= WEA LT OOM ARES ag bear ue Te it il a | i [Racor pote Sater “! at nt if THE METRIC SYSTEM 10 GENTIMETER RULE The upper edge is in millimeters, the lower in centimeters, and half centimeters. UNITS. The most commonly used divisions and multiples. ( Centimeter (cm.), 0.01 Meter; Millimeter (mm.), 0.001 Meter: Micron (), 0.001 Millimeter ; the Micron is the unit in Micrometry (? 166). Kilometer, 1000 Meters ; used in measuring roads and other long distances. THE GRAM FOR { Milligram (mg.), 0.001 Gram. WEIGHT. . . ( Atlogram, tooo Grams, used for ordinary masses, like groceries, etc. THE LITER FOR { Cubic Centimeter (cc.),0.001 Liter. Thisis more common than the correct CAPACITY. form, Milliliter. Divisions of the Unzts are indicated by the Latin prefixes: dect, 0.1; centz, 0.01 ; Milli, o.oo1 ; Micro, one millionth (0.oo0001) of any unit. Multiples are designated by the Greek prefixes: deka, 10 times; ecto, 100 times ; £70, 1000 timse ; myrza, 10,000 times ; AZega, one million (1,000,000) times any unit. THE METER FOR. LENGTH. . TABLE OF METRIC AND ENGLISH MEASURES Meter (Unit of length )—100 centimeters ; 1,000 millimeters ; 1,000,000 microns (//) 39.3704 inches ; 3.2808 feet ; 1.0936 yards. Centimeter (cm. )—10 millimeters ;,10,000 microns (//) 0.01 meter ; 0.3937 (2) inch. Millimeter (mm.)—1,000 microns (/) ; 0.I cm. ; 0.001 meter ; 0.03937 (ae) inch. Micron (u) (Unit of measure in micrometry (%166)—o.001 millimeter ; one mil- lionth of a meter ; 0.00003937 (ss}y,5) inch. Yard=3 feet ; 36 inches ; 0.91439 meter ; 91.4399 centimeters. Foot=12 inches ; 30.4799 centimeters ; 304.799 millimeters. Inch=,, foot ; ;4 yard ; 25.3999 millimeters (2.54 centimeters). Liter (Unit of capacity )—1,000 cubic centimeters (milliliters) ; (1 quart—. ) Cubic centimeter=o.oo1 liter (milliliter); (1; cub. inch.) Fluid ounce (8 fluidrachms )—29.574 cubic centimeters (30 cc. ). Gram (Unit of weight)= 1 cc. of water; 15.432 grains. Kilogram=1,000 grams ; 2.2046 (2+) lbs. avoirdupois. Ounce avoirdupois—437} grains ; 28.349 grams. Ounce Troy or apothecaries=480 grains ; 31.103 grams TEMPERATURE To change Centigrade to Farenheit : (Cc. <%)+32=F. For example, to find the equivalent of 10° Centigrade, C.= 10° 2+ 32 = 50° F. To change Farenheit to Centigrade : (F.—32°) x 3=C. For example to re- duce 50° Farenheit to Centigrade, F.= 50°, and (50°— 32°) 3 =10C.; or 54° Farenheit to Centigrade, F.=-— 40° (— 40°— 32°)=— 72°, whence — 7 Oo =— 40°C. \ 30 grams, approx. x3 Address of American Opticians: For the price of microscopes and microscopical supplies the student is advised to obtain a catalog of one or more of the opticians. Nearly all of them import foreign apparatus. Forall institutions entitled to duty free importation, American microscopes are sold at duty freerates. For the foreign opticians see the table of tube-length p. 14. § The Bausch and Lomb Optical Co., ------.--------.-.--------- New York, Rochester, and Chicago. Eimer and Amend, ------._.------------------- 205-211 3d Ave., New York. The Franklin Laboratory Supply Co ,-----.------------ Harcourt St., Boston, Mass. The Gundlach Optical Company, -------- ee er ee ee ee Rochester, N. ¥. Wm. Krafft (Representative of Leitz in America), Bieeeseces 411 West soth St., New York. Edward. Pennock, ...--.-----ssss25-5-sss---5 — -3609 Woodland Ave., Philadelphia, Pa. Queen & Company Se ee ee ee toro Chestnut St., Philadelphia, Pa. Richards & Co., -------- -12 East 18th St., New York, and 108 Lake St., Chicago, Ills. Spencer Lens Company, s eres dye Se ringas 373 Seventh St., Buffalo, N. Y. Williams, Brown & Earle, -- S g18 Chestnut St., Philadelphia, Pa. G.S. Woolman (Queen & Co. in New “York), - = __._...-116 Fulton St., New York. Joseph Zentmayer, --------------------------- = --226-228 South sth St., Philadelphia, Pa. Besides the names here given, nearly every large city has one or more dealers in microscopes and microscope supplies. Cornell oo Library QM 551.G13 190 he microscope; an introduction to micro ‘ll NL A 3 1924 001 037 0 Cornell University Library The original of this book is in the Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://archive.org/details/cu31924001037062 THE MICROSCOPE AN INTRODUCTION TO MICROSCOPIC METHODS AND TO HISTOLOGY BY SIMON HENRY GAGE PROFESSOR OF MICROSCOPY, HISTOL- OGY AND EMBRYOLOGY IN CORNELL UNIVERSITY, AND THE NEW YORK STATE VETERINARY COLLEGE. EIGHTH EDITION REVISED, ENLARGED AND ILLUSTRATED BY OVER TWO HUNDRED FIGURES COMSTOCK PUBLISHING COMPANY ITHACA, NEW YORK IQOI PREFACE TO THE EIGHTH EDITION iP the preface to the sixth edition of this work expresses accurately what should be said to-day it is appended : ‘The rapid advance in microscopical knowledge, and the great strides in the sci- ences employing the microscope as an indispensable tool, have reacted upon the microscope itself, and never before were microscopes so excellent, convenient and cheap. Indeed, the financial reason for not possessing a microscope can no longer be urged by any high school or academy, or by any person whose profession de- mands it. Naturally, to get the greatest good from instruments, tools, or machines of any kind, the one who uses them must understand the principles upon which their action depends, their possibilities and limitations. That the student may acquire a just comprehension of some of the fundamental principles of the microscope, and gain a working acquaintance with it and its ap- plications, this book has been prepared. It is a growth of the laboratory, and has been modified from time to time to keep pace with optical improvements and ad- vancing knowledge.’’ In rewriting this edition the different chapters have been recast, new figures added and in most cases the matter considerably increased. A new chapter, has been added upon class demonstrations. The general availability of the constant electric current, and the improvement in apparatus have made micro-projection practicable and satisfactory. It has served the writer so well in his teaching of histology and embryology that it seemed worth while to give the benefit of his ex- perience to his fellow workers. It is hoped that the book as it now stands will serve more completely than ever before the needs of the class-room and of the laboratory. “Simply reading a work on the microscope, and looking a few times into an instrument completely adjusted by another, is of very little value in giving real knowledge. In order that the knowledge shall be made alive, it must become a part of the student’s experience by actual experiments carried out by the student himself. Consequently, exercises illustrating the principles of the microscope and the methods of its employment have been made an integral part of the work. “In considering the real greatness of the microscope, and the truly splendid service it has rendered, the fact has not been lost sight of that the microscope is, after all, only an aid to the eye of the observer, only a means of getting a larger image on the retina than would be possible without it; but the appreciation of lv PREFACE the retinal image, whether it is made with or without the aid of a microscope, must always depend upon the character and training of the seeing and appreciat- ing brain behind the eye. The microscope simply aids the eye in furnishing raw material, so to speak, for the brain to work upon. (From 3d ed. ) Grateful acknowledgment is made to the opticians and instrument makers for the loan of cuts and for courteous and complete answers to numerous question ; to the directors of laboratories in different parts of the country, to his colleagues in the departments of Physics, Chemistry and Electrical Engineering in Cornell University ; and finally to his pupils past and present who have given their sup- port and encouragement. In closing I would like to urge those who are interested in Microscopy to take same microscopical journal, and if possible to become a member of some microscopical club or society. One can do very little alone, but by helping others and being helped in return, the workers in any field of human endeavor can accomplish great things. SIMON HENRY GAGE, CORNELL UNIVERSITY, October 1, 1907. PoBACA NEY 2 Lois. A.. x xX xs x ENG oS x 4 CONTENTS CHAPTER I I='59—The Microscope and its:parts.. 2.8 29-0 222 -- e2-sp-5 Sees Sees CHAPTER II 60-128—Lighting and Focusing; Manipulation of Dry, Adjustable and Immersion Objectives; Care of the Microscope and of the Eyes. Laboratory Microscopes-__-_-_------. ------------ CHAPTER III 129-153—Interpretation of Appearances______- __... ---. ---. .--- --~--- CHAPTER IV 154-176—Magnification and Micrometry_____-_-_-___-..-_ -.---- ------ CHAPTER V 177-187—Drawing with the Microscope ._______ -_-_ ---_ ---..-. -------- CHAPTER VI 188-233—The Microspectroscope and Polariscope ; Micro-Chemistry ; Textile Fibers and Food Products ; Micro-Metallography ___ CHAPTER VII 234-335—Slides and Cover-Glasses; Mounting; Isolation ; Sectioning by the Collodion and the Paraffin Methods ; Serial Sections ; Labeling and Storing Microscopical Preparations ; Reagents andath eine ere pabatlOm. sae ee ie ee ee eee eee eee CHAPTER VIII 336-392— Photographing objects with a Vertical Camera ; Photograph- ing Large Transparent Objects; Photographing with a Mi- croscope ; (A) Transparent Objects ; (B) Opaque Objects, and the Surfaces of Metals and Alloys ; Enlarging Nega- tives; Photographing Petri Dishes and Culture Tubes _____ CHAPTER IX 393-426—Class-Room Demonstrations with the Microscope ; With the Projection Microscope ; with the Kpiscope-___ -_..--. -_____ CHAPTER X 427-438—The Abbe Test-Plate and Apertometer ; Equivalent Focus of Objectives and Oculars ; Drawings for Photo-Engravings ; Wax Models ; Some Apparatus for Imbedding and Section- LDS ye re oe ae ee ee a ea ee RO en eee oe ees BOOKSERANDMEE RIODICATDS ete ys assem ae eee ee DL ELSY Eh) 5) ea PAGE I- 33 34- 89 go-102 103-121 122-133 134-160 161-204 205-242 243 -267 268-281 282-288 289 Hi | | | | THE MICROS Huygenian ocular (see p. 102 for positive ocular). Draw-tube by which the tube is length- ened or shortened. Main tube or body containing the draw- tube, and attached to the pillar by the arm Society screw in the lower end of the draw-tube. Society screw in the lower end of the tube. Objective in position. Stage, under which is the substage with the substaye condenser. Spring clip for holding the specimen, (exe) PE IN SECTION 9. Serew for centering,and handle for the iris diaphragm in the achromatic con- denser (see Fig. 41). Iris diaphragm outside the principal focus of the condenser for use in cen- tering (781) 3 Mirror with plane and concave face Horse-shoe base. D Rack and pinion for the substage con- denser Jointed pillar. Part of pillar with spiral spring of fine adjustment. Serew of fine adjustment Milled head of coarse adjustment. THE WIGROSEOPE AND MICROSCOPICAL METHODS CHAPTER I THE MICROSCOPE AND ITS PARTS APPARATUS AND MATERIAL FOR THIS CHAPTER A simple microscope (4 2, rr) ; A compound microscope with nose-piece (Figs. 7o-80); eye-shade (Fig. 60), achromatic (720), apochromatic (722), dry (417), immersion (7% 15), unadjustable and adjustable objectives (4% 23, 24) ; Huygenian or negative (4 38), positive (737) and compensation oculars (439); stage microme- ter (Ch. IV) ; homogeneous immersion liquid (718) ; mounted letters or figures (453); ground-glass and lens paper (% 53). A MICROSCOPE 71. A Microscope is an optical apparatus with which one may obtain a clear image of a near object, the image being always larger than the object ; that is, it enables the eye to see an object under a greatly increased visual angle, as if the object were brought very close to the eye without affecting the distinctness of vision. Whenever the microscope is used for observation, the eye of the observer forms an integral part of the optical combination (Figs. 16, 21). 72. A Simple Microscope.—With this an enlarged, erect image of an object may be seen. It always consists of one or more converging lenses or lens-systems (Figs. 16-20), and the object must be placed within the principal focus (#11). The simple microscope may be held in the hand or it may be mounted in some way to facilitate its use (Figs 17-20). 2 MICROSCOPE AND ACCESSORIES [CHAL Fics. 1-9, showing the Principal Optic Axis and the Optical Center of various forms of Lenses. Axis. The Principal Oplic Axis. c-c’. Centers of curvature of the two sur- faces of the lens. c. tl. Optical center of the lens. 1-7’. Radi of curvature of the two lens surfaces. t-l’. Tangents in Fig. 4. 23. Principal Optic Axis.—In spherical lenses, 7. ¢., lenses whose surfaces are spherical, the Axis is a line joining the centers of curvature and indefinitely extended. In the lens it is the unbroken part of the line c-c’ in the figures. In lenses with one plane surface (Figs. 3, 6, 7) the radius of the plane surface is any line at right angles to it, but in determining the axis it must be the one which is continuous with the radius of the curved surface, consequently the axis in such lenses is on the radius of the curved surface which meets the plane surface at right angles. 44. Optical Center.—The optical center of a lens is the point through which rays pass without angular deviation, that is, the emergent ray is parallel to the (CH. L MICROSCOPE AND ACCESSORIES 3 incident ray. It is determined geometrically by drawing parallel radii of the curved surfaces, 7-7’ in Figs. 4-9, and joining the peripheral ends of the radii. The optical center is the point on the axis cut by the line joining the peripheral ends of the parallel radii of the two lens surfaces. In Figs. 4-5 it is within the lens ; in 6-7 it is at the curved surface, and in the meniscus (8, 9) it is wholly out- side the lens, being situated on the side of the greater curvature. In determining the center in a lens with a plane surface, the conditions can be satisfied only by using the radius of the curved surface which is continuous with the axis of the lens, then any line at right angles to the plane surface will be parallel with it, and may be considered part of the radius of the plane surface. . (That is, a plane surface may be considered part of a sphere with infinite radius, hence any line meeting the plane surface at right angles may be considered asthe peripheral part of the radius.) In Figs. 6, 7, (7’) is the radius of the curved sur- face and (7) of the plane surface ; and the point where a line joining the ends of these radii crosses the axis is at the curved surface in each case. By a study of Fig. 4 it will be seen that if tangents be drawn at the peripheral ends of the parallel radii, the tangents will also be parallel and a ray incident at one tangential point and traversing the lens and emerging at the other tangential point acts as if traversing, and is practically traversing a piece of glass which has parallel sides at the point of incidence and emergence, therefore the emergent ray will be parallel with the incident ray. This is true of all rays traversing the center of the lens. Thick Lenses.—In all of the diagrams of lenses and the course of rays through them in this book the lenses are treated as if they were infinitely thin. In thick lenses like those figured, while there would be no angular deviation for rays trav- ersing the center of the lens, there would be lateral displacement. This is shown in Fig. 57 illustrating the effect of the cover-glass. 25. Secondary Axis.—Every ray traversing the center of the lens, except the principal axis, is a secondary axis ; and every secondary axis is more or less oblique to the principal axis. In Fig. 14, line (2), is a secondary axis, and in Fig. 15, line (1). See also Fig. 58. Fics. 10, 11.—Sectional views of a concave or diverging and a convex or converging lens to show that in the con- cave lens the principal focus is virtual as indicated by the dotted lines, while with the convex lens the focus ts real and on the side of the lens opposite to that from which the light comes. 10 II 76. Principal Focus.—This is the point where rays parallel with the axis and traversing the lens cross the axis ; and the distance from the focus to the center of the lens measured along the axis is the Principal Focal Distance. In the diagrams, Fig. 10 is seen to be a diverging lens and the rays cross the axis only by being pro- jected backward. Such a focus is said to be virtual, as it hasno real existence. In 4 MICROSCOPE AND ACCESSORIES LGA el Fig. rr the rays do cross the axis and the focus is said to be real. If the light came from the opposite direction it would be seen that there is a principal focus on the other side, that is there are two principal foci, one on each side of the lens. These two foci are both principal foci, but they will be equally distant from the center of the lens only when the curvature of the two lens surfaces are equal. There may be foci on secondary axes also, each focus on a secondary axis has its conjugate. In the formation of images the image is the conjugate of the object and conversely the object is the conjugate of the image. w f\ axis Fic. 12.—Double Convex Lens, Showing Chromatic Aberration. The ray of white light (w) is represented as dividing into the short waved, blae (6) and the long waved, red (rv) light. The blue (b) ray comes to a focus nearer the lens and the red ray (r) farther from the lens than the principal focus (/). Principal focus ( f) for rays very near the axis, f/ and f’’, foci of blue and red light coming from near the edge of the lens. The intermediate wave lengths would have foct all the way between f/ and f’’. 27. Chromatic Aberration —This is due to the fact that ordinary light con- sists of waves of varying length, and as the effect of a lens is to change the direc- tion of the waves, it changes the direction of the short waves more markedly than the long waves. Therefore, the short waved, blue light will cross the axis sooner than the long waved, red light, and there will result a superposition of colored images, none of which are perfectly distinct (Fig. 12). Fic. 13. The ray (0) near the edge of the lens is brought to a Socus nearer the lens than the ray (t). Both are brought to a focus sooner than rays very near the axts. (f) Principal focus for rays very near the axis; (f’) Focus for the ray (7), and ( f’’) Focus for the ray (0). Intermediate rays would cross the axts all the way from (FCt07 7.8. Spherical Aberration.—This is due to the unequal turning of the light in different zones of a lens. The edge of the lens refracts proportionally too much and hence the light will cross the axis or come to a focus nearer the lens than a ray which is nearer the middle of the lens. Thus, in Fig. 13, if the focus Fic. 13. Double Convex Lens, showing Spherical Aberration. GHuT | MICROSCOPE AND ACCESSORIES 5 of parallel rays very near the axis is at /, rays (o 2), nearer the edge, would come to a focus nearer the lens, the focus of the ray nearest the edge being nearest the lens. 29. Correction of Chromatic and of Spherical Aberration.—Every simple lens has the defect of both chromatic and spherical aberration, and to overcome this, kinds of glass of different refractive _power and different dispersive power are combined, concave lenses neutralizing the defects of convex lenses. If the concave lens is not sufficiently strong to neutralize the aberrations of the convex lens, the combination is said to be wnder-corrected, while if it is too strong and brings the marginal rays or the blue rays to a focus beyond the true principal focus, the combination is over-corrected. In Newton’s time there was supposed to be a direct proportion between the refractive power of any transparent medium and its dispersive power (7. é. its power to separate the light into colors). If this were true then the contention of Newton that it would be impossible to do away with the color without at the same time doing away with the refraction would be true and useful achromatic combinations would be impossible. It was found by experiment, however, that there is not a direct ratio between the refractive and dispersive powers for the different colors in different forms of glass, so that it is possible to do away largely with chromatic aberration and retain sufficient refraction to make the combination serve for the production of images. (See also the discussion under apochromatic objectives 7 22 ) Probably no higher technical skill is used in any art than is requisite in the preparation of microscopical objectives, oculars and illuminators. FIGs. 14 AND 15. 14. Convea lens showing the position of the object( A-B) pt \>—___> 4 r outside the principal focus (f°), and \\ | the course of the rays in the formation 1 \\ of real images. To avoid confusion the \ rays aredrawn from only one point. AB. Object outside the principal \ \ focus. B’ A’. Real, enlarged image NEN on the opposite side of the lens. Axts. Principal opticaxts. 1, 2,3. Rays after traversing the lens. They are converging, and consequently form a realimage. The dotted line and the line (2) give the direction of the rays as if unaffected by the lens. (F). The principal focus. Fic. 15.—Convex lens, showing the position of the object (A B) within the principal focus and the course of rays in the formation of a virtual image. ae 15 AB. The object placed between the lens and its focus ; A’ B’ virtual image formed by tracing the rays backward. It appears on the same side of the lens as the object, and 1s erect (4 11). Axis. The principal opticaxts of the lens. F. The principal focus. 1, 2,3. Rays from the point B of the object. They are diverging after trav- ersing the lens, but not so divergent as if no lens were present, as ts shown by the Axis 6 MICROSCOPE AND ACCESSORIES (CH el dotted lines. Ray (1) traverses the center of the lens, and is therefore not deflected. It ts a secondary axis (3 5). 210, Geometrical Construction of Images.—As shown in Figs. 14-15, for the determination of any point of an image, or the image being known, to determine the corresponding part of the object, it is necessary to know the position of the principal focus (and there is one on each side of the lens, % 6), and the optical center (Figs. 1-9 of the lens). Then a secondary axis (2) in Fig. 14, (1) in Fig. 15, is drawn from the extremity of the object and prolonged indefinitely above the lens, or below it for virtual images. A second line is drawn from the extremity of the object, (3) in Fig. 14, (2) in Fig. 15, to the lens parallel with the principal axis. After traversing the lens it must be drawn through the principal focal point. If now it is prolonged it will cross the secondary axis above the lens for a real image and below for a virtual image. The crossing point of these lines determines the position of the corresponding part of the image. Commencing with any point of the object the corresponding point of the image may be determined as just described, and conversely commencing with the image, corresponding points of the object may be determined. SIMPLE MICROSCOPE : EXPERIMENTS S11. Employ a tripod or other simple microscope, and for object a printed page. Hold the eye about two centimeters from the upper surface of the magnifier, then alternately raise and lower the magnifier until a clearimage may be seen. (This mutual arrangement of micro- scope and object so that a clear image is seen, is called focusing). When a clear image is seen, note that the letters appear as with the unaided eye except that they are larger, and the letters appear erect or right side up, instead of being inverted, as with the compound microscope (§ 12). Fic. 16. Diagram of the simple microscope show- ing the course of the rays and all the images, and that the eye forms an integral part of it. sl’ BY, The object within the principal focus. A3 Bs. The virtual image on the same side of the lens as the object. It ts indicated with dotted lines, as it has no actual existence. LB? A*. Retinal image of the object (A* B'). The virtual image is simply a projection of the retinal image in the field of vision, Axis. The principal optic axis of the micro- scope and of the eye. Cr. Cornea of the eye. L. Crystalline lens of the eve. R. Ideal refracting surface at which all the refractions of the eve may be assumed to take place. CH, T) MICROSCOPE AND ACCESSORIES . Fi Hold the simple microscope directly toward the sun and move it away from and toward a piece of printed paper until the smallest bright point on the paper is obtained. This is the burning point or focus, and as the rays of the sun are nearly parallel, the burning point represents approximately the principal focus (Fig. 11). With- out changing the position of the paper or the magnifier, look into the magnifier and note that the letters are very in- distinct or invisible. Move the magnifier a centimeter or two farther from the paper and no image can be seen. Now move the magnifier closer to the paper, that is, so that it is less than the focal distance from the paper, and the letters will appear distinct. This shows that in order to see a distinct image with a simple microscope, the object must always be nearer to it than its principal focal point. Or, in other words, the object must be within the principal focus. Com- pare (§ 53). After getting as clear an image as possible with a simple micro- scope, do not change the position of the microscope but move the eye nearer and farther from it, and note that when the eye is in one posi- tion, the largest field may be seen. This position corresponds to the eye-point (Fig. 30) of an ocular, and is the point at which the largest number of rays from the microscope enter the eye. Note that the image appears on the same side of the magnifier as the object. FIG. 17. Tripod Magnifier. Fic. 19. Lens-holder (The Bausch & Fic. 18. Zhe Hastings Aplan- Lomb Optical Co.) atic Triplet. (The Bausch & Lomb Optical Co.) 8 MICROSCOPE AND ACCESSORIES Marz er’ el mi Fic. 20. Dissecting Microscope. This is simply a device for holding the lens and the object to be observed. (The Bausch & Lomb Optical Co.) ih Simple microscopes are very convenient when only a small mag- nification (Ch. IV) is desired, as for dissecting. Achromatic triplets are excellent and convenient for the pocket. For use in conjunction with a compound microscope, the tripod magnifier (Fig. 17) 1s one of the best forms. For many purposes a special mechanical mounting is to be preferred. COMPOUND MICROSCOPE 212. A Compound Microscope.—This enables one to see an enlarged, in- verted image. It always consists of two optical parts—an odjective, to produce an enlarged, inverted, real image of the object, and an ocu/ar acting in general like a simple microscope to magnify this real image (Fig. 21). There is also usually present a mirror, or both a mirror and some form of condenser or illuminator for lighting the object. The stand of the microscope consists of certain mechanical arrangements for holding the optical parts and for the more satisfactory use of them. (See frontispiece. ) 713. The Mechanical Parts of a laboratory, compound microscope are shown in‘the frontispiece, and are described in the explanation of that figure. The stu- GAL MICROSCOPE AND ACCESSORIES 9 dent should study the figure with a microscope before him and become thoroughly familiar with the names of all the parts. See also the cuts of microscopes at the end of Ch. II. OPTICAL PARTS 214. Microscopic Objective.—This consists of a converging lens or of one or more converging lens-systems, which give an enlarged, inverted, real image of the object (Figs. 14,21). Andas for the formation of real images in all cases, the object must be placed outside the principal focus, instead of within it, as for the simple microscope. (See 7% II, 53, Figs. 16, 21.) Modern microscopic objectives usu- ally consist of two or more systems or combinations of lenses, the one next the object being called the /vonl com- bination or lens, the one farthest from the object and nearest the ocular, the back combination or system. There may be also one or more intermediate sys- tems. Each combination is, in general, composed of a convex and a concave lens. The combined action of the sys- tem serves to produce an image free from color and from spherical distor- tion. In the ordinary achromatic ob- jectives the convex lenses are of crown and the concave lenses of flint glass (Figs. 22, 23). Fic. 21. Diagram showing the principle of a compound microscope with the course of the rays from the object (A B) through the objective to the real image ( B’ A’), thence through the ocu- lar and into the eye to the retinal image (.42B?), and the projection of the retinal image into the field of vision as the virtual image (B33). AB. The object. A?B?. The retinal image of the inverted realimage, (BA), formed by the objective. B?A3. The inverted virtual image, a projection of the retinal image. 10 MICROSCOPE AND ACCESSORIES (CH. I Axis. The principal optic axis of the microscope and of the eye. Cr. Cornea of the eye. L. Crystalline lens of the eve. R. Single, ideal, re- fracting surface at which all the refractions of the eye may be assumed to take place. F. F. The principal focus of the positive ocular and of the objective. Mirror. The mirror reflecting parallel rays to the object. The light ts central. See Ch. LL. Pos. Ocular. An ocular in which the real image is formed outside the ocular. Compare the positive ocular with the simple microscope (Fig. 16). NOMENCLATURE OR TERMINOLOGY OF OBJECTIVES 215. Equivalent Focus.—In America, England, and sometimes also on the Continent, objectives are designated by their equivalent focal length. This length is given either in inches (usually contracted to in.) or in millimeters (mm.) Thus: An objective designated ;'; in. or 2mm., indicates that the objective produces a real image of the same size as is produced by a simple converging lens whose principal focal distance is ;'; inch or 2 millimeters (Fig. 11). An objective marked 3 in. or 75 mm., produces approximately the same sized real image as a simple converging lens of 3 inches or 75 millimeters focal length. And in accord- ance with the law ¢hat the relative size of object and image vary directly as their distance from the center of the lens (Figs. 14, 15, see Ch. IV,) it follows that the less the focal distance of the simple lens or of the equivalent focal distance of the objective, the greater is the sizeof the real image, as the tube-length remains con- stant and the image in all casesis found at about 160 or 250 mm. from the objective. 716. Numbering or Lettering Objectives.—Instead of designating objectives by their equivalent focus, many Continental opticians use letters or figures for this purpose. With this method the smaller the number, or the earlier in the alpha- bet the letter, the lower is the power of the objective. (See further in Ch. IV, for the power or magnification of objectives). This method is entirely arbitrary and does not, like the one above, give direct information concerning the objective. 417. Air or Dry Objectives.—These are objectives in which the space be- tween the front of the objective and the object or cover-glass is filled with air (Fig. 22). Most objectives of low and medium power (7. ¢,, } in. or 3 mm. and lower powers) are dry. Fic. 22. Section of a dry objective showing working distance and lighting by rejiected light. Axis. The principal optic axis of the ob- jective. BC. Back Combination, composed of a plano-concave lens of flint glass (Ff), and a double convex lens of crown glass (c). FC. Front Combination. C, O, sl. The cover-glass, object and slide. Mirror. The mirror is represented as above the stage, and as reflecting parallel rays from its plane face upon the object. Stage. Section of the stage of the microscope. CH, I) MICROSCOPE AND ACCESSORIES II W. The Working Distance, that is the distance from the front of the objective to the object when the objective ts in_focus. 218. Immersion Objectives.—An immersion objective is one with which there is some liquid placed between the front of the objective and the object or cover-glass. The most common immersion objectives are those (A) in which water is used as the immersion fluid, and (B) where some liquid is used having the same refractive and dispersive power as the front lens of the objective. Such a liquid is called homogeneous, as it is optically homogeneous with the front glass of the objective. It may consist of thickened cedar wood oil or of glycerin contain- ing some salt, as stannous chlorid in solution. When oil is used as the immersion fluid the objectives are frequently called oil immersion objectives. The disturb- ing effect of the cover-glass (Fig. 57) is almost wholly eliminated by the use of homogeneous immersion objectives, as the rays undergo very little or no refraction on passing from the cover-glass through the immersion medium and into the ob- jective ; and when the object is mounted in balsam there is practically no refrac- tion in the ray from the time it leaves the balsam till it enters the objective. Fic. 23. Sectional view of an Immersion, Ad- justable Objective, and the object lighted with axial or centraland with oblique light. alts. The principal opticaxts of the objective. BC, MC,FC. The back, middle and front | combination of the objective. In this case the Sront 1s not a combination, but a single plano- convex lens. A,B. Parallel rays reflected by the mirror axially or centrally upon the object. C. Ray reflected to the object obliquely. LI. Immersion fluid between the front of the objective and the cover glass or object (O). Mirror. The mirror of the microscope. O. Object. [tis represented without a cover- glass. Ordinarily objects are covered whether ex- amined with immersion or with dry objectives. Stage. Section of the stage of the microscope. 719. Non-Achromatic Objectives.—These are objectives in which the chro- matic aberration is not corrected, and the image produced is bordered by colored fringes. They show also spherical aberration and are used only on very cheap microscopes. (%% 7, 8, Figs. 12, 13). 720. Achromatic Objectives.—In these the chromatic and the spherical aber- ration are both largely eliminated by combining concave and convex lenses of dif- ferent kinds of glass ‘‘so disposed that their opposite aberrations shall correct each other.’’ All the better forms of objectives are achromatic and also apianan. That is the various spectral colors come to the same focus. 421. Aplanatic Objectives, etc.—These are objectives or other pieces of optical apparatus (oculars, illuminators, etc. ), in which the spherical distortion is 12 MICROSCOPE AND ACCESSORIES (CAoL wholly or nearly eliminated, and the curvatures are so made that the central and marginal parts of the objective focus rays at the same point or level. Such pieces of apparatus are usually achromatic also. 722. Apochromatic Objectives.—A term used by Abbe to designate a form of objective made by combining new kinds of glass with a natural mineral (Calcium fluorid, Fluorite, or Fluor spar). The name, Apochromatic, is used to indicate the higher kind of achromatism in which rays of three spectral colors are com- bined at one focus, instead of rays of two colors as in the ordinary achromatic ob- jectives. At the present time (rgor) several opticians make apochromatic ob- jectives without using the fluorite. Some of the early apochromatics deteriorated rather quickly in hot moist climates. Those now made are quite permanent. The special characteristics of these objectives, when used with the ‘‘compen- sating oculars”’ are as follows : (1) Three rays of different color are brought to one focus, leaving a small ter- tiary spectrum only, while with objectives as formerly made from crown and flint glass, only /wo different colors could be brought to the same focus. (2) In these objectives the correction of the spherical aberration is obtained for éwo different colors in the brightest part of the spectrum, and the objective shows the same degree of chromatic correction for the marginal as for the central part of the aperture. In the old objectives, correction of the spherical aberration was confined to rays of ove color, the correction being made for the central part of the spectrum, the objective remaining wder-corrected spherically for the red rays and over-corrected for the blue rays (7 9). (3) The optical and chemical foci are identical, and the image formed by the chemical rays is much more perfect than with the old objectives, hence the new objectives are well adapted to photography. (4) These objectives admit of the use of very high oculars, and seem to bea considerable improvement over those made in the old way with crown and flint glass. According to Dippel (Z.w.M. 1886, p. 300) dry apochromatic objectives give as clear images as the same power water immersion objectives of the old form. 223. Non-Adjustable or Unadjustable Objectives.