177, 178. 
 
 Nicol prism, 43 et seq. 
 
 Nitric acid, 67, 101, 153, 206, 207. 
 
 Nose-piece, 15. 
 
 Nucleolus, 73, 82. 
 
 Nucleus, 73, 77, 82. 
 
 Numerical aperture, 28. 
 
 O. 
 
 Objectives, 13, 15. 
 achromatic, 11. 
 adjustable, 26, 60. 
 apocliromatic, 11, 170. 
 homogeneous immersion, 24, 26, 
 59. 
 
 precautions in handling, 18. 
 water immersion, 24, 59. 
 
 Objects, fresh, methods of studying, 
 63 et seq. 
 
 Objects, opaque, 57. 
 Ocular, 13. 
 
 Oil globules, 58, 64. 
 Omega prints, 181. 
 Opal blue, 204. 
 Origanum, oil of, 112. 
 
 P. 
 
 Paper, blotting, 69. 
 filter, 69. 
 
 Japanese, 18. 
 
 Paraffin, 78, 89, 91, 107, 108, 109, 113, 
 119, 121, 122. 
 
 imbedding in, 107, 108, 119. 
 moulds, 108. 
 
 Pedesis, 59. 
 
 Pellicle, 149, 150. 
 
 Penicillium glaucum, 67. 
 
 Peptone, 160. 
 
 Phosphorescence, 146. 
 Photo-micrography, 170 et seq. 
 
 Pith, 68, 78, 107. 
 
 Pipette, 69, 76, 101, 159, 165. 
 
 Plate cultures, 163, 166. 
 
 Plates, dry, 173. 
 
 mica, 47, 48, 52, 53,129, 132, 165. 
 Pleurosigma angulatum, 54, 57, 59. 
 Polarized light, 41 et seq., 71, 75, 127, 
 129, 132, 135, 137. 
 uses of, 51, 52. 
 
 Polarizer, 44. 
 
 Pollen, 66. 
 
 Potassium acetate, 207. 
 chlorate, 67, 103, 126, 206. 
 dichromate, 89,101, 102, 126, 206. 
 hypochlorite, 206. 
 iodide, 70, 126, 208. 
 
 Practice mounting, 96 et seq. 
 Preparations, cover-glass, 80, 151, 165. 
 Prints, bromide, 181. 
 omega, 181. 
 silver, 178,179, 180. 
 
 Prism, 3 et seq. 
 
 Nicol, 43 et seq. 
 
 Protoplasm, 72, 73. 
 
 in bacteria, 145. 
 
 Pure cultures, 165. 
 
 Putrefaction bacteria, 149. 
 
218 
 
 INDEX, 
 
 R. 
 
 Raphides, 75. 
 
 Razor, 63, 68, 106. 
 
 Reflection, total, 7, 43. 
 
 Refraction, double, 43. 
 index of, 5, 6, 7. 
 of light, 3. 
 
 Rhubarb, scalariform vessels in, 68. 
 
 Rock sections, 52,131 et seq. 
 
 Rolling sand, 140. 
 
 S. 
 
 Salicine, 51, 126, 128. 
 
 Salt solution, 77. 
 
 Schallibaum’s collodion fixative, 40, 
 100, 120, 122, 123, 204. 
 
 Schizomycetes, 144. 
 
 Schultze’s macerating mixture, 67,206. 
 reagent, 71, 72, 208. 
 
 Section-cutter, 36. 
 
 Section-cutting, 68,120. 
 
 Section-lifter, 68. 
 
 Section-smoother, 120. 
 
 Sections of animal tissues, 78, 100. 
 of vegetable substances, 68, 99, 100, 
 105 et seq. 
 radial, 105. 
 rock, 52, 131 et seq. 
 tangential, 106. 
 transverse, 105. 
 
 Sediment, water, 76. 
 
 Selenite, 52, 129, 132. 
 
 Shellac cement, 127, 205. 
 fixative, 124. 
 
 Silicified wood, 134. 
 
 Silver prints, 178, 179,180. 
 
 Single-refracting objects, 53. 
 
 Slides, cleaning of, 87. 
 finishing of, 92, 93. 
 lantern, 181. 
 
 Soap, imbedding in, 110. 
 
 Soda, caustic, 102, 206. 
 
 Sodium acetate, 179, 211. 
 carbonate, 161, 166, 207. 
 hypochlorite, 69, 96, 111. 
 hyposulphite, 177, 178, 180, 210. 
 
 Spherical aberration, 10,14. 
 
 Spiral vessels, 67. 
 
 Spirilla dentium, 149. 
 
 Spirillum, 145, 148, 155. 
 
 Spirochxta, 145,148. 
 denticula, 149. 
 plicatalis, 150. 
 
 Spirogyra, 65, 66, 150. 
 
 Spores, of bacteria, 158. 
 of ferns, 66. 
 resting, 151. 
 
 Spring clips, 15, 87. 
 
 Staining, 78, 110, 111, 118. 
 
 Stains, 201 et seq. 
 aniline, 203. 
 
 Starch, 54, 70, 74, 90, 97. 
 
 test for, 70. 
 
 Sterilization, 158. 
 
 Sterilizer, hot-air, 159. 
 
 steam, 159. 
 
 Stomata, 67. 
 
 Sub-stage, 15. 
 
 condenser, 29, 30, 59. 
 
 Sugar, 126. 
 
 Sulplional, 51, 128. 
 
 Sulphuric acid, 72, 77, 102, 103, 153, 
 207, 209. 
 
 T. 
 
 Teasing, 67, 100. 
 
 Teeth, bacteria of, 148. 
 
 Test-tube cultures, 162. 
 
 Thin fluids, mounting in, 89, 91. 
 
 Thin plates, colors in, 47, 48. 
 
 Torula cerevisise, 66. 
 
 Tradescantia Virginica, 72. 
 
 Tube, draw-, 15, 22, 56. 
 length, 22. 
 
 Tuberculosis, bacillus, 153. 
 
 Turn-table, 36, 89, 91, 93, 120. 
 Turpentine, 7, 58, 78, 86, 96, 98, 101, 
 108, 113, 119, 121, 122, 152. 
 Typhoid bacillus, 154,159. 
 
 U. 
 
 Undulatory Theory of Light, 1. 
 Uniaxial crystals, 41. 
 
 V. 
 
 Vacuole, 73. 
 
 Vampyrella spirogyrss, 66. 
 
INDEX. 
 
 219 
 
 Vaseline, 166, 167. 
 
 Vegetable sections, 64, 68, 99, 105 
 et seq. 
 
 Vibrio, 145, 147. 
 
 W. 
 
 Water bath, 39, 87, 119, 121, 122, 124. 
 Water-immersion objectives, 24. 
 Water plants, 65. 
 sediment, 76. 
 
 Wave-lengths, of light, 1, 2. 
 
 Wood, silicified, 134. 
 
 X. 
 
 Xylol, 85, 152, 206. 
 
 balsam, 85, 96, 113, 206. 
 
 Y. 
 
 Yeast, 66. 
 
 Z. 
 
 Zielil-Neelsen Carbol-Fuclisin Method 
 for Bacillus tuberculosis, 153. 
 Zoogloea, 151. 
 
 Zygnemaceae, 65, 66, 74. 
 
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