—Objectives in which the lenses or lens systems are permanently fixed in their mounting so that their rela- tive position always remains the same. Low power objectives and those with homogenous immersion are mostly non-adjustable. For beginners and those un- skilled in manipulating adjustable objectives (7% 24), non-adjustable ones are more satisfactory, as the optician has put the lenses in such a position that the most satisfactory results may be obtained when the proper thickness of cover-glass and tube-length are employed. (See table of tube-length and thickness of cover-glass below, p. 14.) 424. Adjustable Objectives.—An adjustable objective is one in which the dis- tance between the systems of lenses (usually the front and the back systems) may be changed by the observer at pleasure. The object of this adjustment is to cor- rect or compensate for the displacement of the rays of light produced by the mounting medium and the cover-glass after the rays have left the object. It is also to compensate for variations in ‘‘tube-length.’’ See % 29. As the displace- ment of the rays by the cover-glass is the most constant and important, these ob- jectives are usually designated as having cover-glass adjustment or correction, (Fig. 23. See also practical work with adjustable objectives, Ch. IT). CH, 1) MICROSCOPE AND ACCESSORIES 13 225. Parachromatic, Pantachromatic and Semi-apochromatic Objectives.— These are trade names for objectives, most of them containing one or more lenses of the new glass (% 22). They are said to approximate much more closely to the apochromatics than to the ordinary objectives. 226. Variable Objective.—This is a low power objective of 36 to 26mm. equivalent focus, depending upon the position of the combinations. By means of a screw collar the combinations may be separated, diminishing the power, or ap- proximated and thereby increasing it. 427. Projection Objectives.—These are designed especially for projecting an image on a screen and for photo-micrography. They are characterized by having a flat, sharp field brilliantly lighted. (See Ch. IV, IX.) 228, Wluminating or Vertical Illuminating Objectives.—These are designed for the study of opaque objects with good reflecting surfaces, like the rulings on metal bars and broken or polished and etched surfaces of metals employed in micro-metallography. The light enters the side of the tube or objective and is reflected vertically downward through the objective and thereby is concentrated upon the object. The object reflects part of the light back into the microscope thus enabling one to see a clear image. %29. Tube-Length and Thickness of Cover-Glasses.—‘‘In the construction of microscopic objectives, the corrections must be made for the formation of the image at a definite distance, or in other words the tube of the microscope on which the objective is to be used must have a definite length. Consequently the microscopist must know and use this distance or ‘microscopical tube-length’ to obtain the best results in using any objective in practical work.’’ Unfortunately different opticians have selected different tube-lengths and also different points between which the distance is measured, so that one must know what is meant by the tube-length of each optician whose objectives are used. See table. The thickness of cover-glass used on an object (See Ch. VII, on mounting), except with homogeneous immersion objectives, has a marked effect on the light passing from the object (Fig. 57). To compensate for this the position of the sys- tems composing the objective are closer together than they would be if the object were uncovered. Consequently, in non-adjustable objectives some standard thick- ness of cover-glass is chosen by each optician and the position of the systems arranged accordingly. With such an objective the image of an uncovered object would be less distinct than a covered one, and the same result would follow the use of a cover-glass much too thick. I4 MICROSCOPE AND ACCESSORIES [GHsL Length in Millimeters and Parts included in ‘‘Tube-Length”’ by Various Oplicians.* ' Pts. included : in ‘*Tube- ‘Tube-Length”’ in G ! oe length. Millimeters = _.-b See Diagram. 8 henge PB Weitz, Wetzlar.2--u52222 > y an 42° (aye ae 3584 | 2=1 NAS I X0.3583 = 0.358 ae 100° Sin = = 0.7660 = NA I X0.7660 = 0.766 i | 6 ; 3 Ixy ’ Cy.) | 755 Sin 78. — 0.6087 a=1 |N.A.= 1 X0.6087 =0.609 | 6 mm | 136° 136 m=1 |N.A.= 1X0.9272=0.92 (Dry.) | 1% Sin > = 0.9272 | = N. A. +9272 = 0.927 | | tory.) | 115° | Sin 8 0.8434 | m—=1 |N.A.= 1X0.8434 = 0.843 | epry.) 163° Sin 2s =0.9890 | *=1 N.A.= 1X0.9890 = 0.989 2mm 96°12/ ee Water. 96°12’| Sin ~—— —0.7443| #7 = 1.33)N. A.=1.33X 0.7443 = 0.99 Immersion. 2 | | Zaha | 110°38/ | : Homogeneous 110°38’| Sin a —=0,8223 #=1.52|N. A.=1.52X0,8223 = 1.25 Immersion. 2 mim. | 134°10/ | Homogeneous | 134°10’| Sin oa pee nm —= 1.52|\N. A.=1.52X0.9210 = 1.40 Immersion. | 20 MICROSCOPE AND ACCESSORIES PCEERE $34. Significance of Aperture.—As to the real significance of aperture in microscopic objectives, it is now an accepted doctrine that— the corrections in spherical and chromatic aberration being the same— (1) Objectives vary directly as their numerical aperture in their ability to define or make clearly visible minute details (resolving power). For example an objective of 4 mm. equivalent focus and a numerical aper- ture of 0.50 would define or resolve only half as many lines to the millimeter or inch as a similar objective of 1.00 N.A. So also an objective of 2 mm. focus and 1.40 N.A. would resolve only twice as many lines to the millimeter as a 4 mm. objective of 0.70 N.A. Thus it is seen that defining power is not a result of magnification but of aperture, otherwise the 2 mm. objective would resolve far more than twice as many lines as the 4 mm. objective. Taking the results of the researches of Abbe as a guide to visibility with the microscope, one has the general formula 2\xN.A. That is twice the number of wave lengths of the light used multiplied by the numerical aperture of the objective. From this general statement it will be seen that the shorter the wave lengths of the light, the more there ’ will be in an inch or centimeter and therefore the greater the number of lines visible in a given space. That is the kind of light used is one ele- ment and the objective the other in determining the number of lines visible under the microscope. Following Mr. E. M. Nelson (Jour. Roy. Micr. Soc., 1893, p. 15) it is believed that not more than 3,ths of the numerical aperture of an objective is really available for microscopic study, with a central, solid cone of light. To determine the number of lines visible in a given space witha given light the formula would become 2A x 34ths N.A.=3/2AN.A. To determine the working-resolving power of any objective it is only necessary to know the number of light waves in a given space, say an inch or a centimeter and to multiply this number by 3/2 N. A. For example suppose one uses ordinary daylight and assumes the average wave length is 1/46666 in., then there must be 46,666 per inch and 46,666 X 3/2 = 70,000 approximately. If the N. A. is 1, then the objective will resolve or make visible 70,000 lines to the inch, or ap- proximately 28,000 to the centimeter. If blue light were used the number would be 32,000 per centimeter, or 80,000 per inch. It will be seen that the number of lines here given is smaller than that in the table of Carpenter-Dallinger, because in the latter the full aperture is supposed to be employed and the light is of the greatest available obliquity. CH. I) MICROSCOPE AND ACCESSORIES 21 (2) The illuminating power of an objective of a given focus is found to vary directly as the square of the numerical aperture (N.A. )’. Thus if two 4 mm. objectives of N.A. 0.20 and N. A. 0.40 were compared as to their illuminating power it would be found from the above that they would vary as 0.20°:0.40°= 0.0400:0.1600 or 1:4. That is the objective of o.20 N.A. would have but th the illuminating power of the one of 0.40 N.A. (3) The penetrating power, that is the power to see more than one plane, is found to vary as the reciprocal of the numerical aperture “ so that in an objective of a given focus the greater the aperture the less the penetrating power. Of course when equivalent focus and numerical aperture both differ the problem becomes more complex. While all microscopists are agreed that the fineness of detail which can be seen depends directly on the numerical aperture of the objective used, the general theory of microscopic vision has two interpretations : (A.) That itis as with the unaided eye, the telescope and the photographic camera. Thisis the original view and the one which many are favoring at the present day (see Mercer, Proceedings of the Amer. Micr. Soc. 1896, pp. 321-396). (B) The other view originated with Professor Abbe, and in the words of Carpenter—Dallinger, pp. 62, 43: ‘‘What this is becomes ex- plicable by the researches of Abbe. It is demonstrated that micro- scopic vision is sw generis. There isand can be, 20 comparison between microscopic and macroscopic vision. The images of minute objects are not delineated microscopically by means of the ordinary laws of refraction ; they are not dzoptrical results, but depend entirely on the laws of diffraction. These come within the scope of and demonstrate the undulatory theory of light, and involve a characteristic change which material particles or fine structural details, in proportion to their minuteness, effect in transmitted rays of light. The change consists generally in the breaking up of an incident ray into a group of rays with large angular dispersion within the range of which periodic alter- nations of dark and light occur.”’ For a consideration of the aperture question, its history and sig- nificance, see J. D. Cox, Proc. Amer. Micr. Soc., 1884, pp. 5-39 ; Jour. Roy. Micr. Soc., 1881, pp. 303, 348, 365, 388 ; 1882, pp. 300, 460 ; 1883, p. 790; 1884, p. 20; 1896, p. 681 ; 1897, p. 71 ; 1898, pp. 354, 362, 592; Mercer, Proceedings Amer. Micr. Soc., 1896, pp. 321- 22 MICROSCOPE AND ACCESSORIES (CH. I 396; Lewis Wright, Philos. Mag., June, 1898, pp. 480-503 ; Carpen- ter-Dallinger, Chapter II; Nelson, Jour. Quekett Micr. Club, VI, pp. 14-38. THE OCULAR 235. A Microscopic Ocular or Eye-Piece consists of one or more converging lenses or lens systems, the combined action of which is, like that of a simple microscope, to magnify the real image formed by the objective. \ Fic. 30. Sectional view of a Huygenian ocular to show Nee) the formation of the Eye-Point. Axis .Opticaxis of the ocular, D. Diaphragm of the ocular, E.L. Evye-Lens. F.L. Field-Lens. E. P. Eye-point. As seen in- section, 1t appears some- thing like an hour-glass. When seen as looking into the \ 4 ocular, 1. e., in transection, wt appears asa circle of light. It ~| | 7s at the point where the most rays cross. ee \ A Depending upon the relation and action of the different ‘ lenses forming oculars, they are divided into two great i | 7 L groups, zegative and posziive. 236. Negative Oculars are those in which the real, inverted image is formed within the ocular, the lower or field-lens serving to collect the image-forming rays somewhat, so that the real image is smaller than as if the field-lens were absent (Fig. 21). As the field-lens of the ocular aids in the formation of the real image it is considered by some to form a part of the objective rather than of the ocular. The upper or eye-lens of the ocular magnifies the real image. 237. Positive Oculars are those in which the real, inverted image of the object is formed outside the ocular, and the entire system of ocular lenses magnifies the real image like a simple microscope (Fig. 16). Positive and negative oculars may be readily distinguished, as a positive ocular may be used as a simple microscope, while a negative ocular cannot be so used when its field-lens is in the natural position toward the object. By turning the eye-lens toward the object and looking into the field-lens an image may be seen, however. In works and catalogs concerning the microscope and microscopic apparatus, and in articles upon the microscope in periodicals, various forms of oculars or eye- pieces are so frequently mentioned, without explanation or definition, that it seems worth while to give a list, with the French and German equivalents, and a brief statement of their character. Achromatic Ocular ; Fr. Oculaire achromatique ; Ger. achromatisches Okular. Oculars in which chromatic aberration is wholly or nearly eliminated. —Aplanatic Ocular ; Fr. Oculaire aplanatique ; Ger. aplanatisches Okular (see 2 21 ).—Brnocu- lar, stereoscopic Ocular ; Fr. Oculaire binoculaire stereoscopique ; Ger. stereosko- pisches Doppel-Okular. An ocular consisting of two oculars about as far apart as the two eyes. These are connected with a single tube which fits a monocular mi- croscope. By an arrangement of prisms the image forming rays are divided, half GH f| MICROSCOPE AND ACCESSORIES 23 being sent to each eye. The most satisfactory form was worked out by Tolles and is constructed on true stereotomic principles, both fields being equally illuminated. His ocular is also erecting.—Campani’s Ocular (see Huygenian Ocular ).—Com- pound Ocular ; Fr. Oculaire composé; Ger. zusammengesetztes Okular. An ocu- lar of two or more lenses, e. g., the Huygenian (see Fig. 30).—Condinental Ocular. An ocular mounted in a tube of uniform diameter as in Fig. 31.-—Deep Ocular, see high ocular.—Zvrecting Ocular; Fr. Oculaire redresseur ; Ger. bildumkeh- rendes Okular. An ocular with which an erecting prism is connected so that the image is erect as with the simple microscope. Such oculars are most common on dissecting microscopes.—Filar micrometer Ocular ; Screw m. 0., Cobweb m. o., Ger. Okular-Schraubenmikrometer. A modification of Ramsden’s Telescopic Cob- web micrometer ocular.—Goniometer Ocular; Fr. Oculaire 4 goniométre ; Ger. Goniometer-Okular. Anocular with goniometer for measuring the angles of minute crystals.—A/igh Ocular, sometimes called a deep ocular. One that magnifies the real image considerably, 7. e., 10 to 20 fold.—Huygenian Ocular, Huygens’ O., Campani’s O., Airy’s O.; Fr. Oculaire d’Huygens, o. de Campani; Ger. Huy- gens’sches Okular, Campaniches Okular, see 7 38.—/ndeax Ocular; Ger. Spitzen- O. An ocular with a minute pointer or two pointers at the level of the real image. The points are movable and serve for indicators and also, although not satisfac- torily, for micrometry.—el/ner’s Ocular, see orthoscopic ocular—ZLow ocular, also called shallow ocular. An ocular which magnifies the real image only moder- ately, z.¢., 2 to 8 fold.—A/icrometer or micrometric Ocular ; Fr. Oculaire microme- trique ou a micrométre ; Ger. Mikrometer-Okular, Mess Okular, Bénéches O., Jackson m.o., see 7 41.—Microscopic Ocular ; Fr. Oculaire microscopique ; Ger. mi- kroskopisches Okular. An ocular for the microscope instead of one for a telescope. —Negative Ocular, see 7 36.—Nelson’s screw-micrometer ocular. A modification of the Ramsden’s screw or cob-web micrometer in which positive compensating ocu- lars may be used.—Orthoscopic Oculars; also called Kellner’s Ocular ; Fr. Ocu- laire orthoscopique ; Ger. Kellner’sches oder orthoskopisches Okular. An ocular with an eye-lens like one of the combinations of an objective (Figs. 22, 23) anda double convex field lens. The field-lens is in the focus of the eye-lens and there is no diaphragm present. The field is large and flat.—/ur-focal Oculars, a series of oculars so arranged that the microscope remains in focus when the oculars are interchanged (Pennock, Micr. Bulletin, vol. iti, p. 9, 31).—/eriscopic Ocular ; Fr. Oculaire periscopique ; Ger. periskopisches Okular. A positive ocular devised by Gundlach. It consists of a double convex field-lens and a triplet eye-lens. It gives a large, flat field.—F/ositive Ocular, see % 37.—Projection Ocular; Fr. Ocu- laire de projection ;-Ger. Projections-Okular, see % 40.—Aamsden’s Ocular; Fr. Oculaire de Ramsden ; Ger. Ramsden’sches Okular. A positive ocular devised by Ramsden. It consists of two plano-convex lenses placed close together with the convex surfaces facing each other. Only the central part of the field is clear. Searching Ocular; Fr. Oculaire d’orientation; Ger. Sucher-Okular, see § 39, Shallow Ocular, see low ocular.—Solid Ocular, holosteric O.; Fr. Oculaire holo- stére ; Ger. holosterisches Okular, Vollglass-Okular. A negative eye-piece de- vised by Tolles. It consists of a solid piece of glass with a moderate curvature at one end for a field-lens, and the other end with a much greater curvature for an eye-lens. For a diaphragm, a groove is cut at the proper level and filled with black pigment. It is especially excellent where a high ocular is desired. —Spectral 24 MICROSCOPE AND ACCESSORIES (CH. I or spectroscopic Ocular ; Fr. Oculaire spectroscopique ; Ger, Spectral-Okular, see Microspectroscope, Ch. VI.—Stawroscopic Ocular ; Fr. Oculaire Stauroscopique. Ger. Stauroskop-Okular. An ocular with a Bertrand’s quartz plate for mineralog- ical purposes.— Working Ocular; Fr. Oculaire de travail; Ger. Arbeits-Okular, see % 39.. 238. Huygenian Ocular —A negative ocular designed by Huygens for the telescope, but adapted also to the microscope. It is the one now most commonly employed. It consists of a field-lens or collective (Fig. 30), aiding the objective in forming the real image, and an eye-lens which magnifies the real image. While Ocular Wo. 2 4 6 8 12 18 Fic. 31. Compensating Oculars of Zeiss, with section removed to show the con- struction. The line A-A is at the level of the upper end of the tube of the micro- scope while B-B represents the lower focal points. Tt will be seen that the mount- ing is soarranged that the lower focal points in all are in the same plane and therefore the microscope remains in focus upon changing oculars. ( The oculars are par-focal). The lower oculars, 2, gand 6 are negative, and the higher ones, 8, 12, 18, are positive. The numbers 2, 4, 6, 8, 12, 18, indicate the magnification of the ocular. From Zeiss’ Catalog.) the field-lens aids the objective in the formation of the real, inverted image, and increases the field of view, it also combines with the eye-lens in rendering the image achromatic. (See % 46). 439. Compensating Oculars.—These are oculars specially constructed for use with the apochromatic objectives. They compensate far aberrations outside the axis which could not be so readily eliminated in the objective itself. An ocu- lar of this kind, magnifying but twice, is made for use with high powers, for the sake of the large field in finding objects; it is called a searching ocular; those ordinarily used for observation are in contradistinction called working oculars. Part of the compensating oculars are positive and part negative. (Fig. 31. ) 7440. Projection Oculars.—These are oculars especially designed for project- ing a microscopic image on the screen for class demonstrations, or for photo- graphing with the microscope. While they are specially adapted for use with apochromatic objectives, they may also be used, with ordinary achromatic objectives of large numerical aperture. CH. I) MICROSCOPE AND ACCESSORIES 25 Fic. 32. Projection Oculars with section re- moved to show the construction. Below are shown the upper ends with graduated circle to indicate the amount of rotation found necessary to focus the diaphragm on the screen. No. 2, No. 4. The numbers indicate the amount the ocular magnifies the image formed by the objective as with the compensation oculars. (Zetss’ Catalog.) % 41. Micrometer Ocular.—This is an ocular connected with an ocular micrometer. The micrometer may be removable, or it may be permanently in connection with the ocular, and arranged with a spring and screw, by which it may be moved back and forth across the field. (See Ch. IV.) 1 i i iii FIGS. 33-34. Ocular Micrometer and movable scale. Fig. 33 1s a side view of the ocular while Fig. 34 gives a sectional end view, and shows the ocular micrometer in position. In both the screw which moves the micrometer ts shown at the left. (From Bausch & Lomb Opt. Co.) %4.42. Spectral or Spectroscopic Ocular.—(See Micro-Spectroscope, Ch. VI). DESIGNATION OF OCULARS 7443. Equivalent Focus.—As with objectives, some opticians designate the oculars by their equivalent focus (415). With this method the power of the ocular, as with objectives, varies inversely as the equivalent focal length, and therefore the greater the equivalent focal length the less the magnification. This seems as desirable a mode for oculars as for objectives and is coming more and more into use by the most progressive opticians. It is the method of designation advo- cated by Dr. R. H. Ward for many years, and was recommended by the committee of the American Microscopical Society,(Proc. Amer. Micr. Soc., 1883, p. 175, 1884, p. 228). 26 MICROSCOPE AND ACCESSORIES (CH. I Fic. 35. Ocular Screw-Micrometer with compensation ocular 6. The upper figure shows a sectional view of the ocular and the screw for moving the micrometer al the right. a At the left is shown a clamping screw to | fasten the ocular to the upper part of the mi- croscope tube. Below is a face view, showing the graduation on the wheel. An ocular micrometer like this is in general like the cob-web micrometer and may be used for measuring objects of varying sizes very accu- rately. With the ordinary ocular micrometer very small objects frequently fil but a part of an interval of the micrometer, but with this the movable cross lines traverse the object (or rather its real image) regardless of the minute- ness of the object. ( Zeiss’ Catalog’). 244. Numbering and Lettering.—Oculars like objectives may be numbered or lettered arbitrarily. When so designated, the smaller the number, or the earlier the letter in the alphabet, the lower the power of the ocular. 245. Magnification.—The compensating oculars are marked with the amount they magnify the realimage. Thus an ocular marked & 4, indicates that the real image of the objective is magnified four fold by the ocular. The projection oculars are designated simply by the amount they multiply the real image of the objective. Thus for the short or 160 mm. tube-length they are, <2, 4; and for the long or 250 mm. tube, they are x3 and «6. That is, the final image on the screen or the ground glass of the photographic camera will be 2, 3, 4, or 6 times greater than it would be if no ocular were used. See Ch. VIII. 446. Standard Size Oculars.—The Royal Microscopical Society of London took a very important step (Dec. 20, 1899) in establishing standard sizes for ocu- lars and sub-stage condensers. To quote from the Journal of the Royal Micro- scopical Society for 1900, p. 147: Resolved, ‘‘That the standard size for the inside diameter of the substage fit- ting be 1.527 in.—38.786 mm. That tbe gauges for standardizing eye-pieces be the internal diameters of the draw-tubes, the tightness of the fit being left to the discretion of the manufacturers.”’ The sizes for oculars are four in number, 1 and 2 being most common. (1) 0.9173 inch = 23.300 mm. This is the Continental size. (2) 1.04 inch= 26.416mm. This is the size used by the English Opticians for student and small microscopes. (3) 1.27. inch = 32.258 mm. Medium size binoculars (English. ) (4) 1.41 inch=35.814 mm. Long tube binoculars. For the history of the Huygenian Ocular, and a discussion of formulae for its construction, see Nelson, J. R. M. S., 1900, p. 162-169. EXPERIMENTS S47. Putting an Objective in Position and Removing it.— Elevate the tube of the microscope by means of the coarse adjustment CELA] MICROSCOPE AND ACCESSORIES 27 (frontispiece) so that there may be plenty of room between its lower end and the stage. Grasp the objective lightly near its lower end with two fingers of the left hand, and hold it against the nut at the lower end of the tube. With two fingers of the right hand take hold of the milled ring near the back or upper end of the objective and screw it into the tube of the microscope. Reverse this operation for removing the objective. By following this method the danger of dropping the objective will be avoided. § 48. Putting an Ocular in Position and Removing it.—Ele- vate the body of the microscope with the coarse adjustment so that the objective will be 2 cm. or more from the object—grasp the ocular by the milled ring next the eye-lens (Fig. 21), and the coarse adjustment or the tube of the microscope and gently force the ocular into position. In removing the ocular, reverse the operation. If the above precau- tions are not taken, and the oculars fit snugly, there is danger in in- serting them of forcing the tube of the microscope downward and the objective upon the object. $49. Putting an Object under the Microscope.—This is so placing an object under the simple microscope, or on the stage of the compound microscope, that it will be in the field of view when the microscope is in focus (§ 50). With low powers, it is not difficult to get an object under the microscope. The difficulty increases, however, with the power of the microscope and the smallness of the object. It is usually necessary to move the object in various directions while looking into the micro- scope, in order to get it into the field. Time is usually saved by get- ting the object in the center of the field with a low objective before putting the high objective in position. This is greatly facilitated by using a nose-piece, or revolver. (See Figs. 36-36a, and the pictures of microscopes, Ch. II.) Fic. 36. Triple nose-piece or revol- Fic. 36a. 7yriple nose-piece or re- ver for quickly changing objectives. (The volver for quickly changing objectives. Spencer Lens Co.) (The Bausch & Lomb Optical Co.) 28 MICROSCOPE AND ACCESSORIES [GAL $50. Field or Field of View of a Microscope.—This is the area visible through a microscope when it is in focus. When properly lighted and there is no object under the microscope, the field appears asacircle of light. When examining an object it appears within the light circle, and by moving the object, if it is of sufficient size, differ- ent parts are brought successively into the field of view. In general, the greater the magnification of the entire microscope, whether the magnification is produced mainly by the objective, the ocular, or by increasing the tube length, or by a combination of all three (see Ch. IV, under magnification ), the smaller is the field. The size of the field is also dependent, in part, without regard to magnification, upon the size of the opening in the ocular diaphragm. Some oculars, as the orthoscopic and periscopic, are so constructed as to eliminate the ocular diaphragm, and in consequence, although this is not the sole cause, the field is considerably increased. The exact size of the field may be read off directly by putting a stage micrometer under the microscope and noting the number of spaces required to measure the diameter of the light circle. 2mm \ @ 2 s Pg 75mm a Ss5 mm Fic. 37. Figures showing approximately the actual size of the field with ob- jectives of 85 mm., 45 mm., 17 mmMm., 5 mm., and 2 mm., equivalent focus, and ocular of 373 mm., equivalent focus in cach case. This figure shows graphically what ts also very clearly indicated in the table (4% 52). S51. The size of the field of the microscope as projected into the field of vision of the normal human eye (7. ¢., the virtual image) may be determined by the use of the camera lucida with the drawing surface placed at the standard distance of 250 millimeters (Ch. IV.) $52. Table showing the actual size in millimeters of the field of a group of commonly used objectives and oculars. Compare with the graphic representationin Fig. 37. See also § 50. CA. 1) MICROSCOPE AND ACCESSORIES 29 in | : . | | Equivalent Diameter sie ait Kind of | | Focus and | : NAY of of Field Focus of | Objective | in mm. Ocular Ocular SH wt | 15.4 37% mm. | Shysudbscheeees ie 25 a Huygenian 3 Tagg ee 7.0 3714 mm 45: min) 3 222 5.0 25 oe Huygenian 4.0 12% ‘ 3.0 3712 mm.) PSR eee 2:0) 25 ‘““ Huygenian 1.6 Tay || | 5-7 1So mm. | Tie eee. | 2.8 45 ‘‘ | Compensation NiAL = | 1d 15 « | 0.97 10 ae | 0.541 37% mm. | acer eo eee || 0.371 25 a | Huygenian 0.290 yay | 0.850 | 180 mm, | 0.501 “ ; N. A. =0.92 | eB ne 2 «| Compensation | 0.173 10 ie, | 0.270 37% mm 2 mm._____. | 0.186 25 xe Huygenian | 0.147 12% ‘ 0.450 18s mm 0.251 45 ere | ‘ NicA = 1,25) 0.125 15 “ Compensation 0.088 Io DY | FUNCTION OF AN OBJECTIVE §$ 53. Puta 50 mm. objective on the microscope or screw off the front combination of a 16 mm.,(23-in.), and put the back combination on the microscope for a low objective. Place some printed letters or figures under the microscope, and light well. In place of an ocular put a screen of ground glass, or a piece of lens paper, over the upper end of the tube of the microscope. * *Ground glass may be very easily prepared by placing some fine emery or carborundum between two pieces of glass, wetting it with water and then rubbing the glasses together for a few minutes. If the glass becomes too opaque, it may be rendered more translucent by rubbing some oil upon it. 30 MICROSCOPE AND ACCESSORIES (CH. I Lower the tube of the microscope by means of the coarse adjust- ment until the objective is within 2-3 cm. of the object on the stage. Look at the screen on the top of the tube, holding the head about as far from it as for ordinary reading, and slowly elevate the tube by means of the coarse adjustment until the image of the letter appears on the screen. The image can be more clearly seen if the object is in a strong light and the screen in a moderate light, z. e., if the top of the micro- scope is shaded. The letters will appear as if printed on the ground glass or paper, but will be inverted (Fig. 21). If the objective is not raised sufficiently, and the head is held too near the microscope, the objective will act as a simple microscope. If the letters are erect, and appear to be down in the microscope and _ not on the screen, hold the head farther from it, shade the screen, and raise the tube of the microscope until the letters do appear on the ground glass. To demonstrate that the object must be outside the principal focus with the compound microscope, remove the screen and turn the tube of the microscope directly toward the sun. Move the tube of the micro- scope with the coarse adjustment until the burning or focal point is found (§6). Measure the distance from the paper object on the stage to the objective, and it will represent approximately the principal focal distance (Figs. ro, 11). Replace the screen over the top of the tube, noimage can be seen. Slowly raise the tube of the microscope and the image will finally appear. If the distance between the object and the objective is now taken, it will be found considerably greater than the principal focal distance (compare § 11). $54. Aerial Image.—After seeing the real image on the ground- glass, or paper, use the lens paper over about half of the opening of the tube of the microscope. Hold the eye about 250 mm. from the microscope as before and shade the top of the tube by holding the hand between it and the light, or in some other way. The real image can be seen in part asif on the paper and in part in the air. Move the paper so that the image of half a letter will be on the paper and half in the air. Another striking experiment is to have a small hole in the paper placed over the center of the tube opening, then if a printed word extends entirely across the diameter of the tube its central part may be seen in the air, the lateral parts on the paper. The advantage of the paper over part of the opening is to enable one to accommodate the CH a7 | MICROSCOPE. AND ACCESSORIES 31 eyes for the right distance. If the paper is absent the eyes adjust themselves for the light circle at the back of the objective, and the aerial image appears low in the tube. Furthermore it is more difficult to see the aerial image in space than to see the image on the ground- glass or paper, for the eye must be held in the right position to receive the rays projected from the real image, while the granular surface of the glass and the delicate fibres of the paper reflect the rays irregularly, so that the image may be seen at almost any angle, as if the letters were actually printed on the paper or glass. $55. The Function of an Objective, as seen from these experi- ments, is to form an enlarged, inverted, real image of an object, this image being formed on the opposite side of the objective from the object (Fig. ar). FUNCTION OF AN OCULAR $56. Using the same objective as for § 53, get as clear an image of the letters as possible on the lens paper screen. Look at the image with a simple microscope (Fig. 17 or 18) as if the image were an object. Observe that the image seen through the simple microscope is merely an enlargement of the one on the screen, and that the letters remain inverted, that is they appear as with the naked eye (§ 11). Remove the screen and observe the aerial image with the tripod. Put a 50mm. (A, No. 1 or 2 in.), ocular 7. e., an ocular of low magnification) in position (§ 48). Hold the eye about ro to 20 milli- meters from ‘the eye-lens and look into the microscope. ‘The letters will appear as when the simple microscope was used (see above), the image will become more distinct by slightly raising the tube of the microscope with the coarse adjustment. $57. The Function of the Ocular, as seen from the above, is that of a simple microscope, viz.: It magnifies the real image formed by the objective as if that image were an object. Compare the image formed by the ocular (Fig. 21), and that formed by a simple microscope (Fig. 38). It should be borne in mind, however, that the rays from an object as usually examined with a simple microscope, extend from the object in all directions, and no matter at what angle the simple microscope is held, provided it is sufficiently near and points toward the object, an image may be seen. The rays froma real image, however, are continued in certain definite lines and not in all directions; hence, in order to see this aerial image with an ocular or simple microscope, or 2 MICROSCOPE AND ACCESSORIES (CH. I w in order to see the aerial image with the unaided eye, the simple micro- scope, ocular or eye must be in the path of the rays (Fig. 21.) Sais At Fic. 38. Diagram of the simple microscope showing the course of the rays and all the images, and that the eye forms an integral part of i. A Bt. The object within the principal focus. AB. The virtual image on the same side of the lens as the object. It is indicated by dotted lines, as it has no actual existence. B: A?. Retinal image of the object (A* B*). saAZYEA Wa The virtual image is simply a projection of the retinal image into the field of vision. . . eo rr , 3 Woy Axis. The principal optic axis of the micro- ia See fAllee Nace scope and of the eye. Cr. Cornea of the eve. L. He / F a i Crystalline lens of the eye. R. Ideal refracting nee uy oa surface at which all the refractions of the eye may we 2 Nt be assumed to take place. yi eee fe $58. The field-lens of a Huygenian ocular makes the real image smaller and consequently increases the size of the field ; it also makes the image brighter by contracting the area of the real image. (Fig. 30.) Demonstrate this by screwing off the field-lens and using the eye-lens alone as an ocular, refocusing if necessary. Note also that the image is bordered by a colored haze (§ 7). When looking into the ocular with the field-lens removed, the eye should not be held so close to the ocular, as the eye-point is consider- ably farther away than when the field-lens is in place. $59. The eye-point.—This is the point above the ocular or simple microscope where the greatest number of emerging rays cross. Seen in profile, it may be likened to the narrowest part of an hour glass. Seen in section (Fig. 30), it is the smallest and brightest light circle above the ocular. This is called the eye-point, for if the pupil of the eye is placed at this level, it will receive the greatest number of rays from the microscope, and. consequently see the largest field. Demonstrate the eye-point by having in position an objective and ocular as above (§ 53). Light the object brightly, focus the micro- scope, shade the ocular, then hold some ground-glass or a piece of the lens paper above the ocular and slowly raise and lower it until the smallest circle of light is found. By using different oculars it will be CH. I, MICROSCOPE AND ACCESSORIES 33 seen that the eye-point is nearer the eye-lens in high than in low ocu- lars, that is the eye-point is nearer the eye-lens for an ocular of small equivalent focus than for one of greater focal length. REFERENCES FOR CHAPTER I In chapter X will be given a bibliography, with full titles, of the works and periodicals referred to. For the subjects considered in this chapter, general works on the microscope may be consulted with great advantage for different or more exhaustive treatment. The most satisfactory work in English is Carpenter-Dallinger, Sth Ed. For the history of the microscope, Mayall’s Cantor Lectures on the microscope are very satisfactory. For a continuation of the history begun by Mayall in the Cantor Lectures see Nelson, Journal of the Queckett Micr. Club, and the Jour. Roy. Micr. Soc., 1897-1g01+. Carpenter-Dallinger, 8th Ed. Petri, Das Mikroskop. The following special articles in periodicals may be examined with advantage : Apochromatic Objectives, etc. Dippel in Zeit. wiss. Mikr., 1886, p. 303 ; also in the Jour. Roy. Micr. Soc., 1886, pp. 316, 849, II10, ; same, 1890, p. 480; Zeit. f. Instrumentenk., 1890, pp. 1-6 ; Micr. Bullt., 1891, pp. 6-7. Tube-length, etc. Gage, Proc. Amer. Soc. Micrs., 1887, pp. 168-172; also in the Microscope, the Jour. Roy. Micr. Soc., and in Zeit, wiss. Mikr., 1887-8. Bausch, Proc. Amer. Soc. Micrs., 1890, pp. 43-49; also in the Microscope, 18go, pp. 289-296. Aperture. J.D. Cox, Presidential Address, Proc. Amer. Soc. Micrs., 1884, pp, 5-39, Jour. Roy. Micr. Soc., 1881, pp. 303, 348, 365, 388; 1882, Ppp. 300, 460; 1883, p. 790; 1884, p. 20. Czapski, Theorie der optischen Instrumente nach Abbe. See also references in 7 34. — The Barnes Dissecting Microscope (The Bausch & Lomb Optical Company). CHAPTER II LIGHTING AND FOCUSING; MANIPULATION OF DRY, ADJUSTABLE AND IMMERSION OBJECTIVES ; CARE OF THE MICROSCOPE AND OF THE EYES ; LABORATORY MICROSCOPES APPARATUS AND MATERIAL FOR THIS CHAPTER Microscope supplied with plane and concave mirror, Achromatic and Abbe condensers, dry, adjustable and immersion objectives, oculars, triple nose-piece. Microscope lamp and movable condenser (bull’s eye or other form, Fig. 53), Homogeneous immersion liquid ; Benzin, alcohol, distilled water ; Mounted prep- aration of fly’s wing (% 70); Mounted preparation of Plewrosigma (477, 78). Stage or ocular micrometer (4% 92); Glass slides and cover-glasses (Ch. VII); 10 per ct. solution of salicylic acid in 95 per ct. alcohol (7 92); Preparation of stained bac- teria (7 108); Vial of equal parts olive or cotton seed oil or liquid vaselin and benzin (% 112): Double eye shade (Fig. 60); Screen for whole microscope (Fig. 59). FOCUSING ¢ 60, Focusing is mutually arranging an object and the microscope so that a clear image may be seen. With a simple microscope (7 11) either the object or the microscope or both may be moved in order to see the image clearly, but with the compound microscope the object more conveniently remains stationary on the stage, and the tube or body of the microscope is raised or lowered (frontispiece). In general, the higher the power of the whole microscope whether simple or compound, the nearer together must the object and objective be brought. With the compound microscope, the higher the objective, and the longer the tube of the microscope, the nearer together must the object and the objective be brought. If the oculars are not par-focal, the higher the magnification of the ocular, the nearer must the object and objective be brought. 461. Working Distance.—By this is meant the space between the simple mi- croscope and the object, or between the front lens of the compound microscope and the object, when the microscope is in focus. This working distance is always con- siderably less than the equivalent focal length of the objective. For example, the front-lens of a6 mm. or (th in. objective would not be 6 millimeters or (th inch from the object when the microscope is in focus, but considerably less than that distance. If there were no other reason than the limited working distance of high objectives, it would be necessary to use a very thin cover-glass over the CA. II) LIGATING AND FOCUSING 39 object. (See 724, 29.) If too thick covers are used it may be impossible to get an objective near enough an object to get itin focus. For objects that admit of examination with high powers it is always better to use thin covers. LIGHTING WITH DAYLIGHT 262. Unmodified sunlight should not be employed except in special cases. North light is best and most uniform. When the sky is covered with white clouds the light is most favorable. To avoid the shadows produced by the hands in manipulating the mirror, etc., it is better to face the light ; but to protect the eyes and to shade the stage of the microscope some kind of screen should be used. The one figured in (Fig. 62) is cheap and efficient. If one dislikes to face the window or lamp it is better to sit so that the light will come from the left as in reading. It is of the greatest importance and advantage for one who is to use the microscope for serious work that he should comprehend and appreciate thoroughly the various methods of illumination, and the special appearances due to different kinds of illumination. Depending on whether the light illuminating an object traverses the object or is reflected upon it, and also whether the object is symmetrically lighted, or lighted more on one side than the other, light used in microscopy is designated as reflected and transmitted, axial and oblique. 39 40 Fics. 39-go. For full explanation see Figs. 22 and 23. 763. Reflected, Incident or Direct Light.—By this is meant light reflected upon the object in some way and then irregularly reflected from the object to the microscope. By this kind of light objects are ordinarily seen by the unaided eye, and the objects are mostly opaque. In Vertebrate Histology, reflected light is but 36 LIGHTING AND FOCUSING (CH. 11 little used; but in the study of opaque objects, like whole insects, etc., it is used agreat deal. For low powers, ordinary daylight that naturally falls upon the object, or is reflected or condensed upon it with a mirror or condensing lens, answers very well. For high powers and for special purposes, special illuminating apparatus has been devised (% 28). (See also Carpenter-Dallinger, Ch. IV). 264. Transmitted Light.—By this is meant light which passes through an object from the opposite side. The details of a photographic negative are in many cases only seen or best seen by transmitted light, while the print made from it is best seen by reflected light. Almost all objects studied in Vertebrate Histology are lighted by transmitted light, and they are in some way rendered transparent or semi-transparent. The light traversing and serving to illuminate the object in working with a compound microscope is usually reflected from a plane or concave mirror, or from a mirror to a condenser (788), and thence transmitted to the object from below (Figs. 48-51). 265. Axial or Central Light.—By this is understood light reaching the object, the rays of light being parallel to each other and to the optic axis of the micro- scope, or a diverging or converging cone of light whose axial ray is coincident with the optic axis of the microscope. In either case the object is symmetrically illuminated. 266. Oblique Light.—This is light in which parallel rays from a plane mirror form an angle with the optic axis of the microscope (Fig. 4o). Orif a concave mirror or a condenser is used, the light is oblique when the axial ray of the cone of light forms an angle with the optic axis (Fig. 40). DIAPHRAGMS 267. Diaphragms and their Proper Employment.—Diaphragmis are opaque disks with openings of various sizes, which are placed between the source of light or mirror and the object. In some cases an iris diaphragm is used, and then the same one is capable of giving a large range of openings. The object of a dia- phragm in general, is to cut off all adventitious light and thus enable one to light the object in such a way that the light finally reaching the microscope shall all come from the object or its immediate vicinity. The diaphragms of a condenser serve to vary its aperture to the needs of each object and each objective. 768, Size and Position of Diaphragm Opening.—When no condenser is used the size of the opening in the diaphragm should be about that of the front lens of the objective. For some objects and some objectives this rule may be quite widely departed from ; one must learn by trial. When lighting with a mirror the diaphragm should be as close as possible to the object in order, (a) that it may exclude all adventitious light from the object ; (b) that it may not interfere with the most efficient illumination from the mirror by cutting off a part of the illuminating pencil. If the diaphragm is a considera- ble distance below the object, (1) it allows considerable adventitious light to reach the object and thus injures the distinctness of the microscope image; (2) it pre- vents the use of very oblique light unless it swings with the mirror ; (3) it cuts off a part of the illuminating cone from a concave mirror. On the other hand, even with a small diaphragm, the whole field will be lighted. CH. IT] LIGHTING AND FOCUSING 37 With an illuminator or condenser (Figs. 41, 48), the diaphragm serves to narrow the pencil to be transmitted through the condenser, and thus to limit the aperture (see 7 84). Furthermore, by making the diaphragm opening eccentric, oblique light may be used, or by using a diaphragm with a slit around the edge (central stop diaphragm), the center remaining opaque, the object may be lighted with a hollow cone of light, all of the rays having great obliquity. In this way the so- called dark-ground illumination may be produced (4% 92; Fig. 51). ARTIFICIAL ILLUMINATION 769. For evening work and for certain special purposes, artificial illumina- tion is employed. A good petroleum (kerosene) lamp with flat wick has been found very satisfactory, also an incandescent electric or Welsbach light, but for brilliancy and for the actinic power necessary for very rapid photo-micrography (see Ch. VIII) the electric arc lamp or an acetylene lamp serves well. Whatever source of artificial light is employed, the light should be brilliant and steady. LIGHTING AND FOCUSING : EXPERIMENTS § 70. Lighting with a Mirror.—As the following experiments are for mirror lighting only, remove the substage condenser if present (see § 79, for condenser). Place a mounted fly’s wing under the microscope, put the 16 mm.( 24 in.) or other low objective in position, also a low ocular. With the coarse adjustment lower the tube of the microscope to within about 1 cm. of the object. Use an opening in the diaphragm about as large as the front lens of the objective ; then with the plane mirror try to reflect light up through the diaphragm upon the object. One can tell when the field (§ 50) is illuminated, by looking at the object on the stage, but more satisfactorily by looking into the microscope. It sometimes requires considerable manipulation to light the field well. After using the plane side of the mirror turn the concave side into position and light the field with it. As the con- cave mirror condenses the light, the field will look brighter with it than with the plane mirror. It is especially desirable to remember that the excellence of lighting depends in part on the position of the diaphragm (§ 68). Ifthe greatest illumination is to be obtained from the concave mirror, its position must be such that its focus will be at the level of the object. This distance can be very easily determined by finding the focal point of the mirror in full sunlight. $71. Use of the Plane and of the Concave Mirror.—The mir- ror should be freely movable, and have a plane and aconcave face. The concave face is used when a large amount of light is needed, the plane face when a moderate amount is needed or when it is necessary to have parallel rays or to know the direction of the rays. 38 LIGHTING AND FOCUSING (CH. I re $72. Focusing with Low Objectives.—Place a mounted fly’s wing under the microscope; put the 16 mm. (%% in.) objective in position, and also the lowest ocular. Select the proper opening in the diaphragm and light the object well with transmitted light (§ 64, 68). Hold the head at about the level of the stage, look toward the window, and between the object and the front of the objective ; with the coarse adjustment lower the tube until the objective is within about half a centimeter of the object. Then look into the microscope and slowly elevate the tube with the coarse adjustment. The image will appear dimly at first, but will become very distinct by raising the tube still higher. If the tube is raised too high the image will become indistinct, and finally disappear. It will again appear if the tube is lowered the proper distance. When the microscope is well focused try both the concave and the plane mirrors in various positions and note the effect. Put a high ocular in place of the low one ($43). If the oculars are not par- focal it will be necessary to lower the tube somewhat to get the micro- scope in focus.* Pull out the draw-tube 4-6 cm., thus lengthening the body of the microscope ; it will be found necessary to lower the tube of the micro- scope somewhat. (For reason, see Fig. 58.) $73. Pushing in the Draw-Tube.—To push in the draw-tube, grasp the large milled ring of the ocular with one hand, and the milled head of the coarse adjustment with the other, and gradually push the draw-tube into the tube. If this were done without these precautions the objective might be forced against the object and the ocular thrown out by the compressed air. $74. Focusing with High Objectives.—Employ the same object as before, elevate the tube of the microscope and, if no revolving as nose-piece is present, remove the 16 mm. (23 in.) objective as indi- cated. Put the 3 mm.( 1% in.) or a higher objective in place, and use a low ocular. *Par-focal oculars are so constructed, or so mounted, that those of different powers may be interchanged without the microscopic image becoming wholly out of focus (Fig. 31). When high objectives are used, while the image may be seen after changing oculars, the instrument nearly always needs slight focusing. With low powers this may not be necessary. Objectives are also now commonly mounted in the triple or double revolving nose-pieces ( Figs. 36, 36 a) so that if one of the objectives is in focus either of the others will be approximately in focus when turned into position. This is a very great convenience, CH, IT) LIGHTING AND FOCUSING 39 Light well, and employ the proper opening in the diaphragm, etc. ($ 68). Look between the front of the objective and the object as before (§ 72), and lower the tube with the coarse adjustment till the objective almost touches the cover-glass over the object. Look into the microscope, and with the coarse adjustment, raise the tube very slowly until the image begins to appear, then turn the milled head of the fine adjustment (frontispiece), first one way and then the other, if necessary, until the image is sharply defined. In practice it is found of great advantage to move the preparation slightly while focusing. This enables one to determine the approach to the focal point either from the shadow or the color, if the object is col- ored. With high powers and scattered objects there might be no object in the small field (see § 50, Fig. 37 for size of field). By moving the preparation an object will be moved across the field and its shadow gives one the hint that the objective is approaching the focal point. It is sometimes desirable to focus on the edge of the cement ring or on the little rng made by the marker (see Figs. 61-66). Note that this high objective must be brought nearer the object than the low one, and that by changing to a higher ocular (if the ocu- lars are not par-focal) or lengthening the tube of the microscope it will be found necessary to bring the objective still nearer the object, as with the low objective. (For reason see Fig. 58.) $75. Always Focus Up, as directed above. If one lowers the tube only when looking at the end of the objective as directed above, there will be no danger of bringing the objective in contact with the object, as may be done if one looks into the microscope and focuses down. When the instrument is well focused, move the object around in order to bring different parts into the field. It may be necessary to re-focus with the fine adjustment every time a different part is brought into the field. In practical work one hand is kept on the fine adjust- ment constantly, and the focus is continually varied. §$ 76. Determination of Working Distance.—) - If the incident ray were in glass and the refracted ray in sin 7 water : (2 \- ( <3.) - And similarly for any two media; and as stated Ee r 1.52 above if any three of the factors are given the fourth may be readily found. 2100. Critical Angle and Total Reflection.—In order to understand the Wol- laston camera lucida (Ch. IV) and other totally reflecting apparatus, it is necessary briefly to consider the critical angle. The critical angle is the greatest angle that a ray of light in the denser of two contiguous media can make with the normal and still emerge into the less refrac- tive medium. On emerging it will form an angle of 90° with the normal, and if the substances are liquids, the refracted ray will be parallel with the surface of the denser medium. Total Reflection.—In case the incident ray in the denser medium is at an angle with the normal greater than the crttical angle, it will be ¢otally reflected at the surface of the denser medium, that surface acting as a perfect mirror. By consult- ing the figures it will be seen that there is no such thing as a critical angle and total reflection in the varer of two contiguous media. To find the critical angle in the denser of two contiguous media :— Make the angle of refraction (7. e., the angle in the rarer of the two media) : (i z index r go° and solve the general equation : is G oe ) . Let the two sub- (a Yr index 7 stances be water and air, then the sine of 7 (g0°) is 1, the index of air is 1, that of sin 7 water 1.33, whence (4 Ve -(= ee ) or sinz—751+. This is the sine of 3 48°+-, and whenever the ray in the water is at an angle of more than 4S° it will not emerge into the air, but be totally reflected back into the water. The case of a ray passing from crown glass into the water : ( sin z _ ( index water (1.33) sin 2 sin r (sin go°=1) ) = (ae glass (1.52) Je (3 a l= (ss 1 52 alk whence sin 7=.875 sine of critical angle in glass covered with water. The corresponding angle is approximately 61°. 4101. Color Images.—These are images of objects which are strongly col- ored and lighted with so wide an aperture that the refraction images are drowned in the light. Such images are obtained by removing the diaphragm or by using a larger opening. This method of illumination is especially applicable to the study of deeply stained bacteria. (See below. ? 108). ADJUSTABLE WATER AND HOMOGENEOUS OBJECTIVES EXPERIMENTS 7102, Adjustment for Objectives.—As stated above (? 24), the aberration produced by the cover-glass (Fig. 57), is compensated for by giving the combina- tions in the objective a different relative position than they would have if the objective were to be used on uncovered objects. Although this relative position cannot be changed in unadjustable objectives, one can secure the best results of CH. IT} LIGHTING AND FOCUSING 55 which the objective is capable by selecting covers of the thickness for which the objective was corrected. (See table p. 14.) Adjustment may be made also by tucreasing the tube-length for covers ¢Ainner than the standard and by shortening the tube-length for covers /Azcker than the standard (Fig. 58). Fic. 57. Effect of the cover-glass on the rays from the object to the objective (Ross). Axis. The projection of the optic axts of the microscope. F. Focal or axial point of the objective. £’ and F’’. Points on the axis where rays2and 3 appear to originate if traced backward after emerging Srom the upper side of the cover-glass. In learning to adjust objectives, it is best for the student to choose some object whose structure is well agreed upon , and then to practice lighting it, shad- ing the stage and adjusting the objective, until the proper appearance is obtained. The adjustment is made by turning a ring or collar which acts on a screw and increases or diminishes the distance between the systems of lenses, usually the front and the back systems (Fig. 4o). $103. General Directions.—(A) The thinner the cover-glass, the further must the system of lenses be separated, 7. e., the adjusting collar is turned nearer the zero or the mark ‘‘uncovered,’’ and conversely; (B) the thicker the cover-glass the closer together are the systems brought by turning the adjusting collar from the zero mark. This also increases the magnification of the objective (Ch. IV). The following specific directions for making the cover-glass adjust- ment are given by Mr. Wenham (Carpenter, 7th Ed., p. 166). ‘‘Select any dark speck or opaque portion of the object, and bring the outline into perfect focus; then lay the finger on the milled-head of the fine motion, and move it briskly backwards and forwards in both directions from the first position. Observe thé expansion of the dark outline of the object, both when within and when without the focus. If the greater expansion or coma is when the object is w7thow?t the focus, or farthest from the objective [7. ¢., in focusing up], the lenses must be placed further asunder, or toward the mark uncovered [the adjusting collar is turned toward the zero mark as the cover-glass is too thin for the present adjustment]. If the greater expansion is when the object is within the focus, or nearest the objective [7. e., in focusing down], the lenses must be brought closer together, or toward the mark covered [z. e., the adjusting collar should be turned away from the zero mark, 56 LIGHTING AND FOCUSING (CH. LI the cover-glass being too thick for the present adjustment].’’ 2 most objectives the collar ts graduated arbitrarily, the zero (O) mark represent- ing the position for uncovered objects. Other objectives have the collar graduated to correspond to the various thicknesses of cover-glasses for which the objective may be adjusted. This seems to be an admirable plan ; then if one knows the thickness of the cover-glass on the preparation (Ch. VIIT) the adjusting collar may be set at a corresponding mark, and one will feel confident that the adjustment will be approximately correct. It ts then only necessary for the observer to make the slight adjustment to compensate for the mounting medium or any variation from the standard length of the tube of the microscope. In adjusting for variations -of the length of the tube from the standard it should be remembered that: (A) If the tube of the microscope is longer than the standard for which the ob- jective was corrected, the effect is approximately the same as thicken- ing the cover-glass, and therefore the system of the objective must be brought closer together, z.¢., the adjusting collar must be turned away from the zero mark. (B) If the tube is shorter than the standard for which the objective is corrected, the effect is approximately the same as diminishing the thickness of the cover-glass, and the systems must therefore be separated (Fig. 4o). In using the tube-length for cover correction Shorten the tube for too thick covers and Lengthen the tube for too thin covers. Furthermore, whatever the interpretation by different opticians of what should be included in ‘‘tube-length,’’ and the exact length in mil- limeters, its importance is very great ; for each objective gives the most perfect image of which it is capable with the ‘‘tube-length’’ for which it is corrected, and the more perfect the objective the greater the ill- effects on the image of varying the ‘‘tube-length’’ from this standard. The plan of designating exactly what is meant by ‘‘tube-length,’’ and engraving on each objective the ‘‘tube-length’’ for which it is corrected, is to be commended, for it is manifestly difficult for each worker with the microscope to find out for himself for what ‘‘tube-length’’ each of his objectives was corrected. (See Ch. X). S 104. Water Immersion Objectives.—Put a water immersion objective in position (§ 47) and the fly’s wing for object under the microscope. Place a drop of distilled water on the cover-glass, and with the coarse adjustment lower the tube till the objective dips into the water, then light the field well and turn the fine adjustment one way and another till the image is clear. Water immersions are exceed- ingly convenient in studying the circulation of the blood, and for CA. 1T) LIGHTING AND FOCUSING 57 many other purposes where aqueous liquids are liable to get on the cover-glass. If the objective is adjustable, follow the directions given in § 103. Y nr R: Objestive Obyect—4 Object-a Fic. 58. Figure to show that in lengthening the tube of the microscope the ob- ject must be brought nearer the principal focus or center of the lens. It will be seen by consulting the figure that in shortening the tube of the microscope the object must be removed farther from the center of the lens. By consulting the figure showing the effect of the cover-glass (Fig. 57) it will be seen that the effect of the cover-glass ts to bring the object nearer the objective, and the thicker the cover the nearer ts the object brought to the objective. As shortening the tube serves to remove the object, it neutralizes the effect of the thick cover, and if the cover ts so thin that it does not elevate the olject enough for the corrections of the objective, then an in- crease in the tube-length will correct the defect. LIGHTING AND FOCUSING (CH. Il nn ioe) When one is through using a water immersion objective, remove it from the microscope and with some lens paper wipe all the water from the front lens. Unless this is done dust collects and sooner or later the front lens will be clouded. It is better to use distilled water to avoid the gritty substances that are liable to be present in natural waters, as these gritty particles might scratch the front lens. HOMOGENEOUS IMMERSION OBJECTIVES : EXPERIMENTS $105. As stated above, these are objectives in which a liquid of the same refractive index as the front lens of the objective is placed between the front lens and the cover-glass. $106. Tester for Homogeneous Liquid.—In order that full advantage be derived from the homogeneous immersion principle, the liquid employed must be truly homogeneous. To be sure that such is the case, one may use a tester like that constructed by the Gundlach Optical Co., then if the liquid is too dense it may be properly diluted and wee versa. For the cedar oil immersion liquid, the density may be diminished by the addition of pure cedar wood oil. The density may be increased by allowing it to thicken by evaporation. (See H. L. Smith, Proc. Amer. Soc. Micr., 1885, p. 83, and Ch. X). $107. Refraction Images.—Puta 2 mm.(;);th in.) homogeneous immersion objective in position, employ an illuminator. Use some histological specimen like a muscular fiber as object, make the dia- phragm opening about 3 mm. in diameter, add a drop of the homo- geneous immersion liquid and focus as directed in § 74. The object will be clearly seen in all details by the unequal refraction of the light traversing it. The difference in color between it and the surrounding medium will also increase the sharpness of the outline. If an air bub- ble preparation (§ 77) were used, one would get pure, refraction images. § 108. Color Images.—Use some stained bacteria as Bacillus tuberculosis for object. Put a drop of the immersion liquid on the cover-glass or the front lens of the homogeneous objective. Remove the diaphragms from the illuminator or in case the iris diaphragm is used, open to its greatest extent. Focus the objective down so that the immersion fluid is in contact with both the front lens and the cover- glass, then with the fine adjustment get the bacteria in focus. They will stand out as clearly defined colored objects on a bright field. CH, IT) LIGHTING AND FOCUSING 59 Fic. 59. Screen for shading the microscope and _}~—————————— the face of the observer. This is very readily con- nae Hy structed as shown in the figure by supporting a wire ina disc of lead, tron, or heavy wood. The screen is then completed by hanging over the bent wire, cloth or manilla paper 30x 4o cm. The lower edge of the screen should be a little below the stage of the micro- scope and the upper edge high enough to screen the eves of the observer. go cm 50 om $ 109. Shading the Object.—To get the = clearest image of an object no light should pan reach the eye except from the object. A hand- CBX kerchief or a dark cloth wound around the objective will serve the purpose. Often the proper effect may be ob- tained by simply shading the top of the stage with the hand or with a piece of bristol board. Unless one has a very favorable light the shading of the object is of the greatest advantage, especially with homogeneous immersion objectives. The screen (Fig. 59) is the most satisfactory means for this purpose, as the entire microscope above the illuminating apparatus is shaded. $110. Cleaning Homogeneous Objectives.—After one is through with a homogeneous objective, it should be carefully cleaned as follows: Wipe off the homogeneous liquid with a piece of the lens paper ($ 114), then if the fluid is cedar oil, wet one corner of a fresh piece in benzin or chloroform and wipe the front lens with it. Imme- diately afterward wipe with a dry part of the paper. The cover-glass of the preparation can be cleaned in the same way. If the homogen- eous liquid is a glycerin mixture proceed as above, but use water to remove the last traces of glycerin. CARE OF THE MICROSCOPE §$ 111. The microscope should be handled carefully and kept per- fectly clean. The oculars and objectives should never be allowed to fall. When not in use keep it ina place as free as possible from dust. All parts of the microscope should be kept free from liquids, especially from acids, alkalies, alcohol, benzin, turpentine and chloroform. $112. Care of the Mechanical Parts.—To clean the mechan- ical parts put a small quantity of some fine oil (olive oil or liquid vas- 60 LIGHTING AND FOCUSING (CA. Lf elin and benzin, equal parts), on a piece of chamois leather or on the lens paper, and rub the parts well, then with a clean dry piece of the chamois or paper wipe off most of the oil. If the mechanical parts are kept clean in this way a lubricator is rarely needed. Where op- posed brass surfaces ‘‘cut,’’ 7. ¢., when from the introduction of some gritty material, minute grooves are worn in the opposing surfaces, giv- ing a harsh movement, the opposing parts should be separated, care- fully cleaned as described above and any ridges or prominences scraped down with a knife. Where the tendency to ‘‘cut’’ is marked, a very slight application of equal parts of beeswax and tallow, well melted together, serves a good purpose. In cleaning lacquered parts, benzin alone answers well, but it should be quickly wiped off with a clean piece of the lens paper. Do not use alcohol as it dissolves the lacquer. § 113. Care of the Optical Parts.—These must be kept scrupu- lously clean in order that the best results may be obtained. Glass surfaces should never be touched with the fingers, for that will soil them. The glass of which the lenses are made is quite soft, consequently it is necessary that only soft, clean cloth or paper be used in wiping them. Whenever an objective is left in position on a microscope, or when several are attached by means of a revolving nose-piece, an ocular should be left in the upper end of the tube to prevent dust from falling down upon the back lens of the objective. §$ 114. Lens Paper.—The so-called Japanese filter paper, which from its use with the microscope, I have designated lens paper, has been used in the author’s laboratory for the last sixteen years for clean- ing the lenses of oculars and objectives, and especially for removing the fluid used with immersion objectives. Whenever a piece is used once it is thrown away. It has proved more satisfactory than cloth or chamois, because dust or sand is not present ; and from its bibulous character it is very efficient in removing liquid or semi-liquid substances. S115. Dust may be removed witk a camel’s hair brush, or by wiping with the lens paper. Cloudiness may be removed from the glass surfaces by breathing on them, then wiping quickly with a soft cloth or the lens paper. Cloudiness on the inner surfaces of the ocular lenses may be re- moved by unscrewing them and wiping as directed above. A high objective should never be taken apart by an inexperienced person. GET LIGHTING AND FOCUSING 61 Lf the cloudiness cannot be removed as directed above, moisten one corner of the cloth or paper with 95 per cent. alcohol, wipe the glass first With this, then with the dry cloth or the paper. Water may be removed with soft cloth or the paper. Glycerin may be removed with cloth or paper saturated with dis- tilled water ; remove the water as above. Blood or other albumtnous material may be removed while fresh with a moist cloth or paper, the same as glycerin. If the material has dried on the glass, it may be removed more readily by adding a small quantity of ammonia to the water in which the cloth is moistened, (water 100 cc., ammonia I cc). Canada Balsam, damar, paraffin, or any oily substance may be re- moved with a cloth or paper wet with chloroform, benzin or xylene. The application of these liquids and their removal with a soft dry cloth or paper should be as rapid as possible, so that none of the liquid will have time to soften the setting of the lenses. Shellac Cement may be removed by the paper or a cloth moistened in 95 per cent. alcohol. Brunswick Black, Gold Size, and all other substances soluble in chloroform, etc., may be removed as directed for balsam and damar. In general, use a solvent of the substance on the glass and wipe it off quickly with a fresh piece of the lens paper. It frequently happens that the upper surface of the back combina- tion of the objective becomes dusty. This may be removed in part by a brush, but more satisfactorily by using a piece of the soft paper loosely twisted. When most of the dust is removed some of the paper may be put over the end of a pine stick (like a match stick) and the glass sur- face carefully wiped. CARE OF THE EYES $ 116. Keep both eyes open, using the eye-screen if necessary (Figs. 60, 60a); and divide the labor between the two eyes, 7. é., use one eye for observing the image awhile and then the other. In the begin- ning it is not advisable to look into the microscope continuously for more than half an hour at atime. One never should work with the microscope after the eyes feel fatigued. After one becomes accustomed to microscopic observation he can work for several hours with the microscope without fatiguing the eyes. This is due to the fact that the eyes become inured to labor like the other organs of the body by judicious exercise. It is also due to the fact that but very slight ac- commodation is required of the eyes, the eyes remaining nearly in a condition of rest as for distant objects. The fatigue incident upon 62 “LIGHTING AND FOCUSING [CAH lL using the microscope at first is due partly at least to the constant effort on the part of the observer to remedy the defects of focusing the microscope by accommodation of the eyes. This should be avoided and the fine adjustment of the microscope used instead of the muscles of accommodation. With a microscope of the best quality, and suita- ble light—that is light which is steady and not so bright as to dazzle the eyes nor so dim as to strain them in determining details—micro- scopic work should improve rather than injure the sight. 7 X4A4 em. Fic. 60. Double Eye Shade. This is readily made by taking some thick bris- tol board 7 x ry centimeters and making an oblong opening with rounded ends ee: S EX (0-0) and of such a diameter that it goes | | ; re . . J readily over the tube of the microscope. Sco hates tea = This 1s then covered on both sides with velveteen and a central slit (s) made in the cloth. This admits the tube of the microscope and holds the screen in posi- tion. Lt may readily be pulled from side to side and thus serves for either eye, or for the use of the eyes alternately. Ve < ? Fic. 60a. Adjusting Eye-Shade. This is prepared like the preceding by cov- ering acard about 6 x 12 centimeters with black velveteen. A copper wire about 3 mm.(¥ in.) and of the right length is curved as shown in the figure. Its ends are rounded, and finally it is put under the cloth and sewed carefully all around. The card and cloth are then cut as shown. The flexible wire makes it possible to put the screen on the tube at any level. $117. Position and Character of the Work-Table.—The work-table should be very firm and large (60x 120 cm.; 24x 48 in.), so that the necessary apparatus and material for work may not be too crowded. The table should also be of the right height to make work by it comfortable. An adjustable stool, something like a piano stool is convenient, then one may vary the height corresponding to the neces- GHATL| LIGHTING AND FOCUSING 63 sities of special cases. It is a great advantage to sit facing the window if daylight is used, then the hands do not constantly interfere with the illumination. To avoid the discomfort of facing the light a screen like that shown in Fig. 59 is very useful (see also under lighting, § 62). TESTING THE MICROSCOPE 2118. Testing the Microscope.—To be of real value this must be accom- plished by a person with both theoretical and practical knowledge, and also with an unprejudiced mind. Such a person is not common, and when found does not show over anxiety to pass judgment. Those most ready to offer advice should as a rule be avoided, for in most cases they simply ‘‘have an ax to grind,’’ and are sure to commend only those instruments that conform to the ‘‘fad’’ of the day. From the writer’s experience is seems safe to say that the inexperienced can do no better than to state clearly what he wishes to do with a microscope and then trust to the judgment of one of the optical companies. The makers of microscopes and objectives guard with jealous care the excellence of both the mechanical and optical part of their work, and send out only instruments that have been carefully tested and found to conform to the standard. This would be done as a matter of business prudence on their part, but it is believed by the writer that microscope makers are artists first and take an artist’s pride in their work, they therefore have a stimulus to excellence greater than business prudence alone could give. % 119. Mechanical Parts.—All of the parts should be firm, and not too easily shaken. Bearings should work smoothly. The mirror should remain in any position in which it is placed. Focusing Adjustments.—The coarse or rapid adjustment should be by rack and pinion, and work so smoothly that even the highest power can be easily focused with it. In no case should it work so easily that the body of the microscope is liable to run down and plunge the objective into the object. If any of the above defects appear in a microscope that has been used for some time, a person with moderate mechanical instinct will be able to tighten the proper screw, etc. The Fine Adjustment is more difficult to deal with. From the nature of its’ purpose unless it is approximately perfect, it would be better off the microscope entirely. It has been much improved recently. It should work smoothly and be so balanced that one cannot tell by the feel- ing when using it whether the screw is going up or down. Then there should be absolutely no motion except in the direction of the optic axis, otherwise the image will appear to sway even with central light. Compare the appearance when using the coarse and when using the fine adjustment. There should be no swaying of the image with either if the light is central (4 77). 7120. Testing the Optical Parts.—As stated in the beginning, this can be done satisfactorily only by an expert judge. It would be of very great advantage to the student if he could have the help of such a person. J” no case 1s a micro- scope to be condemned by an inexperienced person, If the beginner will bear in mind that his failures are due mostly to his own lack of knowledge and lack of skill; and will truly endeavor to learn and apply the principles laid down in this and in the standard works referred to, he will learn after a while to estimate at their true value all the pieces of his microscope. (See Ch. X). 64 LABORATORY MICROSCOPES (CH. IL LABORATORY AND HIGH-SCHOOL COMPOUND MICROSCOPES 2121, Optical Parts.—A great deal of beginning work with the microscope in biological laboratories is done with simple and inexpensive apparatus. Indeed if one contemplates the large classes in the high schools,’the universities and med- ical schools, it can be readily understood that microscopes costing from $25-50 each and magnifying from 25 to 500 diameters, are all that can be expected. But for the purpose of modern histological investigation and of advanced microscopical work in general, a microscope should have something like the following character : Its optical outfit should comprise, (a) dry objectives of 50 mm.(2 in.), 16-18 mm. (% in.) and 3mm. (% in.) equivalent focus. There should be present also a 2mm. (;, in.) or 1.5 mm. (7; in.) homogeneous imniersion objective. Of oculars there should be several of different power. A centering substage condenser, and an Abbe camera lucida are also necessities, and a micro-spectroscope and a micro-polarizer are very desirable. Even in case all the optical parts cannot be obtained in the beginning, it is wise to secure a stand upon which all may be used when they are finally secured. As to the objectives. The best that can be afforded should be obtained. Cer- tainly at the present, the apochromatics stand at the head, although the best achromiatic objectives approach them very closely. 4.122. Mechanical Parts or Stand.—The stand should be low enongh so that it can be used in a vertical position on an ordinary table without inconvenience ; it should have a jointed (flexible) pillar for inclination at any angle to the hori- zontal. The adjustments for focusing should be two,—a coarse adjustment or rapid movement with rack and pinion, and a fine adjustment by means of a mi- crometer screw. Both adjustments should move the entire tube of the microscope. The body or tube should be short enough for objectives corrected for the short or 160 millimeter tube-length. It is an advantage to have the draw-tube graduated ‘in centimeters and millimeters. The lower end of the draw tube and of the tube should each possess a standard screw for objectives (frontispiece). The stage should be quite large for the examination of slides with serial sections and other large objects. The substage fittings should be so arranged as to enable one to use the condenser or to dispense entirely with diaphragms. The condenser mount- ing should allow up and down motion. STANDARD SIZES RECOMMENDED BY THE ROYAL MICROSCOPICAL SOCIETY 2123. Society Screw.—Owing to the lack of uniformity in screws for micro- scope objectives, the Royal Microscopical Society of London, in 1857, made an earnest effort to introduce a standard size. The specifications of this standard are CHeTI LABORATORY MICROSCOPES 65 as follows: ‘Whitworth thread, 7. ¢., a V shaped thread, sides of thread inclined to angle of 55° to each other, one-sixth of the V depth of the thread being rounded off at the top of the thread, and one-sixth of the thread being rounded off at the bottom of the thread. Pitch of screw, 36 to the inch ; length of thread on object-glass, 0.125 inch; plain fitting above thread of object-glass, 0.15 inch long, to be about the size of the bottom of male thread ; length of thread of nose-piece [on the lower end of the tube of the microscope], not less than 0.25 inch ; diam- eter of the object-glass screw at the bottom of the screw, 0.7626 inch ; diameter of the nose-piece screw at the bottom of the thread, 0.8 inch.”’ In order to facilitate the introduction of this universal screw, or as it soon came to be called ‘‘ The Society Screw,’’ the Royal Microscopical Society undertook to supply standard taps. From the mechanical difficulty in making these taps perfect there soon came to be considerable difference in the ‘‘Society Screws,’ and the object of the society in providing a universal screw was partly defeated. In 1884 the American Microscopical Society appointed Mr. Edward Bausch and Prof. William A. Rogers upon a committee to correspond with the Royal Microscopical Society, with a view to perfecting the standard ‘‘Society Screw,”’ or of adopting another standard and of perfecting methods by which the screws of all makers might be truly uniform. Although this matter was earnestly consid- ered at the time by the Royal Microscopical Society, the mechanical difficulties were so great that the improvements were abandoned. Fortunately, however, during the year (1896) that society again took hold of the matter in earnest, and the ‘‘Society Screw’’ is now accurate, and facilities for obtaining the standard are so good that there is a reasonable certainty that the universal screw for microscopic objectives may be realized. It is astonishing to see how widely the ‘‘Society Screw’’ has been adopted. Indeed there is not a maker of first-class microscopes in the world who does not supply the objectives and stands with the ‘Society Screw,”’ and an objective in England or America which does not have this screw should be looked upon with suspicion, That is, it is either old, cheap, or not the product of one of the great opticians. For the Stand- ard, or ‘‘Society Screw,’’ see: Trans. Roy. Micr. Soc., 1857, pp. 39-41 ; 1859, pp. 92-97 ; 1860, pp. 103-104. (All to be found in Quar. Jour. Micr. Sci., 0. s., vols. VI, VII and VIII). Proc. Amer. Micr. Soc. 1884, p. 274; 1886, p. 199 ; 1893, p. 38. Journal of the Royal Microscopical Society, August, 1896. In this last paper of four pages the matter is very carefully gone over and full specifications of the new screw given. It conforms almost exactly with the orig- inal standard adopted by the society, but means have been devised by which it may be kept standard. 2124. Standard Size Oculars and Substage Condensers.—For a considera- tion of these, with measurements, see 7 46, 87. MARKERS AND MECHANICAL STAGES Markers are devices to facilitate the finding of some object or part which it is especially desired to refer to again or to demonstrate to a class. The mechanical stage makes it much easier to follow out a series of objects, to move the slide when using high powers, and for complete exploration of a preparation. Most of the mechanical stages have scales or scales and verniers by which an object once recorded may be readily found again. 66 LABORATORY MICROSCOPES eee IOF 2125. Marker for Preparations. (Figs. 61-66).—This instrument consists of an objective-like attachment which may be screwed into the nose-piece of the micro- scope. It bears on its lower end (Figs. 61-3) a small brush and the brush can be made more or less eccentric and can be rotated, thus making a larger or smaller circle. In using the marker the brush is dipped in colored shellac or other cement and when the part of the preparation to be marked is found and put exactly in the middle of the field the objective is turned aside and the marker turned into posi- tion. The brush is brought carefully in contact with the cover-glass and rotated. This will make a delicate ring of the colored cement around the object. Within this very small area the desired object can be easily found on any microscope. The brush of the marker should be cleaned with 95% alcohol after it is used. (Proc. Amer. Micr. Soc., 1894, pp. 112-118). 61. 62. 63. Fics. 61-63. Sectional Views of the two Forms of the Marker. Fic. 61. The simplest form of marker. It consists of the part SS with the milled edge (M). This part bears the society or objective screw for attaching the marker to the microscope. FR. Rotating part of the marker. This bears the eccen- tric brush (B) at its lower end. The brush is ona wire (W). This wire ts eccen- tric, and may be made more or less so by bending the wire. The central dotted line coincides with the axis of the microscope. The revolving part is connected with the ‘Society Screw’? by the small screw (S ). Fic. 62. SS, R,and B. All parts same as with Fig. 61, except that the brush 1s carried by a sliding cylinder the end view being indicated in Fig. 63. CA. IT) LABORATORY MICROSCOPES 67 66 Fics. 64, 65, 66. Specimens Showing the Use of the Marker. In Fig. 64 a section of a series 1s marked to indicate that this section shows some- thing especially well. In Fig. 65 some blood corpuscles showing ingested carbon very satisfactorily are surrounded by a minute ring, and in Fig. 66 the lines of a micrometer are ringed to facilitate finding the lines. 4126. Pointer in the Ocular.—The Germans have a pointer ocular (Spitzen- Okular), an ocular with one or two delicate rods or pointers at the level of the real image, that is, at the level of the diaphragm (Figs. 21, 30D). For the pur- poses of demonstrating any particular structure or object in the field, a temporary pointer may be easily inserted in any ocular as follows: Remove the eye-lens and with a little mucilage or Canada Balsam fasten an eye-lash (cilium) to the diaphragm (Fig. 30 D) so that it will project about half way across the opening. If one uses this ocular, the pointer will appear in the field and one can place the specimen so that the pointer indicates it exactly, asin using a pointer on a diagram or on the black-board. It is not known to the author who devised this method. It is cer- tainly of the greatest advantage in demonstrating objects like amoebas or white blood corpuscles to persons not familiar with them, as the field is liable to have in it many other objects which are more easily seen. 4127. Mechanical Stage.—For High School and ordinary laboratory work a mechanical stage is not needed ; but for much work, especially where high objec- tives are used a mechanical stage is of great advantage. Itis also advantageous if the mechanical stage can be easily removed (see Figs. 67 to 70). The one found on the most expensive American and English microscopes for the last twenty years and the one now present on the larger continental microscopes, is excellent for high powers and preparations of moderate dimensions, but for the study of serial sections and large sections or preparations in general, mechanical stages like those shown in Figs. 68-69 are more useful. This form of mechanical stage has the advantage of giving great lateral and forward and backward motion. Itis a mod- ification of the mechanical stage of Tolles. The modification consists in doing away with the thin plate and having a clamp to catch the ends of the glass slide. The slide is then moved on the face of the stage proper. This modification was first made by Mayall. It has since been modified by Reichert, Zeiss, Leitz, and others in Europe and by the Bausch & Lomb Optical Co., Queen & Co., and the Spencer Lens Co., in America.—Jour. Roy. Micr. Soc., 1885, p. 122. See also Zeit. Wiss. Mikroskopie (II), 1885, pp. 289-295 ; 1887 (IV, pp. 25-30). Those figured below have the great advantage of ready removal from the stage of the microscope, thus leaving it free. They have also the very excellent feature that with them one can explore an entire slide full of serial sections, as the sec- tions are ordinarily mounted, 7. ¢., under a cover-glass 24 X 50 mm. 68 LABORATORY MICROSCOPES (CH. lI i il , Fic. 67a. Fics. 67, 67a. he removable mechanical stage of the Bausch & Lomb Optr- cal Company. Ln the upper figure tt is in position on the stage af the microscope ; in the lower figure only the clamping part ts in postition, the rest having been removed to leave the stage of the microscope free. ; CH. IT] LABORATORY MICROSCOPES 69 Bacillus tuvercutosis Fic. 68. ( angats ) FA] Stsoinasaqny snyseg z FIG. 68a. Fic. 68, 68a. Two forms of removable mechanical stage by Leitz. 68 is some- what more complex and expensive. Both have the desirable features mentioned im 4 127. 70 LABORATORY MICROSCOPES [CH. I 10 APOANEnT 40 30 20 MITONT AANA ROTTLAOOAAAT BAY cere Fic. 69. The removable mechanical stage of the Spencer Lens Co. tis a modification of a form devised by Winkel. Besides the general features mentioned in 4 127 tthas the advantage of fitting any square stage. Tt is fastened to the stage by the clamps shown at the right. Another form is made having one screw on the side. CH. IT) LABORATORY MICROSCOPES 71 Fic. 70. Avauss’ Method of Marking Objectives on a Revolving Nose-Piece. Als seen tn the figure, the equiv- alent focus of the objective is en- graved on the diaphragm above the back lens and may be very readily seen n rotating the nose-piece. This ts of great advantage, as one can see what objective is coming into place without trouble, Lt is also an aa- vantage in showing where each ob- jective belongs when the microscope comes from the manufacturers. The method ts coming into general use. FIGURES OF LABORATORY AND HIGH SCHOOL MICROSCOPES In order that teachers and students may get a good general idea of the appear- ance of microscopes of various makers for high school and advanced laboratory work a few pictures are appended of the microscopes most used in the United States- This has been rendered possible by the courtesy of the manufacturers or import- ers. It will be seen that the microscope for the advanced laboratory work is on the left and the cheaper one for school and less advanced laboratory work is on right. The microscopes are arranged in alphabetical order. Laboratory microscopes which will answer nearly all the requirements for work in Biology, including Histology, Embryology, Pathology and Bacteriology, are listed in the makers catalogs at about $100.00. The less expensive micro- scopes shown on the right are listed at $25 to $45. There is usually a discount of 10%, or more from these prices. Fortunately in the State of New York the State pays half for high school apparatus, so that there is no reason why every high school should not be properly equipped with microscopes of a good grade. To avoid misunderstanding it should be added that the quality of the oculars and objectives on the high school microscopes figured is the same as for the best laboratory microscopes. The mechanical work also is of excellent quality. During the last ten years great vigor has been shown in the microscopical world. This has been stimulated largely by the activity in biological science and the widespread appreciation of the microscope, not only as a desirable, but as a necessary instrument for study and research. The production of the new kinds of glass, (Jena glass), and the apochromatic objectives has been a no less potent factor in promoting progress. The student is advised to write to one or more of the opticians for complete catalogs. (See list, p. 2 of cover). LABORATORY MICROSCOPES ETT Ta LABORATORY MICROSCOPES 73 Fic. 72. The Bausch & Lomb Continental Microscope. 74 LABORATORY MICROSCOPES (CH. 71 i ‘ll 7 a Fic. 73. 2. & J. Beck's New Continental Microscope, No. 1152 ( Williams, brown & Earle, Philadelphia) CGHT#R) LABORATORY MICROSCOPES 75 | i i re He i Fic. 74. Rk. & J. Beck's New Continental Microscope, No. 1125 (Williams, Brown & Earle, Philadelphia). 76 Mill FIG. 75. LABORATORY MICROSCOPES (CA. LI ti E. Leitz i ae ze Sut it Microscope 11 C. (Win. Krafft, New York). CH. IT] Fic. 76. Leitz Microscc joint for inclination (Wm. Krafft, N. Y.). LABORATORY MICROSCOPES "ii 77 78 LABORATORY MICROSCOPES (CH. lf cH ll Fic. 77. Nachet’s Microscope 6 bis. Old model. Nachet is a successor to Flartnack who introduced the present ‘Continental Model” Mavall, Cantor Lec- tures, p. 68 (Franklin Laboratory Supply Co., Boston). CHS TT] LABORATORY MICROSCOPES 79 Fic. 78. Nachet’s Microscope, No. 11 (Franklin Laboratory Supply Co., Boston). This microscope has no joint for inclination, and no rack and pinion for coarse adjustment. For coarse adjustment, the tube 1s pulled up and down with the hands. This kind of coarse adjustinent was much more common ten years ago than now. (See also Fig. 82.) So LABORATORY MICROSCOPES DELP S77. FIG. 79. A. Queen & Co's Continental Microscope, No. Il, B. Dust-prooy, triple nose-piece. The difference between this an by comparing with Fig. 36. This form of revol many years by Winkel of Goettingen. a the ordinary form can be seen ving nose-prece has been made jor CH. IT] LABORATORY MICROSCOPES Cy: qRTHEL Sinanuains PHIL, Queen & Co’s Acme Microscope for Schools. LABORATORY MICROSCOPES (CH. Fic. 81. Reichert's New M1- croscope, No. III B. ( Richards & Co., N. Y¥. and Chicago). CA. [1] LABORATORY MICROSCOPES 83 Fic. 82. Leichert’s Microscope, No. V. (Richards & Co., New Vork and Chicago). This has no joint for inclination. Fic. 83. The Spencer Lens Company's Microscope No. 1. This has the standard size, Con- tinental oculars, and the object- ives and oculars have the equiv- alent focus in millimeters. ii i it Fic. 84. Zhe Spencer Lens Company's Microscope, No. 2. Pars 86 LABORATORY MICROSCOPES (CH. 1f Fic. 85. Watson & Sons, Edinburgh Students’ Alicroscope (Stand G). This is a good representative of the tripod-base, English models of to-day. Lt is in gen- eral like the Powell and Lealand stands which have held their position with the Soremost English microscopists for the last yo years. (See Carpenter-Dallinger, p. 172.) Microscopes with tripod base something after this pattern are now being made on the Continent. alltention is called to the sub-stage for the condenser. It possesses centering screws so that any apparatus used in it may be accurately centered. Lt is to be hoped that all microscopes of this grade will soon be supplied with a centering sub-stage. CH, [1] LABORATORY MICROSCOPES 87 Fic. 86. Zeiss Microscope 1 with Mechanical Stage. This figure from Zeiss? Catalog No. 30, represents the Continental Model of Mi icroscope in its most perfect form. K. Milled head of the screw for the lateral movements of the stage. L. Screw for fixing the laterally moving mechanism of the stage. By un- screwing this the laterally moving part may be removed, leaving the plain slage. W. Screw for moving the stage forward and backward. LABORATORY MICROSCOPES ui Fic. 87, Zentmeyer's Microscope, No. [CH. Il CH. 11] Fic. 88. LABORATORY MICROSCOPES Zentmayer’s Microscope, No. IV. 89 GHAPTER Sit INTERPRETATION OF APPEARANCES APPARATUS AND MATERIAL FOR CHAPTER III A laboratory, compound microscope (4121); Preparation of fly’s wing ; 50 per cent. glycerin; Slides and covers; Preparation of letters in stairs (Fig. 89); Mucilage for air-bubbles and olive or clove oil for oil-globules (7% 136-139). Solid glass rod, and glass tube (% 144-146); Collodion (4% 146); Carmine, India ink, or lamp black (4% 148-150); Frog, castor oil and micro-polariscope (% 152). INTERPRETATION OF APPEARANCES UNDER THE MICROSCOPE S$ 129. General Remarks.—The experiments in this chapter are given secondarily for drill in manipulation, but primarily so that the student may not be led into error or be puzzled by appearances which are constantly met with in microscopical investigation. Anyone can look into a microscope, but it is quite another matter to interpret cor- rectly the meaning of the appearances seen. It is especially important to remember that the more of the relations of any object are known, the truer is the comprehension of the object. In microscopical investigation every object should be scrutinized from all sides and under all conditions in which it is likely to occur in nature and in microscopical investigation. It is best also to begin with objects of considerable size whose character is well known, to look at them carefully with the unaided eye so as to see them as wholes and in their natural setting ; then a low power is used, and so on, step by step until the highest power available has been employed. One will in this way see less and less of the object as a whole, but every increase in magnifi- cation will give increased prominence to detail, detail which might be meaningless when taken alone and independent of the object as a whole. The pertinence of this advice will be appreciated when the student undertakes to solve the problems of histology ; for even after all the years of incessant labor spent in trying to make out the structure of man and the lower animals, many details are still in doubt, the same visual appearances being quite differently interpreted by eminent observers, CA, IIT) INTERPRETATION OF APPEARANCES gI Appearances which seem perfectly unmistakable with a low power may be found erroneous or very inadequate, for details of structure that were indistinguishable with the low power may become perfectly evi- dent with a higher power or a more perfect objective. Indeed the prob- lems of microscopic structure appear to become ever more complex, for difficulties overcome by improvements in the microscope simply give place to new difficulties, which in some cases render the subject more obscure than it appeared to be with the less perfect appliances. The need of the most careful observation and constant watchful- ness lest the appearances may be deceptive are thus admirably stated by Dallinger (see Carpenter-Dallinger, p. 427): ‘‘The correctness of the conclusions which the microscopist will draw regarding the nature of any object from the visual appearances which it presents to him when examined in the various modes now specified will necessarily depend in a great degree upon his previous experience in microscopic observation and upon his knowledge of the class of bodies to which the particular specimen may belong. Not only are observations of any kind liable to certain fallacies arising out of the previous notions which the observer may entertain in regard to the constitution of the objects or the nature of the actions to which his attention is directed, but even the most practiced observer is apt to take no note of such phenomena as his mind is not prepared to appreciate. Errors and im- perfections of this kind can only be corrected, it is obvious, by general advance in scientific knowledge ; but the history of them affords a use- ful warning against hasty conclusions drawn from a too cursory exain- ination. If the history of almost any scientific investigation were fully made known it would generally appear that the stability and completeness of the conclusions finally arrived at had been only attained after many modifications, or even entire alterations, of doctrine. And it is therefore of such great importance as to be almost essential to the correctness of our conclusions that they should not be finally formed and announced until they have been tested in every conceivable mode. It is due to science that it should be burdened with as few false facts [artifacts] and false doctrines as possible. It is due to other truth-seekers that they should not be misled, to the great waste of their time and pains, by our errors. And it is due to ourselves that we should not commit-our reputation to the chance of impairment by the premature formation and publication of conclusions which may be at once reversed by other observers better informed than ourselves, or may be proved fallacious at some future time, perhaps even by our 92 INTERPRETATION OF APPEARANCES (CA. LIT own more extended and careful researches. The suspension of the judg- ment whenever there seems room for doubt is a lesson inculcated by all those philosophers who have gained the highest repute for practical wisdom ; and it is one which the microscopist cannot too soon learn or too constantly practice.’’ For these experiments no condenser ts to be used except where specifically indicated, $130. Dust or Cloudiness on the Ocular.—Employ the 16 mm. (23 in.) objective, low ocular, and fly’s wing as object. Unscrew the field-lens and put some particles of lint from dark cloth on its upper surface. Replace the field-lens and put the ocular in position (§ 48). Light the field well and focus sharply. The im- age will be clear, but part of the field will be obscured by the irregular outline of the particles of lint. Move the object to make sure this appearance is not due to it. Grasp the ocular by the milled ring, just above the tube of the microscope, and rotate it. The irregular objects will rotate with the ocular. Cloudiness or particles of dust on any part of the ocular may be detected in this way. $131. Dust or Cloudiness on the Objective.—Employ the same ocular and objective as before and the fly’s wing as object. Focus and light well, and observe carefully the appearance. Rub glycerin on one side of a slide near the end. Hold the clean side of this end close against the objective. The image will be obscured, and cannot be made clear by focusing. Then use a clean slide and the image may be made clear by elevating the tube slightly. The obscurity produced in this way is like that caused by clouding the front-lens of the objec- tive. Dust would make a dark patch on the image that would remain stationary while the object or ocular is moved. If a small diaphragm is employed and it is close to the object, only the central part of the field will be illuminated, and around the small light circle will be seen a dark ring (Fig. 42). If the diaphragm is lowered or a sufficiently large one employed the entire field will be lighted. $132. Relative Position of Objects or parts of the same object. The general rule is that objects highest up come into focus Jast in focusing up, fst in focusing down. $ 133. Objects having Plane or Irregular Outlines.—As object use three printed letters in stairs mounted in Canada balsam (Fig. 89). The first letter is placed directly upon the slide, and covered with a CA, 111) INTERPRETATION OF APPEARANCES 93 small piece of glass about as thick as a slide. The second letter is placed upon this and covered in like manner. The third letter is placed upon the second thick cover and covered with an ordinary cover-glass. The letters should be as near together as possible, but not over-lapping. Employ the same ocular and objective as above (§ 130). Fic. 89. Letters mounted tn stairs to show the order of coming into focus. a b c d l ton a,6,c,d. The various letters indi- cated by the oblique row of black marks in sectional view. Slide. The glass slide on which the letters are mounted, Lower the tube till the objective almost touches the top letter, then look into the microscope, and slowly focus up. The lowest letter will first appear, and then, as it disappears, the middle one will appear, and soon. Focus down, and the top letter will first appear, then the mid- dle one, etc. The relative position of objects is determined exactly in this way in practical work. For example, if one has a micrometer ruled on a cover-glass 15-25 hundredths mm. thick, it is not easy to determine with the naked eye which is the ruled surface. But if one puts the micrometer under a microscope and uses a 3-mm.( 1 in.) objective, it is easily determined. The cover should be laid on a slide and focused till the lines are sharp. Now, without changing the focus in the least turn the cover over. If it is necessary to focus up to get the lines of the micrometer sharp, the lines are on the upper side. If one must focus down, the lines are on the under surface. With athin cover and delicate lines this method of determining the position of the rulings is of considerable practical importance. $134. Determination of the Form of Objects.—The procedure is exactly as for the determination of the form of large objects. That is, one must examine the various aspects. For example, if one were placed in front of a wall of some kind he could not tell whether it was a simple wall or whether it was one side of a building unless in some way he could see more than the face of the wall. In other words, in order to get a correct notion of any body, one must examine more than one dimension,—two for plane surfaces, three for solids. So for micro- scopic objects, one must in some way examine more than one face. To do this with small bodies in a liquid the bodies may be made to roll over by pressing on one edge of the cover-glass. And in rolling over the various aspects are presented to the observer. With solid bodies, 94 INTERPRETATION OF APPEARANCES [CH L/L like the various organs, correct notions of the form of the elements can be determined by studying sections cut at right angles to each other. The methods of getting the elements to roll over, and of sectioning in different planes are in constant use in Histology, and the microscopist who neglects to see all sides of the tissue elements has a very inade- quate and often a very erroneous conception of their true form. § 135. Transparent Objects having Curved Outlines.—The success of these experiments will depend entirely upon the care and skill used in preparing the objects, in lighting, and in focusing. Employ a 3 mm. (¥% in.) or higher objective and a high ocular for all the experiments. It may be necessary to shade the object (§ 109) to get satisfactory results. When a diaphragm is used the opening should be small and it should be close to the object. § 136. Air Bubbles.—Prepare these by placing a drop of thin mucilage on the center of a slide and beating it with a scalpel blade until the mucilage looks milky from the inclusion of air bubbles. Put on a cover-glass but do not press it down. Fic. 90. Diagram show- Fine forcens ing how to place a cover- glass upon an object with the Sorceps. $137. Air Bubbles with Central Illumination.—Shade the object ; and with the plane mirror, light the field with central light (Fig. 23). Search the preparation until an air bubble is found appearing about 1 mm. in diameter, get it into the center of the field, and if the light is central the air bubble will appear with a wide, dark, circular margin and a small bright center. If the bright spot is not in the center, adjust the mirror until it is. This is one of the simplest and surest methods of telling when the light is central or axial when no condenser is used ($ 65). Focus both up and down, noting that, in focusing up, the central spot becomes very clear and the black ring very sharp. On elevating the tube of the microscope still more the center becomes dim, and the whole bubble loses its sharpness of outline. $138. Air Bubbles with Oblique Ilumination.—Remove the sub-stage of the microscope and all the diaphragms. Swing the mirror so that the rays may be sent very obliquely upon the object (Fig. 203 CA. LIT) INTERPRETATION OF APPEARANCES 95 C). The bright spot will appear no longer in the center but on the side away from the mirror (Fig. yr). §$ 139. Oil Globules.—Prepare these by beating a small drop of clove oil with mucilage on a slide and covering as directed for air bub- bles (§ 137), or use a drop of milk. § 140. Oil Globules with Central Ilumination.—Use the same diaphragm and light as above ($137). Find an oil globule appearing about 1 mm. in diameter.: Ifthe light is central a bright spot will ap- pear in the center as with air. Focus up and down as with air, and note that the bright center of the oil globule is clearest /as¢ in focus- ing up. Fic. 91. Very small Globule of Oil (O) and an Air Bubble (A) seen by Oblique Light. The arrow indicates the direction of the light rays. § 141. Oil Globules with Oblique Illumination.— Remove the sub-stage, etc., as above, and swing the mir- ror to one side and light with oblique light. The bright spot will be eccentric, and will appear to be on the same side as the mirror (Fig. 91). § 142. Oil and Air Together.—Make a prepara- tion exactly as described for air bubbles (§ 136), and add at one edge a little of the mixture of oil and mucilage 0 (§ 139) ; cover and examine. - The sub-stage need not be used in this experiment. Search the preparation until an air bubble and an oil globule, each appearing about 1 mm. in diameter, are found in the same field of view. Light first with central light, and note that, in focusing up, the air bubble comes into focus first and that the central spot is smaller than that of the oil globule. Then, of course, the black ring will be wider in the air bubble than in the oil globule. Make the light oblique. The bright spot in the air bubble will move away from the mirror while that in the oil globule will move foward zt. See Fig. g1.* §$ 143. Air and Oil by Reflected Light.—Cover the diaphragm or mirror so that no transmitted light (§ 64) can reach the preparation, using the same preparation asin § 142. The oil and air will appear like globules of silver on a dark ground. The part that was darkest in *It should be remembered that the image in the compound microscope is inverted (Fig. 21), hence the bright spot really moves toward the mirror for air, and away from it for oil. 96 INTERPRETATION OF APPEARANCES (CH. 111 each will be lightest, and the bright central spot will be somewhat dark.* § 144. Distinétness of Outline.—In refraction images this depends on the difference between the refractive power of a body and that of the medium which surrounds it. The oil and air were very distinct in outline as both differ greatly in refractive power from the medium which surrounds them, the oil being more refractive than the mucilage and the air less. (Figs. 54-56. ) Place a fragment of a cover-glass on a clean slide, and cover it (see under mounting). The outline will be distinct with the unaided eye. Use it as object and employ the 16 mm. (%3 in.) objective and high ocular. Light with central light. The fragment will be outlined by a dark band. Put a drop of water at the edge of the cover-glass. It will run in andimmerse the fragment. The outline will remain dis- tinct, but the dark band will be somewhat narrower. Remove the cover-glass, wipe it dry, and wipe the fragment and slide dry also. Put a drop of 50% glycerin on the middle of the slide and mount the fragment of cover-glass in that. The dark contour will be much nar- rower than before. Draw a solid glass rod out toa fine thread. Mount one piece in air, and the other in 50% glycerin. Puta cover-glass on each. Em- ploy the same optical arrangement as before. Examine the one in air first. There will be seen a narrow, bright band, with a wide, dark band on each side. The one in glycerin will show a much wider bright central band, with the dark borders correspondingly narrow (Fig. 92, b). The dark contour depends also on the numerical aperture of the objective—being wider with low apertures. This can be readily understood when it is remembered that the greater the aperture the more oblique the rays of light that can be received, and the dark band simply represents an area in which the rays are so greatly bent or refracted (Figs. 54-56) that they cannot enter the objective and contribute to the formation of the image ; the edges are dark simply because no light from them reaches the observer. *It is possible to distinguish oil and air optically, as described above, only when quite high powers are used and very small bubbles are selected for observa- tion. If a 16 mm. (74 in.) is used instead of.a 3 mm. (1s in.) objective, the ap- pearances will vary considerably from that given above for the higher power. It is well to use a low as well as a high power. Marked differences will also be seen in the appearances with objectives of small and of large aperture. CHET) INTERPRETATION OF APPEARANCES 97 Fic. 92. Solid glass rod showing the pores -¢ appearance when viewed with transmit- ted, central light, and with an objective of medium aperture. a. Mounted in air. 6. Mounted in 50 per cent. glycerin. If the glass rod or any other object were mounted in a medium of the same color and refractive power, it could not be distinguished from the medium.* . A very striking and satisfactory demonstration may be made by painting a zone or band of eosin or other transparent color on a solid glass rod, and immersing the rod in a test tube or vial of cedar oil, clove oil or turpentine. Above the liquid the glass rod is very evident, asit is also at the colored zone, but at other levels it can hardly be seen in the liquid. $145. Highly Refractive.—This expression is often used in de- scribing microscopic objects, (medullated nerve fibers, for example), and means that the object will appear to be bordered by a wide, dark margin when it is viewed by transmitted light. And from the above ($144), it would be known that the refractive power of the object, and the medium in which it was mounted must differ considerably. $146. Doubly Contoured.—This means that the object is bounded by two, usually parallel dark lines with a lighter band between them. In other words, the object is bordered by (1) a dark line,(2) a light band, and (3) a second dark line (Fig. 93). This may be demonstrated by coating a fine glass rod (§ 144) with one or more coats of collodion or celloidin and allowing it to dry, and then mounting in 50% glycerin as above. Employ a 3 mm.(¥% in.) or higher objective, light with transmitted light, and it will be seen that where the glycerin touches the collodion coating there is a dark line— next this is a light band, and finally there is a second dark line where the collodion is in contact with the glass rod. * (Fig. 93). en Fic. 93. Solid glass rod coated with oa } lodion to show a double contour. Toward one end the collodion had gathered in a fusi- Se form drop. *Some of the rods have air bubbles in them, and then there results a capillary tube when they are drawn out. It is well to draw out a glass tube into a fine thread and examine it as described. The central cavity makes the experiment much more complex. 98 INTERPRETATION OF APPEARANCES (CH. £17 § 147. Optical Section.—This is the appearance obtained in examining transparent or nearly transparent objects with a microscope when some plane below the upper surface of the object is in focus. The upper part of the object which is out of focus obscures the image but slightly. By changing the position of the objective or object, a different plane will be in focus and a different optical section obtained. The most satisfactory optical sections are obtained with high objectives having large aperture. Nearly all the transparent objects studied may be viewed in optical section. A striking example will be found in studying mammalian red blood-corpuscles on edge. The experiments with the solid glass rods (Fig. 92) furnish excellent and striking examples of optical sections. § 148. Currents in Liquids.—Employ the 16 mm. (% in.) ob- jective, and as object put a few particles of carmine on the middle of a slide, and add a drop of water. Grind the carmine well with a scalpel blade, and then cover it. If the microscope is inclined, a current will be produced in the water, and the particles of carmine will be carried along by it. Note that the particles seem to flow up instead of down— why is this? Lamp-black rubbed in water containing a little mucilage answers well for this experiment. §$ 149. Velocity Under the Microscope.—In studying currents or the movement of living things under the microscope, one should not forget that the apparent velocity is as unlike the real velocity as the apparent size is unlike the real size. If one consults Fig. 37 it will be seen that the actual size of the field of the microscope with the different objectives and oculars is inversely as the magnification. That is, with great magnification only a small area can be seen. The field appears to be large, however, and if any object moves across the field it may appear to move with great rapidity, whereas if one measures the actual distance passed and notes the time, it will be seen that the actual motion is quite slow. One should keep this in mind in study- ing the circulation of the blood. The truth of what has just been said can be easily demonstrated in studying the circulation in the gills *The collodion used is a 6% solution of gun cotton in equal parts of sulphuric ether and 95% alcohol. It is well to dip the rod two or three times in the collo- dion and to hold it vertically while drying. The collodion will gather in drops, and one will see the difference between a thick and a thin membranous covering. (Fig. 93). CA. LIT) INTERPRETATION OF APPEARANCES 99 of Necturus, or in the frog’s foot, by using first a low power in which the field is actually of considerable diameter (Fig. 37, Table, § 51) and then using a high power. With the high power the apparent motion will appear much more rapid. For spiral, serpentine and other forms of motion, see Carpenter-Dallinger, p. 433. $150. Pedesis or Brownian Movement.—Employ the same object as above, but a 3 mm. (14 in.) or higher objective in place of the 16 mm. Make the body of the microscope vertical, so that there may be no currents produced. Use a small diaphragm and light the field well. Focus and there will be seen in the field large motionless masses, and between them small masses in constant motion. This is an indefinite, dancing or oscillating motion. This indefinite but continuous motion of small particles in a hquid is called 72-dé'sts or Brownian movement. Also, but improperly, molec- ular movement, from the smallness of the particles. The motion is increased by adding a little gum arabic solution or a slight amount of silicate of soda or soap; sulphuric acid and various saline compounds retard or check the motion. One of the best objects is lamp-black ground up with a little gum arabic. Carmine prepared in the same way, or simply in water, is excellent ; and very finely powdered pumice-stone in water has for many years been a favorite object. Pedesis is exhibited by all solid matter if it is finely enough di- vided and in a suitable liquid. In the minds of most, no adequate explanation has yet been offered. See Carpenter-Dallinger, p. 431 ; Beale, p. 195; Jevons in Quart. Jour. Science, n. s., Vol. VIII (1878), p. 167. In 1894, Meade Bache published a paper in the Proc. Amer. Philos. Soc., Vol. XX XIII, pp. 163-167, entitled ‘‘The Secret of the Brownian Movement.’’ This paper is suggestive if not very satisfactory. For the orginal account of this see Robert Brown, ‘‘Botanical appendix to Captain King’s voyage to Australia,’ Vol. II, p. 534. (1826). See also Dr. C. Aug. Sigm. Schultze, ‘‘Mikroskopische Unter- suchungen tiber des Herren Robert Brown Entdeckung lebender, selbst im Feuer unzerstorbarer Theilchen in allen Korpern.’’ From ‘‘Die Gesellschaft fiir Betorderung der Naturwissenschaften zu Freiburg.”’ 1828. Compare the pedetic motion with that of a current by slightly in- clining the tube of the microscope. The small particles will continue 100 INTERPRETATION OF APPEARANCES (CH. 17/ their independent leaping movements while they are carried along by the current. The pedetic motion makes it difficult to obtain good photographs of milk globules and other small particles. The difficulty may be overcome by mixing the milk with a very weak solution of gelatin and allowing it to cool (see Ch. IX). $151. Demonstration of Pedesis with the Polarizing Micro- scope (Ch. VI).—The following demonstration shows conclusively that the pedetic motion is real and not illusive. (Ranvier, p. 173.) Open the abdomen of a dead frog (an alcoholic or formalin specimen is satisfactory). Turn the viscera to one side and observe the small, whitish masses at the emergence of the spinal nerves. With fine forceps remove one of these and place it on the middle of a clean slide. Add a drop of water, or of water containing a little gum arabic. Rub the white mass around in the drop of liquid and soon the liquid will have a milky appearance. Remove the white mass, place a cover- glass on the milky liquid and seal the cover by painting a ring of castor oil all around it, half the ring being on the slide and half on the cover-glass. This is to avoid the production of currents by evaporation. Put the preparation under the microscope and examine with, first a low then a high power (3 mm. or !g in.). In the field will be seen multitudes of crystals of carbonate of lime; the larger crystals are motionless but the smallest ones exhibit marked pedetic movement. Use the micro-polariscope, light with great care and exclude all adventitious light from the microscope by shading the object (§ 10g) and also by shading the eye. Focus sharply and observe the pedetic motion of the small particles, then cross the polarizer and analyzer, that is, turn one or the other until the field is dark. Part of the large motion- less crystals will shine continuously and a part will remain dark, but small crystals between the large ones will shine for an instant, then disappear, only to appear again the next instant. This demonstration is believed to furnish absolute proof that the pedetic movement is real and not illusory. $152. Muscae Volitantes.—These specks or filaments in the eyes due to minute shreds or opacities of the vitreous sometimes appear as part of the object as they are projected into the field of vision. They may be seen by looking into the well lighted microscope when there is no object under the microscope. They may also be seen by looking at brightly illuminated snow or other white surface. By studying them carefully it will be seen that they are somewhat movable and float CHOTTT INTERPRETATION OF APPEARANCES IOI across the field of vision, and thus do not remain in one position as do the objects under observation. Furthermore, one may, by taking a little pains, familiarize himself with the special forms in his own eyes so that the more conspicuous at least may be instantly recognized. S153. In addition to the above experiments it is very strongly recommended that the student follow the advice of Beale, p. 248, and examine first with a low then with a higher power, mounted dry, then in water, lighted with reflected light, then with transmitted light, the following : Potato, wheat, rice, and corn starch, easily obtained by scraping the potato and the grains mentioned ; bread crumbs ; portions of feather. Portions of feather accidentally present in histological preparations have been mistaken for lymphatic vessels (Beale, 288). Fibers of ‘cotton, linen and silk. Textile fibers accidentally present have been considered nerve fibers, etc. Human and animal hairs. Study with especial care hairs from various parts of the body of the animals used for dissection in the laboratory where you work. These are hable to be present in histological preparations, and unless their character is understood there is chance for much confusion and erro- neous interpretation. The scales of butterflies and moths, especially the common clothes moth. The dust swept from carpeted and wood floors. Tea leaves and coffee grounds. Dust found in living rooms and places not frequently dusted. In the last will be found a regular museum of objects. For figures (photo-micrographs, etc.) of the various forms of starch, see Bulletin No. 13 of the Chemical Division of the U. S. Department af Agriculture. For Hair and Wool, see Bulletin of the National Asso- ciation of Wool Growers, 1875, p. 470, Proc. Amer. Micr. Soc., 1884, pp. 65-68. Herzfeld, translated by Salter.—The technical testing of yarns and textile fabrics, London, 1898. For different appearances due to the illuminator, see Nelson, in Jour. Roy, Micr. Soc., 1891, pp. 90-105; and for the illusory appear- ances due to diffraction phenomena, see Carpenter-Dallinger, p. 434. Mercer. Trans. Amer. Micr. Soc., pp. 321-396. If it is necessary to see all sides of an ordinary gross object, and to observe it with varying illumination and under various conditions of temperature, moisture, etc., in order to obtain a fairly accurate and satisfactory knowledge of it, so much the more is it necessary not to be satisfied in microscopical observation until every means of investigation and verification has been called into service, and then of the image that falls upon the retina, only such details will be noted as the brain behind the eye is ready to appreciate. 102 INTERPRETATION OF APPEARANCES (CH. 111 To summarize this chapter and leave with the beginning student the result of the experience of many eminent workers : 1. Get all the information possible with the unaided eye. See the whole object and all sides of it, so far as possible. 2. Examine the preparation with a simple microscope in the same thorough way for additional detail. 3. Use a low power of the compound microscope. 4. Use a higher power. 5. Use the highest power available and applicable. In this way one sees the object as a whole and progressively more and more details. Then as the object is viewed from two or more aspects, something like a correct notion may be gained of its form and structure. THE MICROSCOPE IN SECTION 1. Positive ocular. 11. Plane and concave mir- 2. Draw-tube. ror. 3. Main tube or body. 12. Horse-shoe base. 4-5. Society screws in the | 13. Rack and pinion for sdraw-tube and body. condenser. 6. Objective in position. 14. Flexible pillar. 7. Stage. 15. Spiral spring of fine ad- 8. Spring for holding justment. slides, 16. Fine adjustment 9g. Sub-stage condenser. 17. Coarse adjustment. to. Iris diaphragm. CHAPTER IV MAGNIFICATION AND MICROMETRY APPARATUS AND MATERIAL FOR THIS CHAPTER Simple and compound microscope (% 156, 158); Steel scale or rule divided to millimeters and }ths ; Block for magnifier and compound microscope (? 156, 160); Dividers (2 156, 160); Stage micrometer (4% 159); Wollaston camera lucida (% 160); Ocular screw-micrometers (Figs. 106-107); Micrometer ocular (Figs. 104-105). Abbe camera lucida (Fig. 101). Necturus red blood corpuscles (% 168). $154. The Magnification, Amplification or Magnifying Power of a simple or compound microscope is the ratio between the real and the apparent size of the object examined. The apparent size is ob- tained by measuring the virtual image (Figs. 21, 38). The object for determining magnification must be of known length and is designated a micrometer (§ 159). In practice a virtual image is measured by the aid of some form of camera lucida (Figs. 97, 101), or by double vision ($156). As the length of the object is known, the magnification is easily determined by dividing the apparent size of the image by the actual size of the object. For example, if the virtual image measures 4o mm. and the object magnified, 2 mm., the amplification must be 40 + 2 = 20, that is, the apparent size is 20 fold greater than the real size. Magnification is expressed in diameters or times linear, that is, but one dimension is considered. In giving the scale at which a micro- scopical or histological drawing is made, the word magnification is fre- quently indicated by the sign of multiplication thus: X 450, upon a drawing would mean that the figure or drawing is 450 times as large as the object. $155. Magnification of Real Images.—In this case the mag- nification is the ratio between the size of the real image and the size of the object, and the size of the real image can be measured directly. By recalling the work on the function of an objective (§ 53), it will be remembered that it forms a real image on the ground glass placed on the top of the tube, and that this real image could be looked at with the 104 MAGNIFICATION AND MICROMETRY (CH. 1V eye or measured as if it were an actual object. For example, suppose the object were three millimeters long and its image on the ground glass measured 15 mm., then the magnification must be, 15 + 3 = 5, that is, the real image is 5 times as long as the object. The real images seen in photography are mostly smaller than the objects, but the magnifica- tion is designated in the same way by dividing the size of the real im- age measured on the ground glass by the size of the object. For example, if the object is 400 millimeters long and its image on the ground glass is 25 mm. long, the ratio is 25+400=4!;. That is, the image is ;;th as long as the object and is not magnified but reduced. In marking negatives, as with drawings, the sign of multiplication is put before the ratio, and in the example the designation would be x ;i,th. MAGNIFICATION OF A SIMPLE MICROSCOPE $156. The Magnification of a Simple Microscope is the ratio between the object magnified (Fig. 16, A’B’), and the virtual image (A*B"). To obtain the size of this virtual image place the tripod mag- nifier near the edge of a support of such a height that the distance from the upper surface of the mag- nifier to the table is 250 millimeters. Fic. 94. Tripod Magnifier. As object, place a scale of some kind ruled in millimeters on the support under the magnifier. Put some white paper on the table at the base of the support and on the side facing the light. HY MUTI IAAT IIT VELEN AULA BIULL AULT Fic. 95. Zen Centimeter Rule. The upper edge ts divided into millimeters, the lower into centimeters at the left and half centimetersat the right. Close one eye, and hold the head so that the other will be near the upper surface of the lens. Focus if necessary to make the image clear GASLVa MAGNIFICATION AND MICROMETRY 105 (S11). Open the closed eye and the image of the rule will appear as if on the paper at the base of the support. Hold the head very still, and with dividers get the distance between any two lines of the image. This is the so-called method of double vision in which the microscope image is seen with one eye and the dividers with the other, the two images appearing to be fused in a single visual field. $157. Measuring the Spread of Dividers.—This should be done on a steel scale divided to millimeters and iths. As 4mm. cannot be seen plainly by the unaided eye, place one arm of the dividers at a centimeter line, and with the tripod magnifier count the number of spaces on the rule included between the points of the dividers. The magnifier simply makes it easy to count the spaces on the rule included between the points of the dividers—it does not, of course, increase the number of spaces or change their value. As the distance between any two lines of the image of the scale gives the size of the virtual image (Fig. 16, A*B’), and as the size of the object is known, the magnification is determined by dividing the size of the image by the size of the object. Thus, suppose the distance between the two lines of the image is measured by the dividers and found on the steel scale to be 15 millimeters, and the actual size of the space between the two lines of the object is 2 millimeters, then the magnification must be 15+2—71!4. ‘That is, the image is 7!4 times as long or wide as the object. In this case the image is said to be magnified 714 diameters, or 74 times linear. The Cortes of any simple magnifier may be determined experimentally in the way described for the tripod. MAGNIFICATION OF A COMPOUND MICROSCOPE $158. The Magnification of a Compound Microscope is the ratio between the final or virtual image (Fig. 21, B*A*), and the object magnified (A B). The determination of the magnification of a compound microscope may be made as with a simple microscope (§ 156), but this is very fatiguing and unsatisfactory. $159. Stage, Object or Objective Micrometer.—For deter- mining the magnification of a compound microscope and for the purpose of micrometry, it is necessary to have a finely divided scale or rule on glass or on metal. Sucha finely divided scale is called a micrometer, and for ordinary work one mounted on a glass slide (1X3 in, 25X76 mm.) is most convenient. 106 MAGNIFICATION AND MICROMETRY (CH. lV The spaces between the lines should be 7, and 74, mm. (or if in inches, ;}, and za in.) Micrometers are sometimes ruled on the slide, but more satisfactorily on a cover-glass of known thickness, preferably 0.15-0.18 mm. The covers should be perfectly clean before the ruling, and afterwards simply dusted off with a camel’s hair duster, and then mounted, lines downward over a shellac or other good cell. (See Ch. VII). If one rubs the lines the edges of the furrow made by the diamond are liable to be rounded and the sharp- ness of the micrometer is lost. If the lines are on the slide and un- covered one cannot use the micrometer with an oil immersion, as the oil obliterates the lines. Cleaning the slide makes the lines less sharp as stated. If the lines are very coarse, it is an advantage to fill them with plumbago. This may be done either with some very fine plum- bago on the end of asoft cork, or by using an exceedingly soft lead pencil. Lines properly filled may be covered with balsam and a cover- glass as in ordinary balsam mounting (Ch. VII). §$ 160. Determination of Magnification.—This is most readily accomplished by the use of some form of camera lucida (Ch. V), that of Wollaston being most convenient as it may be used for all powers, and the determination of the standard distance of 250 millimeters at which to measure the images is very readily determined (Fig. 97, $ 162)- Employ the 16 mm. (73 in.) objective and a 37 mm. (or X 8 ocu- lar with a stage micrometer as object. For this power the 4; mm. spaces of the micrometer should be used as object. Focus sharply. = Fic. 96. Diagram of a stage micrometer, with a ring on the lines to facilitate finding them. ( : : : é tlh It is somewhat difficult to find the mi- crometer lines. To avoid this it is well to have a small ring enclosing some of the micrometer lines (Fig. 96). The light must also be carefully regu- lated. If too much light is used, 7. ¢., too large an aperture, the lines will be drowned in the light. In focusing with the high powers be very careful. Remember the micrometers are expensive, and one can- notafford to break them. As suggested in § 74, focus on the edge of the cement ring enclosing the lines, then in focusing down to find the lines, move the preparation very slightly, back and forth. After the lines are sharply focused, and the slide clamped in posi- tion make the tube of the microscope horizontal, by bending the flexible pillar, being careful not to bring any strain upon the fine adjustment (frontispiece ). CH. lV! MAGNIFICATION AND MICROMETRY 107 Fic. 97. Wollaston’s Camera Lu- cida, showing the rays from the micro- scope and from the drawing surface, also the position of the pupil of the eve. Axis, Axts. Axial rays from the microscope and from the drawing surface (Ch. V). ; Camera Lucida. A section of the quadrangular prism showing the course of the rays in the prism from the microscope to the eye. As the rays are twice reflected, they have the same relation on entering the eye that they would have by looking directly into the ocular, A.B. The lateral rays from the microscope and their projection upon a the drawing surface. C.D Rays from the drawing surface to ths eve. A.D. A’ LD’. Overlapping portions of the two fields, where both the nticro- scopic image and the drawing surface, pencil, etc., can be seen. Jt 1s represented by the shaded part of the overlapping circles at the right. Ocular. The ocular of the microscope. P. The drawing pencil. ts point is shown in the overlapping fields. Ocular ae SH A | rere Put a Wollaston camera lucida (Fig. 97 and Ch. V) in position, and turn the ocular around if necessary so that the broad flat surface may face directly upward, as shown in Fig. 97. Elevate the micro- scope by putting a block under the base, so that the perpendicular dis- tance from the upper surface of the camera lucida to the table is 250 mm. (§ 162). Place some white paper on the work-table beneath the camera lucida. Close one eye, and hold the head so that the other may be very close to the camera lucida. Look directly down. The image will ap- pear to be on the table. It may be necessary to readjust the focus after the camera lucida is in position. If there is difficulty in seeing dividers and image consult Ch. V. Measure the image with dividers and obtain the power exactly as above ($156-157). Thus : Suppose two of the ;';th mm., spaces were taken as object, and the image is measured by the dividers, and the spread of the dividers is found on the steel rule to be 92 millimeters. If now the object is 2,ths of a millimeter and the magnified image is 9} milli- 10 meters, the magnification (which is the ratio between size of object 108 MAGNIFICATION AND MICROMETRY (CH. IV and image) must beg? +7;—=47. That is, the magnification is 47 diameters, or 47 times linear. If the fractional numbers in the above example trouble the student, both may be reduced to the same denom- ination, thus: If the size of the image is found to be 9? mm. this number may be reduced to tenths mm., so it will be of the same /mage 2 Objective Object-b Object-a FIG. 99. Fics, 98-99. Figures showing that the size of object and image very directly as their distance from the center of the lens. In Fig. 99 one can also see why it is necessary to focus down, 1. e., bring the object and objective nearer together when the tube is lengthened. See also fig. 58. denomination as the object. Ino mm. there are 50 tenths, and in 2 there are 4 tenths, then the whole length of the image is 90 + 4= 94 tenths of a millimeter. The object is 2 tenths of a millimeter, then CH. IV? MAGNIFICATION AND MICROMETRY 10g there must have been a magnification of 94 + 2 = 47 diameters in order to produce an image 94 tenths of a millimeter long. Put the 25 mm. (1 in., C, or X 12) ocular in place of one of 37 mm. focus, and then put the camera lucida in position. Measure the size of the image with dividers and a rule as before. The power will be considerably greater than when the low ocular was used. This is because the virtual image (Fig. 21, B*A*) seen with the high ocular is larger than the one seen with the low one. The real image (Fig. 21, A'B') remains nearly the same, and would be just the same if positive, par-focal oculars (S$ 37, 72, note) were used. Lengthen the tube of the microscope 50-60 mm. by pulling out the draw-tube. Remove the camera lucida, and focus, then replace the camera and obtain the magnification. It will be greater than with the shorter tube. This is because the real image (Fig. 99) is formed farther from the objective when the tube is lengthened, and the objective must be brought nearer the object. The law is: The size of object and image varies directly as their distance from the center of the Jens. The truth of this statement is illustrated by Figs. 98 and 99. $161. Varying the Magnification of a Compound Micro- scope.—It will be seen from the above experiments (§ 160) that in- dependently of the distance at which the microscopic image is measured (§ 162), there are three ways of varying the power of a compound microscope. These are named below in the order of desirability. (1) By using a higher or lower objective. (2) By using a higher or lower ocular. (3) By lengthening or shortening the tube of the microscope (Fig. * 99).* § 162. Standard Distance of 250 Millimeters at which the Virtual Image is Measured.—For obtaining the magnification of both the simple and the compound microscope the directions were to measure the virtual image at adistance of 250 millimeters. This is not *Amplifier.—In addition to the methods of varying the magnification given in 4 161, the magnification is sometimes increased by the use of an amplifier, that is a diverging lens or combination placed between the objective and ocular and serving to give the image-forming rays from the objective an increased divergence. An effective form of this accessory was made by Tolles, who made it as a small achromatic concavo-convex lens to be screwed into the lower end of the draw-tube (frontispiece) and thus but a short distance above the objective. The divergence given to the rays increases the size of the real image about two-fold. I1O MAGNIFICATION AND MICROMETRY (CH. IV i Fic. 109. figure showing the position of the microscope, the camera lucida, the eye, and the difference in size of the 1m- age depending upon the dis- tance at which it is projected from the eye. (a) The size at 25 cm.; (6) at 35 cm., (4 162). ] that the image could not be seen and measured at any other distance, but because some standard must be selected, and this is the most common one. The necessity for the adoption of some common standard will be seen at a glance in Fig. roo, where is represented graphically the fact Fic. 1o1. Sectional view of the Abbe Camera Lucida to show that in measuring the standard distance of 250 millimeters, one must Meas- ure along the axis from the point P,at the left of the prism, to the mirror, and from the mirror to the drawing surface. for a full explanation of this camera lucida, see next chapter. that the size of the virtual image depends directly on the distance at which it is projected, and this size is directly proportional to the ver- tical distance from the apex of the triangle, of which it forms a base. The distance of 250 millimeters has been chosen on the supposition that it is the distance of most distinct vision for the normal human eye. CH, IV'7) MAGNIFICATION AND MICROMETRY II! Demonstrate the difference in magnification due to the distance at which the image is projected, by raising the microscope so that the distance will be 350 millimeters, then lowering to 150 millimeters. In preparing drawings it is often of great convenience to make them at a distance somewhat less or somewhat greater than the stand- ard. In such a case the magnification must be determined for the special distance. (See the next chapter, § 181.) For discussion of the magnification of the microscope, see: Beale, PP. 41, 355 ; Carpenter-Dallinger, p. 288 ; Nageli and Schwendener, p. 176; Ranvier, p. 29; Robin, p. 126; Amer. Soc. Micrs., 1884, p. 183 ; 1889, p. 22; Amer. Jour. Arts and Sciences, 1890, p. 50; Jour. Roy. Micr. Soc., 1888, 1889. OCULAR OCULAR 37 or 50 mr. 25 mm. onyecrive.| Tu# | Sor | Tuer | Gurr [OPM Menon = ——MM. ——MM. JTUBEIN. QUT——MM. x x x x x x x x x x x x x x x x x x x x x x x XX SIMPLE MICROSCOPE. >4 FIG. 102. $ 163. Table of Magnifications and of the Valuations of the Ocular Micrometer.—7he above table should be filled out by each student. In using tt for Micrometry and Drawing tt is necessary to keep clearly in mind the exact conditions under which the determinations were I12 MAGNIFICATION AND MICROMETRY (CH. lV made, and also the ways in which variations in magnification and the val- uation of the ocular micrometer may be produced (S 161. 162, 172, 176). MICROMETRY § 164. Micrometry is the determination of the size of objects by the aid of a microscope. MICROMETRY WITH THE SIMPLE MICROSCOPE $165. With a simple microscope (A), the easiest and best way is to use dividers and then with the simple microscope determine when the points of the dividers exactly include the object. The spread of the dividers is then obtained as above (§ 157). This amount will be the actual size of the object, as the microscope was only used in helping to see when the divider points exactly enclosed the object, and then for reading the divisions on the rule in getting the spread of the dividers. (B) One may put the object under the simple microscope and then, as in determining the power (§ 156), measure the image at the standard distance. If the size of the image so measured is divided by the magnification of the simple microscope, the quotient will give the actual size of the object. Use a fly’s wing or some other object of about that size, and try to determine the width in the two ways described above. If all the work is accurately done the results will agree. MICROMETRY WITH THE COMPOUND MICROSCOPE There are several ways of varying excellence for obtaining the size of objects with the compound microscope, the method with the ocular micrometer (§ 175-176) being most accurate. S$ 166. Unit of Measure in Micrometry.—As most of the ob- jects measured with the compound microscope are smaller than any of the originally named divisions of the meter, and the common or decimal fractions necessary to express the size are liable to be unnecessarily cumbersome, /fartzng, in his work on the microscope (1859), proposed the one thousandth of a millimeter (;,}, mm. or 0.001 mm.) or one inillionth of a meter (;yo}ay7 OF 0.000001 meter) astheunit. He named this unit micro-millimeter and designated it mmm. In 1869, Listing (Carl’s Repetorium fur Experimental-Physik, Bd, X, p. 5) CAVA) = MAGNIFICATION AND MICROMETRY 113 favored the thousandth of a millimeter as unit and introduced the name Mikron or mzcrum. In English it is most often written A/ccron (plural micra oY microns, pronunciation Mik’rdn or Mik’r6n). By universal con- sent the sign or abbreviation used to designate it is the Greek yw. Adopting this unit and sign, one would express five thousandths of a millimeter (;q7 OF 0.005 mm.) thus, 5y.* 2167. Micrometry dy the use of a stage micrometer on which to mount the ob- Ject.—In this method the object is mounted on a micrometer and then put under the microscope, and the number of spaces covered by the object is read off directly. It is exactly like putting any large object on a rule and seeing how many spaces of the rule it covers. The defect in the method is that it is impossible to properly arrange objects on the micrometer. Unless the objects are circular in outline they are liable to be oblique in position, and in every case the end or edges of the object may be in the middle of a space instead of against one of the lines, consequently the size must be estimated or guessed at rather than really measured. $168. Micrometry dy dividing the size of the image by the magnifica- tion of the microscope.—For example, employ the 3 mm.(% in.) objective, 25 mm.(1 in.) ocular, and a Necturus’ red blood-corpuscle preparation as object. Obtain the size of the image of the long and short axes of three corpuscles with the camera lucida and dividers, exactly as in obtaining the magnification of the microscope (§ 160). Divide the size of the image in each case by the magnification, and the result will be the actual size of the blood-corpuscles. Thus, suppose the image of the long axis of the corpuscle is 18 mm. and the magnification of the micro- scope 400 diameters (§ 154), then the actual length of this long axis of the corpuscle is 18 mm.+ 400 0.045 mm. or 45,4 (§ 166). Fic. 103. Preparation of blood witha ring around a group of blood corpuscles. As the same three blood-corpuscles are to be measured in three ways, it is an advantage to put a delicate ring around a group of three or more corpuscles, and make a sketch of the whole enclosed group, sides is entirely inappropriate since the adoption of the definite meanings for the prefixes micro and mega, meaning respectively one-millionth and one million times the unit before which it is placed. A micromillimeter would then mean one-millionth of a millimeter, not one-thousandth. The term micron has been adopted by the great microscopical societies, the international commission on weights and measures, and by original investigators, and is, in the opinion of the writer, the best term to employ. Jour. Roy. Micr. Soc., 1888, p. 502; Nature, Vol. XXXVII (1883), p. 388. 114 MAGNIFICATION AND MICROMETRY [([CZ7TE RY marking on the sketch the corpuscles measured (Figs. 61-66). The different corpuscles vary considerably in size, so that accurate com- parison of different methods of measurement can only be made when the same corpuscles are measured in each of the ways. $169. Micrometry 6y the use of a Stage Micrometer and a Camera Lucida.—Employ the same object, objective and ocular as before. Put the camera lucida in position, and with a lead pencil make dots on the paper at the limits of the image of the blood- corpuscle. Measure the same three that were measured in § 168. Remove the object, place the stage micrometer under the micro- scope, focus well, and draw the lines of the stage micrometer so as to include the dots representing the limits of the part of the image to be measured. As the value of the spaces on the stage micrometer is known, the size of the object is determined by the number of spaces of the micrometer required to include it. This simply enables one to put the image of a fine rule on the image of a microscopic object. It is theoretically an excellent method, and nearly the same as measuring the spread of the dividers with a simple microscope (§ 157, 176). OCULAR MICROMETER § 170. Ocular Micrometer, Eye-Piece Micrometer.—This, as the name implies, isa micrometer to be used with the ocular. It is a micrometer on glass, and the lines are sufficiently coarse to be clearly seen by the ocular. The lines should be equidistant and about ;4th or zjth mm. apart, and every fifth line should be longer and heavier to facilitate counting. If the micrometer is ruled in squares (ef micro- meter) it will be very convenient for many purposes. The ocular micrometer is placed in the ocular, no matter what the form of the ocular (7. e¢., whether positive or negative) at the level at which the real image is formed by the objective, and the image appears to be immediately upon or under the ocular micrometer, and hence the number of spaces on the ocular micrometer required to measure the real image may be read off directly. This, however, is measuring the size of the real image, and the actual size of the object can only be determined by determining the ratio between the size of the real image and the object. In other words, it is necessary to get the valuation of the ocular micrometer in terms of a stage micrometer. S171. Valuation of the Ocular Micrometer.—This is the value of the divisions of the ocular micrometer for the purposes of CH. L1V'7 MAGNIFICATION AND MICROMETRY II5 micrometry, and is entirely relative, depending upon the magnification of the real image formed by the objective, consequently it changes with every change in the magnification of the real image, and must be especially determined for every optical combination (7. ¢., objective and ocular), and for every change in the length of the tube of the micro- scope. That is, it is necessary to determine the ocular micrometer val- uation for every condition modifying the real image of the microscope (§ 161). Any Huygenian ocular (Fig. 30) may, however, be used as a micrometer ocu- lar by placing the ocular micrometer at the level of the ocular diaphragm, where the real image is formed. If there is a slit in the side of the ocular , and the ocular micrometer is mounted in some way it may be introduced through the opening in the side. When no side opening exists the mounting of the eye-lens may be unscrewed and the ocular micrometer, if on a cover-glass can be laid on the upper side of the ocular diaphragm. ee ed wy FIG. 104. FIG. 105. Fics. 104-105. Ocular Micrometer with movable scale. Fig. 104 1s a side view of the ocular while Fig. 105 gives a sectional end view, and shows the ocular mi- crometer in position. In both the screw which moves the micrometer is shown at the left. (Bausch & Lomb Opt. Co.) $172. Obtaining the Ocular Micrometer Valuation for an Ocular Micrometer with Fixed Lines (Figs. 104-105).—Use the stage micrometer as object. Light the field well and look into the microscope. The lines of the ocular micrometer should be very sharply defined. If they are not, raise or lower the eye-lens to make them so ; that is, focus as with the simple magnifer. When the lines of the ocular micrometer are distinct, focus the microscope ($72, 74, 75) for the stage micrometer. "The image of the stage micrometer will appear to be directly under or upon the ocular micrometer. Make the lines of the two micrometers parallel by rotating the ocular or changing the position of the stage micrometer or both if necessary, 116 MAGNIFICATION AND MICROMETRY (CH. LV and then make any two lines of the stage micrometer coincide with any two on the ocular micrometer. To do this it may be necessary to pull out the draw-tube a greater or less distance. See how many spaces are included in each of the micrometers. Divide the value of the included space or spaces on the stage mi- crometer by the number of divisions on the ocular micrometer required to include them, and the quotient so obtained will give the valuation of the ocular micrometer in fractions of the unit of measure of the stage micrometer. For example, suppose the millimeter is taken as the unit for the stage micrometer and this unit is divided into spaces of jth and zipth millimeter. If now, with a given optical combination and tube- length, it requires 10 spaces on the ocular micrometer to include the real image of ;;th millimeter on the stage micrometer, obviously one space on the ocular micrometer would include only one-tenth as much, or jjth mm. +10 = 747th mm. That is, each space on the ocular mi- crometer would include ;t,th of a millimeter on the stage micrometer, or ;1,th millimeter of the length of any object under the microscope, the conditions remaining the same. Or, in other words, it would re- quire 100 spaces on the ocular micrometer to include 1 millimeter on the stage micrometer, then as before, 1 space of the ocular micrometer would have a valuation of ;4,;th millimeter for the purposes of micrometry. The size of any minute object may be determined by multiplying this valuation of one space by the number of spaces required to include it. For example, suppose the fly’s wing or some part of it covered 8 spaces on the ocular micrometer, it would be known that the real size of the part measured is zj;th mm. X 8= 735 mm. or 80 py (§ 166). See Mark, Jour. Applied Microscopy, Vol. I, p. 4. $173. Micrometry with the Ocular Micrometer.—Use the 3 mim.(4 in.) objective with the preparation of Necturus blood-corpuscles as object. Make certain that the tube of the microscope is of the same length as when determining the ocular micrometer valuation. Ina word, be sure that all the conditions are exactly as when the valuation was determined, then put the preparation under the microscope and find the same three red corpuscles that were measured in the other ways ($168-169). Count the divisions on the ocular micrometer required to enclose or measure the long and the short axis of each of the three corpuscles, multiply the number of spaces in both cases by the valuation of the ocular micrometer for this objective, tube-length and ocular, and the results will represent the actual length of the axes of the corpuscles in each case. GHOLY | MAGNIFICATION AND MICROMETRY 117 The same corpuscle is, of course, of the same actual size, when measured in each of the three ways, so that if the methods are correct and the work carefully enough done, the same results should be ob- tained by each method. (§ 176).* Fic. 106. Ocular Screw-Micrometer with compensation ocular x 6. The upper figure Shows a sectional view of the ocular and the screw for moving the micrometer at the right. alt the left is shown a clamping screw to fasten the ocular to the upper part of the mi- croscope tube. Below ts a face view, showing the graduation on the wheel. An ocular micrometer like this 1s im general like the cob-web micrometer and may be used for measuring objects of varying sizes very accu- rately. With the ordinary ocular micrometer very small objects frequently fill but a part of an interval of the micrometer, but with this the movable cross lines traverse the object (or rather tts real image) regardless of the minute- ness of the object. ( Zeiss’ Catalog). 4174. Obtaining the Valuation of the Screw or Filar Micrometer.—This micrometer (Fig. 106-107) usually consists of a Raimsden’s ocular and cross lines. As seen in Fig. 1074 there are three lines. The horizontal and one vertical line are fixed. One vertical line may be moved by the screw back and forth across the field. For obtaining the valuation of this ocular micrometer an accurate stage mi- crometer must be used. Carefully focus the ;};th mm. spaces. The lines of the ocular micrometer should also be sharp. If they are not focus them by moving the top of the ocular up or down (7172). Make the vertical lines of the filar mi- crometer parallel with the lines of the stage micrometer. Take the precautions regarding the width of the stage micrometer lines given in 7 176 (see also Fig. 108). Note the position of the graduated wheel and of the teeth of the recording comb, and then rotate the wheel until the movable line traverses one space on the stage micrometer. Each tooth of the recording comb indicates a total revolution of the wheel, and by noting the number of teeth required and the graduations on the wheel, the revolutions and parts of revolution required to measnre the ;},th mm, of the stage micrometer can be easily noted. Measure in like manner q or 5 spaces and get the average. Suppose this average is 1th revolutions or 125 graduations on the wheel, to measure the ;jj;th mm. or roy: (see % 166), then one of the graduations on the wheel would measure toy divided by 125 =.08. In using this valuation for actual measurement, the tube of the microscope and the objective must be exactly as when obtaining the valuation (see 7 175). Example of Measurement.—Suppose one uses the red blood corpuscles of a dog or monkey, etc., every condition being as when the valuation was determined, one notes very accurately how many of the graduations on the wheel are required to make the movable line traverse the object from edge toedge. Suppose it requires 118 MAGNIFICATION AND MICROMETRY (CH. IV 94 of the graduations to measure the diameter, the actual size of the corpuscle would be 94X.08 “= 7.52 /4. The advantage of the filar micrometer is that the valuation of one graduation being so small, even the smallest object to be measured would require several graduations to measure it. In ocular micrometers with fixed lines, small objects like bacteria might not fill even one space, therefore estimations, not measure- ments, must be made. For large objects, like most of the tissue elements, the ocular micrometers with fixed lines answer very well, for the part which must be estimated is relatively small and the chance of error is correspondingly small. Fic. 107. “ilar Ocular Micrometer with Field A (Bausch & Lomb, Optical Co.). $175. Varying the Ocular Micrometer Valuation.—Any change in the objective, the ocular or the tube-length of the microscope, that is to say, any change in the size of the real image, produces a cor- responding change in the ocular micrometer valuation (§ 161, 171, 176). 2176. Remarks on Micrometry.—In using adjustable objectives (724, 103), the magnification of the objective varies with the position of the adjusting collar, being greater when the adjustment is closed as for thick cover-glasses than when open, as for thin ones. This variation in the magnification of the objective pro- duces a corresponding change in the magnification of the entire microscope, and the ocular micrometer valuation—therefore it is necessary to determine the mag- nification and ocular micrometer valuation for each position of the adjusting collar. While the principles of micrometry are simple, it is very difficult to get the exact size of microscopic objects. This is due to the lack of perfection and uni- *There are three ways of using the ocular micrometer, or of arriving at the size of the objects measured with it : (A) By finding the value of a division of the ocular micrometer for each optical combination and tube-length used, and employing this valuation as a multiplier. This is the method given in the text, and {the one most frequently employed. Thus, suppose with a given optical combination and tube-length it required five divisions on the ocular micrometer to include the image of ths millimeter of the stage micrometer, then obviously one space on the ocular micrometer would in- clude tth of ;4;ths mm. or ,|;th mm.; the size of any unknown object under the (Cra he Ara MAGNIFICATION AND MICROMETRY 11g formity of micrometers, and the difficulty of determining the exact limits of the object to be measured. Hence, all microscopic measurements are only approxi- mately correct, the error lessening with the increasing perfection of the apparatus and the skill of the observer. microscope would be obtained by multiplying the number of divisions on the ocular micrometer required to include its image by the value of one space, or in this case, ,|;th mm. Suppose some object, as the fly’s wing, required 15 spaces of the ocular micrometer to include some part of it, then the actual size of this part of the wing would be 15 & 34, = 2ths, or 0.6 mm. (B) By finding the number of divisions on the ocular micrometer required to include the image of an entire millimeter of the stage micrometer, and using this number as a divisor. This number is also sometimes called the ocular micrometer ratio. Taking the same case as in (A), suppose five divisions of the ocular mi- crometer are required to include the image of ths mmi., on the stage micrometer, then evidently it would require 5 = #4; = 25 divisions on the ocular micrometer to include a whole millimeter on the stage micrometer, and the number of divisions of the ocular micrometer required to measure an object divided by 25 would give the actual size of the object in millimeters or ina fraction of amillimeter. Thus, suppose it required 15 divisions of the ocular micrometer to include the image of some part of the fly’s wing, the actual size of the part included would be 15+ 25 =oro.mm. This method is really exactly like the one in(A), for dividing by 25 is the same as multiplying by 5th. (C) By having the ocular micrometer ruled in millimeters and divisions of a millimeter, and then getting the size of the realimagein millimeters. Inemploy- ing this method a stage micrometer is-used as object and the size of the image of one or more divisions is measured by the ocular micrometer, thus: Suppose the stage micrometer is ruled in ;,th and ;},th mm. and the ocular micrometer is ruled in millimeters and jth mm. Taking ;;th mm. on the stage micrometer as object, as in the other cases, suppose it requires 10 of the ;j>th mm. spaces or I mm. to measure the real image, then the real image must be magnified 1? = 4, = 5 diame- ters, that is, the real image is five times as great in length as the object, and the size of an object may be determined by putting it under the microscope and getting the size of the real image in millimeters with the ocular micrometer and dividing it by the magnification of the real image, which in this case is 5 diameters. Use the fly’s wing as object, as in the other cases, and measure the image of the same part. Suppose that it required 30 of the 7; mm. divisions = }{} mm. or 3 mm. to include the image of the part measured, then evidently the actual size of the part measured is 3 mm, + 5 = 4 mm., the same result as in the other cases. In comparing these methods it will be seen that in the first two (A and B) the ocular micrometer may be simply ruled with equidistant lines without regard to the absolute size in millimeters or inches of the spaces. In the last method the ocular micrometer must have its spaces some known division of a millimeter or inch. In the first two methods only one standard of measure is required, viz, the stage micrometer ; in the last method two standards must be used,—a stage mi- crometer and an ocular micrometer. Of course, the ocular micrometer in the first two cases must have the lines equidistant as well as in the last case, but ruling lines equidistant is quite a different matter from getting them an exact division of a millimeter or of an inch apart. 120 MAGNIFICATION AND MICROMETRY (CH. 1V A difficulty when one is using high powers is the width of the lines of the micrometer, If the micrometer is perfectly accurate half the width of each line belongs to the contiguous spaces, hence one should measure the image of the space from the centers of the lines bordering the space, or as this is somewhat difficult in using the ocular micrometer, one may measure from the inside of one border- ing line and from the outside of the other. If the lines are of equal width this is as accurate as measuring from the center of the lines. Evidently it would not be right to measure from either the inside or the outside of both lines (Fig. 108). It is also necessary in micrometry to use an objective of sufficient power to enable one to see all the details of an object with great distinctness. The necessity of using sufficient amplification in micrometry has been especially remarked upon by Richardson, Monthly Micr. Jour., 1874, 1875, ; Rogers, Proc. Amer. Soc. Micro- scopists, 1882, p. 239; Ewell, North American Pract., 1890, pp. 97, 173. Fic. 108. The appearance of the coarse A B stage micrometer and of the fine ocular mt- oa ia et crometer lines when using ahigh objective. (4). The method of measuring the spaces by putting the fine ocular mi- crometer lines opposite the center of the coarse slage micrometer lines. om S00 (BL). ATethod of measuring the spaces of the stage micrometer by putting one Jine of the ocular micrometer (0. m.) atthe imside and one at the outside of the coarse stage micrometer lines (Ss. mM.). FIG. 108. As to the limit of accuracy in micrometry, one who has justly earned the right to speak with authority expresses himself as follows: ‘‘/ assume that 0.2) 1s the limit of precision in microscopic measures beyond which it is impossible to go with certainty.’ W. A. Rogers Proc. Amer. Soc. Micrs., 1853, p. 198. In comparing the methods of micrometry with the compound microscope given above (% 167, 168, 169, 175), the one given in % 167 is impracticable, that given in 7 168 is open to the objection that two standards are required,—the stage microme- ter, and the steel rule; it is open to the further objection that several different operations are necessary, each operation adding to the probability of error. Theoret- ically the method given in 4 169 is good, but it is open to the very serious objection in practice that it requires so many operations which are especially liable to intro- duce errors. The method that experience has found most safe and expeditious, and applicable to all objects, is the method with the ocular micrometer. If the valuation of the ocular micrometer has been accurately determined, then the only difficulty is in deciding on the exact limits of the objects to be measured and so arranging the ocular micrometer that these limits are inclosed by some divisions of the micrometer. Where the object is not exactly included by whole spaces on the ocular micrometer, the chance of error comes in, in estimating just how far into a space the object reaches on the side not in contact with one of the micrometer [GAL MAGNIFICATION AND MICROMETRY 121 lines. If the ocular micrometer has some quite narrow spaces, and others consid- erably larger, one can nearly always manage to exactly include the object by some two lines. The ocular screw micrometers (Fig. 106-107) obviates this entirely as the cross hair or lines traverse the object or its real image, and whether this distance be great or small it can be read off on the graduated wheel, and uo estimation or guess work is necessary. For those especially interested in micrometry, as in its relation to medical jurisprudence, the following references are recommended. These articles consider the problem in a scientific as well as a practical spirit: The papers of Prof. Wm. A. Rogers on micrometers and micrometry, in the Amer. Quar. Micr. Jour., Vol. I, pp. 97, 208; Proceedings Amer. Soc. Microscopists, 1882, 1883, 1887. Dr. M. D. Ewell, Proc. Amer. Soc. Micrs., r8g0 ; The Microscope, 1889, pp. 43-45 ; North Amer. Pract., 1890, pp. 97, 173. Dr. J. J. Woodward, Amer. Jour. of the Med. Sei., 1875. M. C. White, Article ‘‘Blood-stains,’’ Ref. Hand-Book Med. Sciences, 1885. Medico-Legal Journal, Vol. XII. For the change in magnification due to a change in the adjustment of adjustable objectives, see Jour. Roy. Micr. Soc., 1880, p. 702; Amer. Monthly Micr. Jour., 1880, p. 67. Carpenter-Dallinger, p. 270. If one consults the medico-legal journals; the microscopical journals, the Index Medicus, and the Index Catalog of the Library of the Surgeon General's Office, under Micrometry, Blood, and Jurisprudence, he can get on track of the main work which has been and is being done Dry objectives of 16 mm, (5 im.), gInm. (h22.) and homogeneous immersion objective of 2mm. (q'; in.) tn their mountings. (Bausch & Lomb Opt. Co.). CHAP TE RV, DRAWING WITH THE MICROSCOPE APPARATUS AND MATERIAL FOR THIS CHAPTER Microscope, Abbe and Wollaston’s camera lucidas, drawing board, thumb tacks, pencils, paper, and microscope screen, (Fig. 59), microscopic preparations. DRAWING MICROSCOPIC OBJECTS $177. Microscopic objects may be drawn free-hand directly from the microscope, but in this way a picture giving only the general ap- pearance and relations of parts is obtained. For pictures which shall have all the parts of the object in true proportions and relations, it is necessary to obtain an exact outline of the image of the object, and to locate in this outline all the principal details of structure. It is then possible to complete the picture free-hand from the appearance of the object under the microscope. The appliance used in obtaining out- lines, etc., of the microscope image is known as a camera lucida. $178. Camera Lucida.—This is an optical apparatus for en- abling one to see objects in greatly different situations, as if in one field of vision, and with the same eye. In other words it is an optical device for superimposing or combining two fields of view in one eye. As applied to the microscope, it causes the magnified virtual im- age of the object under the microscope to appear as if projected upon the table or drawing board, where it is visible with the drawing paper, pencil, dividers, etc., by the same eye, and in the same field of vision. The microscopic image appears like a picture on the drawing paper (see note to § 181). This is accomplished in two distinct ways: (A) By a camera lucida reflecting the rays from the microscope so that their direction when they reach the eye coincides with that of the rays from the drawing paper, pencil, etc. In some of the camera lucidas from this group (Wollaston’s, Fig. 112), the rays are reflected twice, and the image appears as when looking directly into the micro- scope. In others the rays are reflected but once, and the image has the inversion produced by a plane mirror. For drawing purposes this CA. V) DRAWING WITH THE MICROSCOPE 123 inversion is a great objection, as it is necessary to similarly invert all the details added free-hand. (B) By a camera lucida reflecting the rays of light from the draw- ing paper, etc., so that their direction when they reach the eye coin- cides with the direction of the rays from the microscope (Fig. 58, 109). In all of the camera lucidas of this group, the rays from the paper are twice reflected and no inversion appears. FIG, 110, FIG. III. Fic. 109. bbe Camera Lucida with the mirror at 45°, the drawing surface horizontal, and the microscope vertical. Axis, Axis. Axialray from the mi- croscope and from the drawing surface. A,B. Marginal rays of the field on the drawing surface. ab. Sectional view of the silvered surface on the upper of the tri- angular prisms composing the cubical prism(P). The silvered surface ts shown as incomplete in the center, thus giving passage to the rays from the microscope. foot. Foot or base of the microscope. G. Smoked glass seen in section. It is placed between the mirror and the prism to reduce the light from the drawing surface. Mirror. The mirror of the camera lucida. A quadrant (Q) has been added to indicate the angle of inclination of the mirror, which in this case ts 45°. Ocular. Ocular of the microscope over which the prism of the camera lucida is placed. P,P. Drawing pencil and the cubical prism over the ocular. Fic. 110. Geometrical figure showing the angles made by the axial ray with the drawing surface and the mirror. A,B. The drawing surface. Fic. 111. Ocular showing eyve-point, E. P. Lt tsatthts point both horizontally and vertically that the hole in the silvered surface should be placed (4 182). FIG. 109. 124 DRAWING WITH THE MICROSCOPE [GK The better forms of camera lucidas (Wollaston’s, Grunow’s, Abbe’s, etc. ), may be used for drawing both with low and with high powers. Some require the microscope to be inclined (Fig. 100) while others are designed to be used on the microscope in a vertical position. As in biological work, it is often necessary to have the microscope vertical, the form for a vertical microscope is to be preferred ; but see Figs. 115- peues Kop § 179. Avoidance of Distortion.—/n order that the picture drawn by the aid of a camera lucida may not be distorted, it ts necessary that the axtal rav from the image on the drawing surface shall be at right angles to the drawing surface (Figs. 112, 114.) 7180. Wollaston’s Camera Lucida.—This is a quadrangular prism of glass put in the path of the rays from the microscope, and it serves to change the direction of the axial ray go degrees. In using it the microscope is made horizon- tal, and the rays from the microscope enter one-half of the pupil while rays from the drawing surface enter the other half of the pupil. As seen in the figure ( Fig. 112), the fields partly overlap, and where they do so overlap, pencil or dividers and microscopic image can be seen together. In drawing or using the dividers with the Wollaston camera lucida it is neces- sary to have the field of the microscope and the drawing surface about equally lighted. If the drawing surface is too brilliantly lighted the pencil or dividers may be seen very clearly, but the microscopic image will be obscure. On the other hand, if the field of the microscope has too much light the microscopic image will be very definite, but the pencil or dividers will not be visible. It is necessary, as with the Abbe camera lucida (7% 182), to have the Wollaston prism properly arranged with reference to the axis of the microscope and the eye-point. If it is not, one will be unable to see the image well, and may be entirely unable to see the pencil and the image at the same time. Again, as rays from the micro- scope and from the drawing surface must enter independent parts of the pupil of the same eye, one must hold the eye so that the pupil is partly over the camera lucida and partly over the drawing surface. One can tell the proper position by trial. This is not a very satisfactory camera to draw with, but it is a very good form to measure the vertical distance of 250 mm. at which the drawing surface should be placed when determining magnification (7 162). $181. *Abbe Camera Lucida.—This consists of a cube of glass cut into two triangular prisms and silvered on the cut surface of the *For some persons the image and drawing surface, pencil, etc., do not appear on the drawing board as stated above, but under the microscope, according to the general principle that ‘objects appear in space where they could be touched along a perpendicular to the retinal surface stimulated,’’—that is in the line of rays entering the eye. This is always the case with the Wollaston camera lucida. The explanation of the apparent location of the image, etc., on the drawing board with the Abbe camera lucida is that the attention is concentrated upon the draw- ing surface rather than upon the object under the microscope (Dr. W. B. Pillsbury ). CHa || DRAWING WITH THE MICROSCOPE 125 upper one. A small oval hole is then cut out of the center of the sil- vered surface and the two prisms are cemented together in the form of the original cube with a perforated 45 degree mirror within it (Fig. 109, ab). The upper surface of the cube is covered by a perforated metal plate. This cube is placed over the ocular in such a way that the light from the microscope passes through the hole in the silvered face and thence directly to the eye. Light from the drawing surface is reflected by a mirror to the silvered surface of the prism and reflect- ed by this surface to the eye in company with the rays from the microscope, so that the two fields appear as one, and the image is seen as ifon the drawing surface (Figs. 109, 114). It is designed for use with a vertical micro- scope, but see § 184. Fic. 112. Wollaston’s Camera Lucida, showing the rays from the microscope and from the drawing sur- face, and the position of the pupil of the eye. For full explanation see Fig. 97. $182. Arrangement of the Camera Lucida Prism.—In plac- ing this camera lucida over the ocular for drawing or the determination of magnification, the center of the hole in the silvered surface is placed in the optic axis of the microscope. This is done by properly arrang- ing the centering screws that clamp the camera to the microscope tube or ocular. The perforation in the silvered surface must also be at the level of the eye-point. In other words the prism must be so arranged vertically and horizontally that the hole in the silvered surface will be in the axis of the microscope and coincident with the eye-point of the ocular. If it is above or below, or to one side of the eye-point, part or all of the field of the microscope will be cut off. As stated above, the centering screws are for the proper horizontal arrangement of the prism. The prism is set at the right height by the makers for the eye-point of a medium ocular. If one desires to use an ocular with the eye-point farther away or nearer, as in using high or low oculars the position of the eye-point may be determined as directd in 126 DRAWING WITH THE MICROSCOPE (CH. V § 59 and the prism loosened and raised or lowered to the proper level ; but in doing this one should avoid setting the prism obliquely to the mirror. In the latest and best forms of this camera lucida special arrange- ments have been made for raising or lowering the prism so that it may be used with equal satisfaction on oculars with the eye-point at differ- ent levels, and the prism is hinged to turn aside without disturbing the mirror. One can determine when the camera 1s in a proper position by looking into the microscope through it. If the field of the microscope appears as a circle and of about the same size as without the camera - lucida, then the prism is ina proper Fic. 113. One of the latest and position. If one side of the field is 4 naaad NGG Beate dark, then the prism is to one side vi of the center; if the field is consid- erably smaller than when the prism is turned off the ocular, it indicates that itis not at the correct level, 7. ¢., it is above or below the eye- point. § 183. Arrangement of the Mirror and the Drawing Surface. —The Abbe camera lucida was designed for use with a vertical micro- scope (Fig. 109). On a vertical microscope if the mirror is set at an angle of 45°, the axial ray will be at right angles with the table top or a drawing board which is horizontal, and a drawing made under these conditions will be in true proportion and not distorted. The stage of most microscopes, however, extends out so far at the sides that with a 45° mirror the image appears in part on the stage of the microscope. In order to avoid this the mirror may be depressed to some point below 45°, say at 4o° or 35°(Fig. 114). But as the axial ray from the mirror to the prism must still be reflected horizontally, it follows that the axial ray will no longer form an angle of go degrees with the drawing sur- face, but a greater angle. Ifthe mirror is depressed to 35°, then the axial ray must take an angle of 110° with a horizontal drawing surface (see the geometrical figure Fig. 114, A). To make the angle 90° again, so that there shall be no distortion, the drawing board must be raised toward the microscope 20°. The general rule is to raise the draw- ing board twice as many degrees toward the microscope as the mirror is depressed below 45°. Practically the field for drawing CH. V7} DRAWING WITH THE MICROSCOPE 127 B A Fic. 114. Cc bbe Camera Lucida in position to avoid distortion. Fic. 114.—The Abbe Camera Lucida with the mirror at 35°. Axis, Axis. Axial ray from the microscope and from the drawing surface. A.B. Drawing surface raised toward the microscope 20°. Foot. The foot or base of the microscope. Mirror with quadrant (OQ). The mirror ts seen to be at an angle of 35°. Ocular. Ocular of the Microscope. P.P. Drawing pencil and the cubical prism over the ocular. W. Wedge to support the drawing board. A. Geometrical figure of the preceding, showing the angles made by the axial ray with the mirror and the necessary elevation of the drawing board to avoid distortion. From the equality of opposite angles, the angle of the axial ray reflected at 35° must make an angle of 110° with a horizontal drawing board. The board must then be elevated toward the microscope 20° 1n order that the axial ray may be perpendicular to tt, and thus fulfill the requirements necessary to avoid distortion (% 179, 153). B. Upper view of the prism of the camera lucida, A considerable portion of the face of the prism ts covered, and the opening in the silvered surface appears oval. C. Quadrant to be attached to the mirror of the Abbe Camera Lucida to in- dicate the angle of the mirror. As the angle is nearly always 45°, go°, or 35°, only those angles are shown. 128 DRAWING WITH THE MICROSCOPE (CH. V can always be made free of the stage of the microscope, at 45°, at 40°, or at 35°. In the first case (45° mirror) the drawing surface should be horizontal, in the second case (40° mirror) the drawing surface should be elevated 10°, and in the third case (35° mirror) the drawing board should be elevated 20° toward the microscope. Furthermore it is necessary in using an elevated drawing board to have the mirror bar project directly laterally so that the edges of the mirror will be in planes parallel with the edges of the drawing board, otherwise there will be front to back distortion, although the elevation of the drawing board would avoid right to left distortion. If one has a micrometer ruled in squares (7et micrometer) the distortion produced by not having the axial ray at right angles with the drawing surface may be very strikingly shown. For example, set the mirror at 35° and use a hori- zontal drawing board. With a pencil make dots at the corners of some of the squares, and then with a straight edge connect the dots. The figures will be considerably longer from right to left than from front to back. Circles in the object would appear as ellipses in the drawings, the major axis being from right to left. The angle of the mirror may be determined with a protractor, but that is troublesome. It is much more satisfactory to have a quadrant attached to the mirror and an indicator on the projecting arm of the mirror. If the quadrant is graduated throughout its entire extent, or preferably at three points, 45°, 4o° and 35°, one can set the mirror at a known angle in a moment, then the drawing board can be hinged and the elevation of 10° and 20° determined with a protractor. The draw- ing board is very conveniently held up by a broad wedge. By marking the position of the wedge for 10° and 20° the protractor need be used but once, then the wedge may be put into position at any time for the proper elevation. §$ 184. Abbe Camera and Inclined Microscope.—It is very fatiguing to draw continuously with a vertical microscope, and many mounted objects admit of an inclination of the microscope, when one can sit and work in a more comfortable position. The Abbe camera is as perfectly adapted to use with an inclined as with a vertical micro- scope. All that is requisite is to be sure that the fundamental law is observed regarding the axialray of the image and the drawing surface, viz., that they should be at right angles. This is very easily accom- plished as follows : The drawing board is raised toward the microscope twice as many degrees as the mirror is depressed below 45° (§ 183), then it is raised exactly as many degrees as the microscope is inclined, CHa | DRAWING WITH THE MICROSCOPE 129 and in the same direction, that is, so the end of the drawing board shall be in a plane parallel with the stage of the microscope. The mirror must have its edges in planes parallel with the edges of the _ drawing board also (Figs. 115, 116.) Fic. 115. Arrangement of — 30° the drawing board for using the Ny i microscope in an inclined position with the Abbe camera lucida (de- rs a \ stgned by Mrs. S. P. Gage, 7887.) Ce ON \ Vans VE 2 $185. Drawing with the Abbe Camera Lucida.—(A) The light from the microscope and from the drawing surface should be of nearly equal intensity, so that the image and the drawing pencil can be seen with about equal distinctness. This may be accomplished with very low powers (16 mm. and lower objectives) by covering the mirror of the microscope with white paper when transparent objects are to be drawn. For high powers it is best to use a substage condenser. Often the light may be balanced by using a larger or smaller opening in the diaphragm. Onecan tell which field is excessively illuminated, for it is the one in which objects are most distinctly seen. If it is the microscopic, then the image of the microscopic object is very distinct and the pencil is invisible or very indistinct. Ifthe drawing surface is too brilliantly lighted the pencil can be seen clearly, but the micro- scopic image will be obscure. When opaque objects, that is objects which must be lighted with reflected light (§ 63), like dark colored insects, etc., are to be drawn the light must usually be concentrated upon the object in some way. The microscope may be placed in a very strong light and the drawing board shaded or the light may be concentrated upon the object by means of a concave mirror or a bull’s eye condenser (Fig. 53). If the drawing surface is too brilliantly illuminated, it may be shaded by placing a book or a ground glass screen between it and the window, also by putting one or more smoked glasses in the path of the rays from the mirror (Fig. 109 G). If the light in the microscope is too intense, it may be lessened by using white paper over the mirror, or by a ground glass screen between the microscope mirror and the source of light (Piersol, Amer. M. M. Jour., 1888, p. 103). It is also an excellent plan to blacken the end of the drawing pencil with carbon 130 DRAWING WITH THE MICROSCOPE (CH. V ink. Sometimes it is easier to draw on a black surface, using a white pencil or style. The carbon paper used in manifolding letters, etc., may be used, or ordinary black paper may be lightly rubbed on one side with a moderately soft lead pencil. Place the black paper over white paper and trace the outlines with a pointed style of ivory or bone. A corresponding dark line will appear on the white paper beneath. (Jour. Roy. Micr. Soc., 1883, p. 423). Fic. 116. Bernhard’s Drawing Board for the Abbe Camera Lucida. This drawing board is adjustable vertically, and the board may be inclined to prevent distortion. It is also arranged for use with an inclined microscope, having the base board hinged. Microscope and drawing surface are then inclined together. (Zeit. wiss. Mikroskopie, vol. vt (1894, p. 298). (Zeiss Catalog). (A) It is desirable to have the drawing paper fastened with thumb tacks, or in some other way. (B) The lines made while using the camera lucida should be very light, as they are liable to be irregular. (C) Only outlines are drawn and parts located with a camera lucida. Details are put in free-hand. (D) It is sometimes desirable to draw the outline of an object with a moderate power and add the details with a higher power. If this is done it should always be clearly stated. It is advisable to do this only with objects in which the same structure is many times duplicated, as a nerve or a muscle. In such an object all CH. V7} DRAWING WITH THE MICROSCOPE 131 the different structures could be shown, and by omitting some of the fibers the others could be made plainer without an undesirable enlarge- ment of the entire figure. (E) If a drawing of a given size is desired and it cannot be ob- tained by any combination of oculars, objectives and lengths of the tube of the microscope, the distance between the camera lucida and the table may be increased or diminished until the image is of the desired size. This distance is easily changed by the use of a book or a block, but more conveniently if one has a drawing board with adjust- able drawing surface like that shownin Fig. 116. The image of a few spaces of the micrometer will give scale of enlargement, or the power may be determined for the special case (§ 186-187). (F) It isof the greatest advantage, as suggested by Heinsius (Zeit. w. Mikr., 1889, p. 367), to have the camera lucida hinged so that the prism may be turned off the ocular for a moment’s glance at the prepa- ration, and then returned in place without the necessity of loosening screws and readjusting the camera. ‘This form is now made by several opticians, and the quadrant is added by some. (Fig. 114.) Any skilled mechanic can add the quadrant. § 186. Magnification of the Microscope and size of Draw- ings with the Abbe Camera Lucida.—In determining the standard distance of 250 millimeters at which to measure the image in getting the magnification of the microscope, it is necessary to measure from the point marked P on the prism (Fig. 109) to the axis of the mirror and then vertically to the drawing board. In getting the scale to which a drawing is enlarged the best way is to remove the preparation and put in its place a stage micrometer, and to trace a few (5 or 10) of its linés upon one corner of the drawing. The value of the spaces of the micrometer being given, thus : Ere iooth mm. Fic. 117. Showing the method of indicating the scale at which a drawing was made. The enlargement of the figure can then be accurately determined at any time by measuring with a steel scale the length of the image of the micrometer spaces and dividing it by their known size. Thus, suppose the 5 spaces of the scale of enlargement given with a drawing were found to measure 25 millimeters and the spaces on the 132 DRAWING WITH THE MICROSCOPE [GA Te micrometer were ;! th millimeter, then the enlargement would be 25 +7%5;—=500. That is, the image was drawn at a magnification of 500 diameters. If the micrometer scale is added to every drawing, there is no need of troubling one’s self about the exact distance at which the drawing is made, convenience may settle that, as the special magnifi- cation in each case may be determined from the scale accompanying the picture. It should be remembered, however, that the conditions when the scale is drawn must be exactly as when the drawing was made. $187. Drawing at Slight Magnification.—Some objects are of considerable size and for drawings should be enlarged but a few diame- ters,—5 to 20. By using sufficiently low objectives and different ocu- lars a great range may be obtained. Frequently, however, the range must be still further increased. For a moderate increase in size the drawing surface may be put farther off, or, as one more commonly needs less rather than greater magnification, the drawing surface may be brought nearer the mirror of the camera lucida by piling books or other objects on the drawing board. If one takes the precaution to draw a scale on the figure under the same conditions, its enlargement can be readily determined (§ 186). A very satisfactory way to draw at low magnifications is to use a simple microscope and arrange a camera lucida over it as over the ocular. In this way one may get drawings at almost any low magnification. If one has many large objects to draw ata = low magnification, then ; some form of embryo- graph is very conven- ient. (Jour. Roy. Micr., Soc., 1899, p. 223.) The writer has made use of a photographic camera and different photo- graphic objectives for the purpose. The object is Fic. 118. Camera lucida for drawing objects natural size. (H. Bausch Jour, Applied Microscopy, vol. tit (1900, p. 89r). illuminated as if for a photograph and in place of the ground glass a plain glass is used and on this some tracing paper isstretched. Noth- CH. V) . DRAWING WITH THE MICROSCOPE 133 ing is then easier than to trace the outlines of the object. See also Ch. VIII. REFERENCES Beale, 31, 355; Behrens, Kossel and Schiefferdecker, 77; Carpenter-Dallinger, 278 ; Van Heurck, 91; American Naturalist, 1886, p- 1071, 1887, pp. 1040-1043 ; Amer. Monthly Micr, Jour., 1888, p. 103, 1890, p. 94; Jour. Roy. Micr. Soc., r88r, p. 819, 1882, p. 402, 1883, pp. 283, 560, 1884, p. 115, 1886, p. 516, 1888, pp. 113, 809, 798; Zeit. wiss. Mikroskopie, 1884, pp. 1-21, 1889, p. 367, 1893, pp. 289-295. Here is described an excellent apparatus made by Winkel. Consult also the latest catalogs of the opticians. TY ATUL UT WVUA|MHE IH) ae ee ei HEEL TIPE WHEE 10 CENTIMETER RULE The upper edge is in millimeters, the lower in centimeters, and half centimeters. THE METRIC SYSTEM UNITS. The most commonly used divisions and multiples. Centimeter (c.m.), P-tooth Meter ; Af7lldmeter (m.m.), 1-toooth Meter: Micron d ff ee eee POR. (mw), I-loooth Millimeter ; the Micron is the unit in Micrometry (7 166). ry ( Kviometer, 1000 Meters; used in measuring roads and other long distances THE GRAM FOR \ Milligram (m.g.), 1-loooth Gram. WEIGHT. . . | Kilogram, 1000 “Grams, used for ordinary masses, like groceries, etc. THE LITER FOR} Cubic Centimeter (c.c.), 1-1oooth Liter. This is more common than the correct CAPACITY. . | form, Milliliter. Divistons of the Unzts are indicated by the Latin prefixes: dec?, I-toth; centt, 1-tooth ; Afzliz, 1-toooth ; Afzcro, 1-1,000,o00th of any unit. Multiples are designated by Greek prefixes: deka, 10 times: hecto, 100 times ; k7/o0, 1000 times myriad, 10,000 times ; A/ega, 1,000,000 times any uuit. CHAPTER VI MICRO-SPECTROSCOPE AND POLARISCOPE, MICRO-CHEMISTRY, MICRO-MATALLOGRAPHY, TEXTILE FIBERS APPARATUS AND MATERIAL REQUIRED FOR THIS CHAPTER Compound microscope ; Micro-spectroscope (7 188) ; Watch-glasses and small vials, slides and covers (% 207) ; Various substances for examination (as blood and ammonium sulphide, permanganate of potash, chlorophyll, some colored fruit, etc., (% 208-217); Micro-polarizer (% 218); Selenite plate (7 227); Various doubly refracting objects, as crystals, textile fibers, starch, section of bone; Various chemicals, metals, etc. MICRO-SPECTROSCOPE 2188. A Micro-Spectroscope, Spectroscopic or Spectral Ocular, is a direct vision spectroscope in connection with a microscope ocular. The one devised by Abbe and made by Zeiss consists of a direct vision spectroscope prism of the Amici pattern, and of considerable dispersion, placed over the ocular of the microscope. This direct vision or Amici prism consists of a single triangular prism of heavy flint glass in the middle and one of crown glass on each side, the edge of the crown glass prisnmis pointing toward the base of the flint glass prism, 7. ¢., the edge of the crown and flint glass prisms point in opposite directions. The flint glass prism serves to give the dispersion or separation into colors, while the crown glass prisms serve to make the emergent rays approximately parallel with the incident rays, so that one looks directly into the prism along the axis of the microscope. The Amici prism is in a special tube which is hinged to the ocular and held in position by a spring. It may be swung free of the ocular. In connection with: the ocular is the slit mechanism and a prism for reflecting horizontal rays verti- cally for the purpose of obtaining a comparison spectrum (% 201). Finally near the top is a lateral tube with mirror for the purpose of projecting an Angstrom scale of wave lengths upon the spectrum (7% 202, Figs. 119-120. ) 7189. Apparent Reversal of the Position of the Colors in a Direct Vision Spectroscope.—In accordance with the statements in 7 188 the dispersion or sepa- ration into colors is given by the flint glass prism or prisms and in accordance with the general law that the waves of shortest length, blue, ete., will be bent most, the colors have the position indicated in the top of Fig. 123, also above Fig. 119. But if one looks into the direct vision spectroscope or holds the eye close to the single prism (Fig. 124), the colors will appear reversed as if the red were more bent. The explanation of this is shown in Fig. 124, where it can be CH, VI] MICRO-SPECTROSCOPE AND POLARISCOPE 135 readily seen that if the eye is placed at E, close to the prism, the different colored rays appear in the direction from which they reach the eye and consequently are crossed in being projected into the field of vision and the real position is in- verted. The same is true in looking into the micro-spectroscope. The actual FIG. 11g. Abbe’s Micro-spectroscope. FIG. 120. 9 Longitudinal Section of Slit Mechanism separately. the whole instrument. (Plan view, Full size.) (1% Full size.) “The eye lens 1s adjustable so as toaccurately focus on the slit situated between the lenses. The mechanism for contracting and expanding the slit ts actuated by the screw Fand causes the laminae to move symmetrically (Merz’s movement). The slit may be made sufficiently wide so as to include the whole visual field. The screw Hf serves to limit the length of the slit so as to completely fill the latter with the image of the object under investigation when the comparison prism 1s inserted. The comparison prism ts provided with a lateral frame and clips to hold the object and the illuminating mirror. All these parts are encased in a drum on the ocular. Above the eve-piece ts placed an Amici prism of great dispersion which may be turned aside about the pivot K, so as to allow of the adjustment of the object. The prism is retained tn its axial position by the spring catch L. A scale ts projected on the spectrum by means of a scale tube and mirror attached to the prism casing. The divisions of the scale indicate in decimals of a micron the wave length of the respective section of the spectrum. The screw P serves to adjust the scale relative to the spectrum. The instrument ts inserted tn the tube in place of the ordinary eye-piece and 1s clamped to the former by means of the screw M in such a position that the mirrors A and O, which respectively serve to illuminate the comparison prism and the scale of wave lengths are stinultaneously illuminated.”’ (Cut loaned by Wm. Keratity Ne Ys) 136 MICRO-SPECTROSCOPE AND POLARISCOPE (CH. VI position of the different colors may be determined by placing some ground glass or some of the lens-paper near the prism and observing with the eye at the distance of distinct vision.* D E F G 4 nA ¢ £0 5,0 40 | (hal eet sl elle (aaron [eee jee = Solar I, © bh Shectru : s Sodium Lo s 1 an rR Perman. i ie ee i hl Ee Rotash, dt AA ra! © Es, Mer har. | 4 ! 5 Ma g robin iW oo a Fic. 121. Various Spectrums.—All except that of sodium were obtained by diffused day-light with the slit of such a width as gave the most distinct Fraunhofer lines. Lt frequently occurs that with a substance giving several absorption bands (e. g., chlorophyll) the density or thickness of the solution must be varied to show all the different bands clearly. Solar Spectrum.— With diffused day-light and a narrow slit the spectrum ts not visible much beyond the fixed line B. In order to extend the visible spectrum in the red to the line A, one should use direct sunlight and a piece of ruby glass in place of the watch-glass in Fig. 123. Sodium Spectrum.—The line spectrum (4191) of sodium obtained by lighting the microscope with an alcohol flame in which some salt of sodium ts glowing. With the micro-spectroscope the sodium line seen in the solar spectrum and with the incandescent sodium appears single, except under very favorable circumstances. (% 792). By using a comparison spectrum of day-light with the sodium spectrum the light and dark D-lines will be seen to be continuous as here shown. Permanganate of Potash.—This spectrum 1s characterized by the presence of five absorption bands in the middle of the spectrum and ts best shown by using a 4's per cent. solution of permanganate tn water in a watch-glass as in Fig. 123. Met-hemogtlobin.— The absorption spectrum of met-hemoglobin is characterized by a considerable darkening of the blue end of the spectrum and of four absorption bands, one in the red near the line C and two between D and E nearly in the place of the two bands of oxy-hemoglobin , finally there 7s a somewhat faint, wide, band near F. Such a met-hemoglobin spectrum is best obtained by making a solution of blood in water of such a concentration that the two oayv-hemoglobin bands run together (4 211), and then adding three or four drops of a +5 per cent. aqueous solution of permanganate of potash ora few drops of hydrogen dioxid (H,0,;). Soon the bright red will change to a brownish color, when tt may be examined. *The author wishes to acknowledge the aid rendered by Professor E. L. Nichols in giving the explanation offered in this section, CH. V1) MICRO-SPECTROSCOPE AND POLARISCOPE 137 VARIOUS KINDS OF SPECTRA By a spectrum is meant the colored bands appearing when the light traverses a dispersing prism or a diffraction grating, or is affected in any way to separate the different wave lengths of light into groups. When daylight or some good artificial light is thus dispersed one gets the appearance so familiar in the rainbow. @ 190. Continuous Spectrum.—In case a good artificial light as the electric light is used the various rainbow or spectral colors merge gradually into one another in passing from end to end of the spectrum. There are no breaks or gaps. @ 191. Line Spectrum.—Ifa gas is made incandescent, the spectrum it pro- duces consists, not of the various rainbow colors, but of sharp, narrow, bright lines, the color depending on the substance. Allthe rest of the spectrum is dark. These line spectra are very strikingly shown by various metals heated till they are in the form of incandescent vapor. 4192. Absorption Spectrum.—By this is meant a spectrum in which there are dark lines or bands in the spectrum. The most striking and interesting of the absorption spectra is the Solar Spectrum, or spectrum of sunlight. If this isexam- ined carefully it will be found to be crossed by dark lines, the appearance being as if one were to draw pen marks across a continuous spectrum at various levels, sometimes apparently between the colors and sometimes in the midst of a color. These dark lines are the so-called Fraunhofer Lines. Some of the principal ones have been lettered with Roman capitals, A, B, C, D, E, F, G, H, commencing at the red end. The meaning of these lines was for a long time enigmatical, but it is now known that they correspond with the bright lines of a line spectrum (§ 191). For example, if sodium is put in the flame of a spirit lamp it will vaporize and become luminous. If this light is examined there will be seen one or two bright yellow bands corresponding in position with D of the solar spectrum (Fig. 121). If now the spirit-lamp flame, colored by the incandescent sodium, is placed in the path of the electric light, and it is examined as before, there will be a continuous spectrum, except for dark lines in place of the bright sodium lines. That is, the comparatively cool yellow light of the spirit lamp cuts off or absorbs the intensely hot yellow light of the electric light ; and although the spirit flame sends a yellow light to the spectroscope it is so faint in comparison with the electric light that the sodium lines appear dark. It is believed that in the sun’s atmosphere there are incandescent metal vapors (sodium, iron, etc.), but that they are so cool in com- parison with the rays of their wave length in the sun that the cooler light of the incandescent metallic vapors absorb the light of corresponding wave length, and are, like the spirit lamp-flame, unable to make up the loss, and therefore the pres- ence of the dark lines. 7193. Absorption Spectra from Colored Substances.—While the solar spec- trum is an absorption spectrum, the term is more commonly applied to the spectra obtained with light which has passed through or has been reflected from colored objects which are not self-luminous. It is the special purpose of the micro-spectroscope to investigate the spectra of colored objects which are not self-luminous, 7. ¢., blood and other liquids, various minerals, as monazite, etc. The spectra obtained by examining the light reflected from these colored bodies or transmitted through them, possess, like the solar 138 MICRO-SPECTROSCOPE AND POLARISCOPE (CH. VI spectrum dark lines or bands, but the bands are usually much wider and less sharply defined. Their number and position depend on the substance or its con- stitution (Fig. 122), and their width, in part, upon the thickness of the body. With some colored bodies, no definite bands are present. The spectrum is simply restricted at one or both ends and various of the other colors are considerably lessened in intensity. This is true of many colored fruits. 2194. Angstr6m and Stokes’ Law of Absorption Spectra.—The waves of light absorbed by a body when light is transmitted through some of its substance Rel a 40r ngep yell =. i Fic. 122. Absorption spectrum of Oxy-hemoglobin or arterial blood (1) and of Homoglobin or venous blood (2). (From Gamgee and McMunn). dA, B,C, D, E, F, G, H. Some of thé Prinipal Fraunhofer lines of the solar spectrum (% 192). .90, SO, .70, .60, .50, .4o. Wave lengths in microns, as shown in Angstrom’s scale (4.202). Lt will be seen that the wave lengths increase toward the red and decrease toward the violet end of the spectrum. Red, Yellow, Orange, etc. Color regions of the spectrum. Indigo should come between the blue and the violet to complete the seven colors usually given. Lt was omitted through inadvertence. . are precisely the waves radiated from it when it becomes self-luminous. For ex- ample, a piece of glass that is yellow when cool, gives out blue light when it is hot enough to be self-luminous. Sodium vapor absorbs two bands of yellow light (D lines); but when light is not sent through it, but itself is luminous and examined as a source of light its spectrum gives bright sodium lines, all the rest of the spectrum being dark (Fig. 121). 2195. Law of Color.—The light reaching the eye from a colored, solid, liquid or gaseous body lighted with white light, will be that due to white light less the light waves that have been absorbed by the colored body. Or in other words, it will be due to the wave lengths of light that finally reach the eye from the ob- ject. For example, a thin layer of blood under the microscope will appear yellowish green, but a thick layer will appear pure red. If now these two layers are examined with a micro-spectroscope, the thin layer will show all the colors, but the red end will be slightly, and the blue end considerably restricted, and some of the colors will appear considerably lessened in intensity. Finally there may ap- pear two shadow-like bands, or if the layer is thick enough, two well-defined dark bands in the green (7 210). If the thick layer is examined in the same way, the spectrum will show only red with a little orange light, all the rest being absorbed. Thus the spectroscope shows which colors remain, in part or wholly, and it is the mixture of this remain- ing or unabsorbed light that gives color to the object. CAH. VI) MICRO-SPECTROSCOPE AND POLARISCOPE 139 2196. Complementary Spectra.—While it is believed that Angstrém’s law (2 194) is correct, there are many bodies on which it cannot be tested, as they change in chemical or molecular constitution before reaching a sufficiently high temperature to become luminous. There are compounds, however, like those of didymium, erbium and terbium, which do not change with the heat necessary to render them luminous, and with them the incandescence and absorption spectra are mutually complementary, the one presenting bright lines where the other presents dark ones (Daniell). ADJUSTING THE MICRO-SPECTROSCOPE $197. The micro-spectroscope, or spectroscopic ocular, is put in the place of the ordinary ocular in the microscope, and clamped to the top of the tube by means of a screw for the purpose. S$ 198. Adjustment of the Slit.—In place of the ordinary dia- phragm with circular opening, the spectral ocular has a diaphragm composed of two movable knife edges by which a slit-like opening of greater or less width and length may be obtained at will by the use of screws for the purpose. To adjust the slit, depress the lever holding the prism-tube in position over the ocular, and swing the prism aside. One can then look into the ocular. The lateral screw should be used and the knife edges approached till they appear about half a millimeter apart. If now the Amici prism is put back in place and the micro- scope well lighted, one will see a spectrum by looking into the upper end of the spectroscope. If the slit is too wide, the colors will overlap in the middle of the spectrum and be pure only at the red and blue ends; and the Fraunhofer or other bands in the spectrum will be faint or invisible. Dust on the edges of the slit gives the appearance of longitudinal streaks on the spectrum. $199. Mutual Arrangement of Shit and Prism.—In order that the spectrum may appear as if made up of colored bands going directly across the long axis of the spectrum, the slit must be parallel with the refracting edge of the prism. If the slit and prism are not thus mutually arranged, the colored bands will appear oblique, and the whole spectrum may be greatly narrowed. If the colored bands are oblique, grasp the prism tube and slowly rotate it to the right or to the left until the various colored bands extend directly across the spec- trum. $200. Focusing the Slit.—In order that the lines or bands in the spectrum shall be sharply defined, the eye-lens of the ocular should be accurately focused on the slit. The eye-lens is movable, and when the prism is swung aside it is very easy to focus the slit as one focused 140 MICRO-SPECTROSCOPE AND POLARISCOPE [CH VI for the ocular micrometer (§ 172). If one now uses daylight there will be seen in the spectrum the dark Fraunhofer lines (Fig. 121 E. F., ete.); [e 1 sleet hr | tl dy Re Reestall Se! [ic 3 | Ct FIG. 123. FIG. 124. FIG. 125. FIG. 123. (1). Section of the tube and slage of the microscope with the spectral ocular or micro-speclroscope in position, Amict Prism (4188).—The direct vision prism of Amici in which the central shaded prism of flint glass gives the dispersion or separation into colors, while the CHAE] MICRO-SPECTROSCOPE AND POLARISCOPE I4I end prisms of crown glass cause the rays to emerge approximately parallel with the axis of the microscope. A single ray is represented as entering the prism and this 1s divided into three groups (Red, Yellow, Blue), which emerge from the prism, the red being least and the blue most bent toward the base of the flint prism (see Fig. 124). Flinge.—The hinge on which the prism tube turns when it ts swung off the ocular. Ocular (2 188)—The ocular in which the slit mechanism takes the place of the diaphragm (§ 198). The eye-lens ts movable as in a micrometer ocular, so that the slit may be accurately focused for the different colors (4% 200). S. Screw for setting the scale of wave lengths (4% 202). S’. Screw for regulating the width of the slit ( 4198). SS’, Screw for clamping the micro-spectroscope to the tube of the microscope. Scale Tube.—The tube near the upper end containing the Angstrom scale and the lenses for projecting the image upon the upper face of the Amici prism, whence it ts reflected upward to the eye with the different colored rays. At the right ts a special mirror for lighting the scale. Slit.—The linear opening between the knife edges. Through the slit the light passes to the prism. It must be arranged parallel with the refracting edge of the prism, and of such a width that the Fraunhofer or Fixed Lines are very clearly and sharply defined when the eye-lens 1s properly focused (% 198-200). Stage.— The stage of the microscope. This supports a watch-glass with sloping sides for containing the colored liquid to be examined. (3) Comparison Prism with tube for colored liquid (C. L.), and mirror. The prism refiects horizontal rays vertically, so that when the prism its made to cover part of the slit two parallel spectra may be seen, one from light sent directly through the entire microscope and one from the light reflected upward from the comparison prism. (4) View of the Slit Mechanism from below.—Slit, the linear space between the knife edges through which the light passes. P. Comparison prisnt beneath the slit and covering part of tt at will. S. SS’. Screws for regulating the length and width of the slit. Fic. 124. Flint-Glass Prism showing the separation or dispersion of white light into the three groups of colored rays (Red, Yellow, Blue), the blue rays being bent the most from the refracting edge (% 189). Fic. 125. Sectional View of a Microscope with the Polariscope in position (4218-227 ). Analyzer and Polarizer.—They are represented with corresponding faces par- allel so that the polarized beam could traverse freely the analyzer. If either Nicol were rotated 90° they would be crossed and no light would traverse the analyzer unless some polarizing substance were used as object. (a) Slot in the analyzer tube so that the analyzer may be raised or lowered to adjust it for difference of level of the eye-point in different oculars (4% 59, 200). Pointer and Scale.—The pointer attached to the analyzer and the scale or divided circle clamped (by the screw S) to the tube of the microscope. The pointer and scale enable one to determine the exact amount of rotation of the analyzer (% 220). Object-— The object whose character 1s to be investigated by polarized light. 142 MICRO-SPECTROSCOPE AND POLARISCOPE (CH. VI To show the necessity of focusing the slit, move the eye-lens down or up as far as possible, and the Fraunhofer lines cannot be seen. While looking into the spectroscope move the ocular lens up or down, and when it is focused the Fraunhofer lines will reappear. As the dif- ferent colors of the spectrum have different wave lengths, it is neces- sary to focus the slit for each color if the sharpest possible pictures are desired. It will be found that the eye-lens of the ocular must be farther from the slit for the sharpest focus of the red end than for the sharpest focus of the lines at the blue end. This is because the wave length of red is markedly greater than for blue light. Longitudinal dark lines of the spectrum may be due to irregular- ity of the edge of the slit or to the presence of dust. They are most troublesome with a very narrow slit. § 201. Comparison or Double Spectrum.—In order to com- pare the spectra of two different substances it is desirable to be able to examine their spectra side by side. This is provided for in the better forms of micro-spectroscopes by a prism just below the slit, so placed that the light entering it from a mirror at the side of the drum shall be totally reflected in a vertical direction, and thus parallel with the rays from the microscope. The two spectra will be side by side with a narrow dark line separating them. If now the slit is well focused and daylight be sent through the microscope and into the side to the reflect- ing or comparison prism, the colored bands and the Fraunhofer dark lines will appear directly continuous across the two spectra. The prism for the comparison spectrum is movable and may be thrown en- tirely out of the field if desired. When it is to be used, it is moved about half way across the field so that the two spectra shall have about the same width. S$ 202. Scale of Wave Lengths.—In the Abbe micro-spectro- scope the scale is in a separate tube near the top of the prism and at right angles to the prism-tube. A special mirror serves to light the scale, which is projected upon the spectrum by a lens in the scale-tube. This scale is of the Angstrom form, and the wave lengths of any part of the spectrum may be read off directly, after the scale is once set in the proper position, that is, when it is set so that any given wave length on the scale is opposite the part of the spectrum known by pre- vious investigation to have that particular wave length. The point most often selected for setting the scale is opposite the sodium line where the wave length is, according to Angstrom, 0.5892 4. In ad- CHNV-AL| MICRO-SPECTROSCOPE AND POLARISCOPE 143 justing the scale, one may focus very sharply the dark sodium line of the solar spectrum and set the scale so that the number 0.589 is oppo- site the sodium or D line, or a method that is frequently used and serves to illustrate § 191-2, is to sprinkle some salt of sodium (carbon- ate of sodium is good) in an alcohol lamp flame and to examine this flame. If this is done in a darkened place with a spectroscope, a narrow bright band will be seen in the yellow part of the spectrum. If now ordinary daylight is sent through the comparison prism, the bright line of the sodium will be seen to be directly continuous with the dark line at D in the solar spectrum (Fig. 121). By reflecting light into the scale-tube the image of the scale will appear on the spectrum, and by a screw just under the scale-tube but within the prism-tube, the proper point on the scale (0.589 mw) can be brought opposite the sodium band. All the scale will then give the wave lengths directly. Sometimes the scale is oblique to the spectrum. This may be remedied by turning the prism-tube slightly one way or the other. It may be due to the wrong position of the scale itself. If so, grasp the milled ring at the distal end of the scale-tube and, while looking into the spectroscope, rotate the tube until the lines of the scale are parallel with the Fraunhofer lines. It is necessary in adjusting the scale to be sure that the larger number, 0.70, is at the red end of the spectrum. The numbers on the scale should be very clearly defined. If they do not so appear, the scale-tube must be focused by grasping the outer tube of the scale-tube and moving it toward or from the prism-tube until the scale is distinct. In focusing the scale, grasp the outer scale- tube with one hand and the prism-tube with the other, and push or pull in opposite directions. In this way one will be less liable to injure the spectroscope. §$ 203. Designation of Wave Length.—Wave lengths of light are designated by the Greek letter 1, followed by the number indicat- ing the wave length in some fraction of a meter. With the Abbe micro-spectroscope the micron is taken as the unit as with other micro- scopical measurements ($ 166). Various units are in use, as the one hundred thousandth of a millimeter, millionths or ten millionths of a millimeter. If these smaller units are taken, the wave lengths will be indicated either as a decimal fraction of a millimeter or as whole num- bers. Thus, according to Angstrom, the wave length of sodium light is 5892 ten millionths mm., or 589.2 millionths, or 58.92 one hundred thousandths, or 0.5892 one thousandth mm., or 0.5892 mt. The last would be indicated thus, 4 D = 0.5892 yp. Id4 MICRO-SPECTROSCOPE AND POLARISCOPE [CH VI §$ 204. Lighting for Micro-spectroscope.—For opaque objects a strong light should be thrown on them either with a concave mirror or a condensing lens. For transparent objects the amount of the sub- stance and the depth of color must be considered. As a general rule it is well to use plenty of light, as that from an Abbe illuminator with a large opening in the diaphragm, or with the diaphragm entirely open. For very small objects and thin layers of liquids it may be better to use less light. One must try both methods in a given case, and learn by experience. The direct and the comparison spectra should be about equally illuminated. One can manage this by putting the object requiring the greater amount of illumination on the stage of the microscope and lighting it with the Abbe illuminator. In lighting it is found in gen- eral that for red or yellow objects, lamp-light gives very satisfactory results. For the examination of blood and blood crystals the light from a petroleum lamp is excellent. For objects with much blue or violet, daylight or artificial light rich in blue light is best. Furthermore, one should be on his guard against confusing the ordinary absorption bands with the Fraunhofer lines when daylight is used. With lamp-light the Fraunhofer lines are absent and, therefore, not a source of possible confusion. §$ 205. Objectives to Use with the Micro-spectroscope.—If the material is of considerable bulk, a low objective (16 to 50 mm.) is to be preferred. This depends on the nature of the object under ex- amination, however. In case of individual crystals one should use sufficient magnification to make the real image of the crystal entirely fill the width of the slit. The length of the slit may then be regulated by the screw on the side of the drum, and also by the comparison prism. If the object does not fill the whole slit the white light enter- ing the spectroscope with the light from the object might obscure the absorption bands. For opaque objects illuminating objectives are useful (§ 28, 233.) In using high objectives with the micro-spectroscope one must very carefully regulate the light ( Ch. II) and sometimes shade the object. § 206. Focusing the Objective.—For focusing the objective the prism-tube is swung aside, and then the slit made wide by turning the adjusting screw at the side. If the slit is open one can see objects when the microscope is focused as with an ordinary ocular. After an object is focused, it may be put exactly in position to fill the slit of the CHE FT] MICRO-SPECTROSCOPE AND POLARISCOPE 145 spectroscope, then the knife edges are brought together till the slit is of the right width ; if the slit is then too long it may be shortened by using one of the mechanism screws on the side, or if that is not sufh- cient, by bringing the comparison prism farther over the field. If one now replaces the Amici prism and looks into the microscope, the spectrum is liable to have longitudinal shimmering lines. To get rid of these focus up or down a little so that the microscope will be slightly out of focus. $207. Amount of Material Necessary for Absorption Spectra and its Proper Manipulation.—The amount of material necessary to give an absorption spectrum varies greatly with different substances, and can be determined only by trial. It a transparent solid is under investigation it is well to have it in the form a wedge, then succes- sive thicknesses can be brought under the microscope. Ifa liquid sub- stance is being examined, a watch glass with sloping sides forms an excellent vessel to contain it, then successive thicknesses of the liquid can be brought into the field as with the wedge-shaped solid. Fre- quently only a very weak solution is obtainable ; in this case it can be placed in a homeceopathic vial, or in some glass tubing sealed at the end, then one can look lengthwise through the liquid and get the effect of a more concentrated solution. For minute bodies like crystals or blood corpuscles, one may proceed as described in the previous section. MICRO-SPECTROSCOPE—EXPERIMENTS* $ 208. Put the micro-spectroscope in position, arrange the slit and the Amici prism so that the spectrum will show the various spec- tral colors going directly across it (§ 198-199) and carefully focus the slit. This may be done either by swinging the prism-tube aside and proceeding as for the ocular micrometer (§ 172), or by moving the eye-lens of the ocular up and down while looking into the micro- spectroscope until the dark lines of the solar spectrum are distinct. If they cannot be made distinct by focusing the slit, then the light is too feeble or the slit is too wide (§ 198). With the lever move the com- parison prism across half the field so that the two spectra shall be of about equal width. For lighting, see § 204. *If one does not possess a micro-spectroscope, quite satisfactory results may be obtained by using a microscope with a 16 to 12 mm. objective and a pocket, direct- vision spectroscope in place of the eye-piece. (Bleile, Trans. Amer. Micr. Soc. 1900, p. 8). 146 MICRO-SPECTROSCOPE AND POLARISCOPE (CH. VI § 209. Absorption Spectrum of Permanganate of Potash.— Make a solution of permanganate of potash in water of such a strength that a stratum 3 or 4 mm. thick is transparent. Put this solution in a watch-glass with sloping sides, and put it under the microscope. Use a 50 mm. or 16 mm. objective, and use the full opening of the illumi- nator. Light strongly. Look into the spectroscope and slowly move the watch-glass into the field. Note carefully the appearance with the thin stratum of liquid at the edge and then as it gradually thickens on moving the watch-glass still farther along. Count the absorption bands and note particularly the red and blue ends. Compare carefully with the comparison spectrum (Figs. 121, 122). For strength of solu- tion see § 207. $210. Absorption Spectrum of Blood.—Obtain blood from a recently killed animal, or flame a needle, and after it is cool prick the finger two or three times in a small area, then wind a handkerchief or a rubber tube around the base of the finger, and squeeze the finger with the other hand. Some blood will ooze out of the pricks. Rinse this off into a watch- glass partly filled with water. Continue to add the blood until the water is quite red. Place the watch-glass of diluted blood un- der the microscope in place of the permanganate, using the same object- ive, etc. Note carefully the spectrum. It would be advantageous to determine the wave length opposite the center of the dark bands. This may easily be done by setting the scale properly as described in § 202. Make another preparation, but use a homeopathic vial instead of a watch-glass. Cork the vial and lay it down upon the stage of the microscope. Observe the spectrum. It will be like that in the watch- glass. Remove the cork and look through the whole length of the vial. The bands will be much darker, and if the solution is thick enough only red and a little orange will appear. Re-insert the cork and incline the vialso that the light traverses a very thin layer, then gradually elevate the vial and the effect of a thicker and thicker layer may be seen. Note especially that the two characteristic bands unite and form one wide band as the stratum of liquid thickens. Compare with the following : Add to the vial of diluted blood a drop or two of ammonium sul- phide, such as is used for a reducing agent in chemical laboratories. Shake the bottle gently and then allow it to stand for ten or fifteen minutes. Examine it and the two bands will have been replaced by a single, less clearly defined band in about the same position. The blood will also appear somewhat purple. Shake the vial vigorously CAH. VI) MICRO-SPECTROSCOPE AND POLARISCOPE 147 and the color will change to the bright red of fresh blood. Examine it again with the spectroscope and the two bands will be visible. After five or ten minutes another examination will show but a single band. Incline the bottle so that a thin stratum may be examined. Note that the stratum of liquid must be considerably thicker to show the absorption band than was necessary to show the two bands in the first experiment. Furthermore, while the single band may be made quite black on thickening the stratum, it will not separate into two bands with a thinner stratum. In this experiment it is very instructive to have a-second vial of fresh diluted blood, say that from the watch- glass, before the opening of the comparison prism. The two banded spectrum will then be in position to be compared with the spectrum of the blood treated with the ammonium sulphide. The two banded spectrum is that of oxy-hemog/obin, or arterial blood, the single banded spectrum of hemoglobin (sometimes called reduced hemoglobin) or venous blood, that is, the respiratory oxygen is present in the two banded spectrum but absent from the single banded spectrum. When the bottle was shaken the hemoglobin took up oxygen from the air and became oxy-hemoglobin, as occurs in the lungs, but soon the ammonium sulphide took away the respiratory oxygen, thus reducing the oxy-hemoglobin to hemoglobin. This may be repeated many times (Fig. 122). § 211. Met-Hemoglobin.—The absorption spectrum of met- hemoglobin is characterized by a considerable darkening of the blue end of the spectrum and of four absorption bands, one in the red near the line C and two between D and E, nearly in the place of the two bands of oxy-hemoglobin ; finally there is a somewhat faint, wide band near F. Sucha met-hemoglobin spectrum is best obtained by making a solution of blood in water of such a concentration that the two oxy-hemoglobin bands run together (§ 210), and then adding three or four drops of a 75 per cent. aqueous solution of permanganate of potash. Soon the bright red will change to a brownish color, when it may be examined (Fig. 121). Instead of the permanganate one may use hydrogen dioxide (H,0O,). § 212. Carbon Monoxide Hemoglobin (CO-Hemoglobin).— To obtain this, kill an animal in illuminating gas, or one may allow illuminating gas to bubble through some blood already taken from the body. The gas should bubble through a minute ortwo. The oxygen will be displaced by carbon monoxide. This forms quite a stable com- pound with hemoglobin, and is of a bright cherry-red color. Its 148 MICRO-SPECTROSCOPE AND POLARISCOPE (CH. V1 spectrum is nearly like that of oxy-hemoglobin, but the bands are farther toward the blue. Add several drops of ammonium sulphide and allow the blood to stand some time. No reduction will take place, thus forming a marked contrast to solutions of oxy-hemoglobin. By the addition of a few drops of glacial acetic acid a dark brownish red color is produced. §$ 213. Carmine Solution.—Make a solution of carmine by put- ting jth gram of carmine in 100 cc. of water and adding 1o drops of strong ammonia. Put some of this in a watch-glass or in a small vial and compare the spectrum with that of oxy-hemoglobin or carbon monoxide hemoglobin. It has two bands in nearly the same position, thus giving the spectrum a striking similarity to blood. If now several drops, 15 or 20, of glacial acetic acid are added to the carmine, the bands remain and the color is not markedly changed, while with either oxy-hemoglobin or CO-hemoglobin the color would be de- cidedly changed from the bright red to a dull reddish brown, and the spectrum, if any could be seen, would be markedly different. Carmine and O-hemoglobin can be distinguished by the use of ammo- nium sulphide, the carmine remaining practically unchanged while the blood shows the single band of hemoglobin (§ 210). The acetic acid serves to differentiate the CO-hemoglobin as well as the O-hemoglobin. $214. Colored Bodies not giving Distinétly Banded Absorp- tion Spectra.—Some quite brilliantly colored objects, like the skin of a red apple, do not give a banded spectrum. Take the skin of a red apple, mount it on a slide, put on a cover-glass and add a drop of water at the edge of the cover. Put the preparation under the micro- scope and observe the spectrum. Although no bands will appear, in some cases at least, yet the ends of the spectrum will be restricted and various regions of the spectrum will not be so bright as the comparison spectrum. Here the red color arises from the mixture of the unab- sorbed waves, as occurs with other colored objects. In this case, however, not all the light of a given wave length is absorbed, conse- quently there are no clearly defined dark bands, the light is simply less brilliant in certain regions and the red rays so predominate that they give the prevailing color. $215. Nearly Colorless Bodies with Clearly Marked Ab- sorption Spectra.—In contradistinction to the brightly colored objects with no distinct absorption bands are those nearly colorless bodies and solutions which give as sharply defined absorption bands as could be desired. The best examples of this are afforded by solutions CH. VI] MICRO-SPECTROSCOPE AND POLARISCOPE 149 of the rare earths, didymium, etc. These in solutions that give hardly a trace of color to the eye give absorption bands that almost rival, the Fraunhofer lines in sharpness. $216. Absorption Spectra of Minerals.—As example take some monazite sand on a slide and either mount it in balsam (see § 256), or cover and add a drop of water. The examination may be made also with the dry sand, but it is less satisfactory. Light well with trans- mitted light, and move the preparation slowly around. Absorption bands will appear occasionally. Swing the prism tube off the ocular, open the slit and focus the sand. Get the image of one or more grains directly in the slit, then narrow and shorten the slit so that no light can reach the spectroscope that has not traversed the grain of sand. The spectrum will be satisfactory under such conditions. It is frequently of great service in determining the character of unknown mineral sands to compare the spectra with known minerals. If the absorption bands are identical, it is strong evidence in favor of the identity of the minerals. For proper lighting see § 204. § 217. While the study of absorption spectra gives one a great deal of accurate information, great caution must be exercised in draw- ing conclusions as to the identity or even the close relationship of bodies giving approximately the same absorption spectra. The rule followed by the best workers is to have a known body as control and to treat the unknown body and known body with the same reagents, and to dissolve them in the same medium. If all the reactions are identical then the presumption is strong that the bodies are ident- ical or very closely related. For example, while one might be in doubt between a solution of oxy- or CO-hemoglobin and carmine, the addition of ammonium sulphide serves to change the double to a single band in the O-hemoglobin, and glacial acetic acid enables one to distinguish between the CO-blood and the carmine, although the ammonium sul- phide would not enable one to make the distinction. Furthermore it is unsafe to compare objects dissolved in different media. The same objects as ‘‘cyanine and aniline blue dissolved in alcohol give a very similar spectrum, but in water a totally different one.’’ ‘‘Totally dif- ferent bodies show absorption bands in exactly the same position (solid nitrate of uranium and permanganate of potash in the blue).’’ (Mac- Munn). The rule given by MacMunn is a good one: ‘‘The recogni- tion of a body becomes more certain if its spectrum consists of several absorption bands, but even the coincidence of these bands with those of another body is not sufficient to enable us to infer chemical identity; 150 MICRO-SPECTROSCOPE AND POLARISCOPE (CH. VI what enables us to do so with certainty is the fact: that the two solu- tions give bands of equal intensities in the same parts of the spectrum which undergo analogous changes on the addition of the same reagent.”’ REFERENCES TO THE MICRO-SPECTROSCOPE AND SPECTRUM ANALYSIS The micro-spectroscope is playing an ever-increasingly important role in the spectrum analysis of animal and vegetable pigments, and of colored mineral and chemical substances, therefore a somewhat extended reference to literature will be given. Full titles of the books and periodicals will be found in the Bibliography at the end. Angstrom, Recherches sur le spectre solaire, etc. Also various papers in periodicals. See Royal Soc’s Cat’l Scientific Papers ; Anthony & Brackett ; Beale, p. 269; Behrens, p. 139; Kossel und Schiefferdecker, p. 63; Carpenter, p. 323; Browning, How to Work with the Spectroscope, and in Monthly Micr. Jour., II, p- 65; Daniell, Principles of Physics. The general principles of spectrum analysis are especially well stated in this work, pp. 435-455; Davis, p. 342; Dippel, p. 277; Frey; Gamgee, p. 91; Halliburton; Hogg, p. 122; also in Monthly Micr. Jour., Vol. II, on colors of flowers; Jour, Roy. Micr. Soc., 1880, 1883, and in various other vols.; Kraus; Lockyer; M’Kendrick; MacMunn; and also in Philos, Trans. R. S., 1886; various vols. of Jour. Physiol.; Nageli und Schwend- ener ; Proctor; Ref. Hand-Book Med. Science, Vol. I, p. 577, VI, p. 516, VII, p. 426; Roscoe; Schellen; Sorby, in Beale, p. 269; also Proc. R. S., 1874, p. 31, 1867, p. 433; see also in the Scientific Review, Vol. V, p. 66, Vol. II, p. 419. The larger works on Physiology, Chemistry and Physics may also be consulted with profit. Vogel, Spectrum analysis ; also in Nature, Vol. xix, p. 495, on absorption spec- tra. The bibliography in MacMunn is excellent and extended. For hemochromogen in medico-legal cases see Bleile, Trans. Amer. Micr. Soc., 1900, p. 9. MICRO-POLARISCOPE @ 218. The micro-polariscope, or polarizer, is a polariscope used in connection with a microscope. The most common and typical form consists of two Nicol prisms, that is, two somewhat elongated rhombs of Iceland spar cut diagonally and cemented together with Canada balsam. These Nicol prisms are then mounted in such a way that the light passes through them lengthwise, and in passing is divided into two rays of plane polarized light. The one of these rays obeying most nearly the ordinary law of refraction is called the ordinary ray, the one departing farthest from the law is called the extra-ordinary ray. These two rays are not only polarized, but polarized in planes almost exactly at right angles to each other. The Nicol prism totally reflects the ordinary ray at the cemented surface as it meets that surface at an angle greater than the critical angle, and only the extraordinary or less refracted ray is transmitted. CH VI) MICRO-SPECTROSCOPE AND POLARISCOPE I51 % 219. Polarizer and Analyzer.—The polarizer is one of the Nicol prisms. It is placed beneath the object and in this way the object is illuminated with polarized light. The analyzer is the other Nicol and is placed at some level above the object, very conveniently above the ocular. When the corresponding faces of the polarizer and analyzer are parallel 7. ¢., when the faces through which the oblique section passes are parallel, light passes freely through the analyzer to the eye. If these corresponding faces are at right angles, that is, if the Nicols are crossed, then the light is entirely cut off and the two transparent prisms become opaque to ordinary light. There are then, in the complete revolution of the analyzer, two points, at o° and 180°, where the corre- sponding faces are parallel and where light freely traverses the analyzer. There are alsostwo crossing points of the Nicols, at 90° and 270°, where the light is extin- guished. In the intermediate points there is a sort of twilight. 4220. Putting the Polarizer and Analyzer in Position.—Swing the diaphragm carrier of the Abbe illuminator out from under the illuminator, remove the disk diaphragm or open widely the iris diaphragm and place the analyzer in the dia- phragm carrier, then swing it back under the illuminator. Remove the ocular, put the graduated ring on the top of the tube and then replace the ocular and put the analyzer over the ocular andring. Arrange the graduated ring so that the indi- cator shall stand at o° when the field is lightest. This may be done by turning the tube down so that the objective is near the illuminator, then shading the stage so that none but polarized light shall enter the microscope. Rotate the analyzer until the lightest possible point is found, then rotate the graduated ring till the index stands at 0°. The ring may then be clamped to the tube by the side screw for the purpose. Or, more easily, one may set the index at 0°, clamp the ring to the microscope, then rotate the draw-tube of the microscope till the field is lightest. 4221. Adjustment of the Analyzer.—The analyzer should be capable of moving up and down in its mounting, so that it can be adjusted to the eye-point of the ocular with which it is used. If on looking into the analyzer with parallel Nicols the edge of the field is not sharp, or if it is colored, the analyzer is not in a proper position with reference to the eye-point, and should be raised or lowered till the edge of the field is perfectly sharp and as free from color as the ocular with the analyzer removed. 2222. Objectives to Use with the Polariscope.— Objectives of the lowest pow- ers may be used, and also all intermediate forms up toa 2 mm. homogeneous im- mersion. Still higher objectives may be used if desired. In general, however, the lower powers are somewhat more satisfactory. A good rule to follow in this case is the general rule in all microscopic work,—uwse the power that most clearly and satisfactorily shows the object under investigation. 4223. Lighting for Micro-Polariscope Work.—Follow the general directions given in Chapter II. It is especially necessary to shade the object so that no un- polarized light can enter the objective, otherwise the field cannot be sufficiently darkened. No diaphragm is used over the polarizer for most examinations. Direct sunlight may be used to advantage with some objects, and as a rule the object would best be very transparent. 7224. Mounting Objects for the Polariscope.—So far as nbseible objects should be mounted in balsam to render them transparent. In many cases objects mounted in water do not give satisfactory polariscope appearances. For example, 152 MICRO-SPECTROSCOPE AND POLARISCOPE (CH. VI if starch is mounted dry or in water, the appearances are not so striking as in a balsam mount (Davis, p. 337; Suffolk). 2 225. Purpose of a Micro-Polariscope.—The object of a micro-polariscope is to determine, in microscopic masses, one or more of the following points: (A) Whether the body is singly refractive, mono-refringent, or 7sotrvopic, that is, opti- cally homogeneous, as are glass and crystals belonging to the cubical system ; (B) Whether the object is doubly refractive or anisotropic, uniaxial or biaxial ; (C) Pleochroism ; (D) The rotation of the plane of polarization, as with solutions of sugar, etc. ; (E) To aid in petrology and mineralogy ; (F) To aid in the determi- nation of very minute quantities of crystallizable substances ; (G) For the pro- duction of colors. For petrological and mineralogical investigations the microscope should possess a graduated, rotating stage so that the object can be rotated, and the exact angle of rotation determined. Fig. 126. It is also found of advantage in investigating object swith polarized light where colors appear, to combine a polariscopic and spectroscope (Spectro-Polariscope ). MICRO-POLARISCOPE—EXPERIMENTS § 226. Arrange the polarizer and analyzer as directed above (§ 220) and use a 16 mm. objective except when otherwise directed. (A) Isotropic or Singly Refracting Objects.—Light the mi- croscope well and cross the Nicols, shade the stage and make the field as dark as possible (§ 219). Asan isotropic substance, put an ordin- ary glass slide under the microscope. The field will remain dark. As an example of a crystal belonging to the cubical system and hence iso- tropic, make a strong solution of common salt (sodium chloride) put a drop on a slide and allow it to crystallize, put it under the microscope, remove the analyzer, focus the crystals and then replace the analyzer and cross the Nicols. The field and the crystals will re- main dark. (B) Anisotropic or Doubly Refracting Objects.— Make a fresh preparation of carbonate of lime crystals like that described for pedesis ($ 151), or use a preparation in which the crystals have dried to the slide, use a 5 or 3 mm. objective, shade the object well, remove the analyzer and focus the crystals, then replace the analyzer. Cross the Nicols. In the dark field will be seen multitudes of shining crystals, and if the preparation is a fresh one in water, part of the smaller crystals will alternately flash and disappear. By observing carefully, some of the larger crystals will be found to remain dark with crossed Nicols, others will shine continuously. If the crystals are in such a position that the light passes through them parallel with the optic i) i for Micro-Chemical Analysis (Jour- a. nal of Applied Microscopy, 1899, p. ; 503). This isa modified and simplified pelrographical microscope and has all the attachments and motions nec- essary for micro-chemical analysis. Hy As the objects studied are mostly id liquid or in liquids the microscope “mK has no joint as it must be used in a vertical position. | Wty Fic. 126. Chamot’s Microscope TT == > = = Cie Soe i 0 aE = ne ' LE plical Eo, ‘Au fl 154 MICRO-SPECTROSCOPE AND POLARISCOPE (CH. V1 axis,* the crystals are isotropic like salt crystals and remain dark. If, however, the light traverses them in any other direction the ray from the polarizer is divided into two constituents vibrating in planes at right angles to each other, and one of these will traverse the an- alyzer, hence such crystals will appear as if self-luminous in a dark field. The experiment with these crystals from the frog succeeds well with a 2 mm. homogeneous immersion. As a further illustration of anisotropic objects, mount some cotton fibers in balsam (§ 256), also some of the lens paper (§ 114). These furnish excellent examples of vegetable fibers. Striated muscle fibers are also very well adapted for polarizing objects. ‘As examples of biaxial crystals, allow some borax solution to dry and crystallize on a slide ; use the crystals as objects. As all doubly refracting objects restore the light with crossed Nicols, they are some- times called depolarizing. (C) Pleochroism.—This is the exhibition of different tints as the analyzer is rotated. An excellent subject for this will be found in blood crystals. §$ 227. Production of Colors.—For the production of gorgeous colors, a plate of selenite giving blue and yellow colors is placed between the polarizer and the object. If properly mounted, the selenite is very conveniently placed on the diaphragm carrier of the Abbe illuminator, just above the polarizer. A thin plate or film of mica also answers well. It is not necessary to use selenite or mica for the production of the most glorious colors in many objects. One of the most beautiful pre- parations, and one of the most instructive also, may be prepared as follows: Heat some xylene balsam on a slide until the xylene is nearly evaporated. Add some crystals of the medicine, sulphonal and warm till the sulphonal is melted and mixes with the balsam. While the balsam is still melted put on acover-glass. If one gets perfect crystals there will be shown not only the most beautiful colors, but the black cross with perfection. (Clark). *The optic axis of doubly refracting crystalsis the axis along which the crystal is not doubly refracting, but isotropic like glass. When there is but one such axis, the crystal is said to be uniaxial, if there are two such axes the crystal is said to be bi-axial. The crystals of carbonate of lime from the frog (see 4 151) are uniaxial crystals. Borax crystals are bi-axial. CHO VL MICRO-CHEMISTRY 155 It is very instructive and interesting to examine many organic and inorganic substances with a micro-polarizer. REFERENCES TO THE POLARISCOPE AND TO THE USE OF POLARIZED LIGHT. Anthony & Brackett, 133 ; Behrens ; Behrens, Kossel und Schiefferdecker ; Car- noy, 61 ; Carpenter-Dallinger, 317, 1097; Clark ; Daniell, 494 ; Davis ; v. Ebener ; Gamgee; Halliburton, 36, 272; Hogg, 133,729 ; Lehmann; M’Kendrick ; Nageli und Schwendener, 299; Quekett ; Suffolk, 125; Valentin. Physical Review, L., p. 127. Daniell, Physics for Medical Students. Nichols, Physics. MICRO-CHEMISTRY §$ 228. During the last decade the microscope has become one of the necessities of the expert chemist, and the signs of the times indicate that in every research laboratory of chemistry the microscope will be- come as familiar as it now is in research laboratories of biology. Its proper place in chemistry has been admirably stated by Chamot : “Tt is rather remarkable how slow American chemists have been in realizing the importance of the microscope as an adjunct to every chemical laboratory. This is, perhaps, largely due to the fact that few of our students in chemistry become familiar with the construction and manipulation of this instrument, just as few of them become sufficiently familiar with the spectroscope and its manifold uses ; and doubtless also because of the prevailing impression that a microscope is primarily an instrument for the biologist and is of necessity a most expensive luxury. The fact is, however, that this instrument is now far from being a luxury to the chemist, and the time is not far distant when it will be conceded to be as much a necessity in every analytical laboratory as is the balance. : ‘‘Nor is the apprenticeship to its use in chemical work long nor intricate. ‘‘ Micro-chemical analysis should appeal to every chemist because of its neat- ness, wonderful delicacy, in which it is not excelled even by the spectroscope, and the expedition with which an analysis can be made. A complete analysis, intricate though it may be, is a matter of a few minutes rather than of a few hours. ‘« While there is no good reason to believe, as do some enthusiasts, that this new system is to displace the old analysis in the wet way, every chemist should, nevertheless, familiarize himself with the microscope, its accessories, and the elegant and time-saving methods of micro-analysis, thus enabling him to examine qualitatively the most minute amounts of material with a rapidity and accuracy which is truly marvelous; not to speak of the many substances for which no other method of identification is known. 2 ‘At present the greatest bar to its general use is the absence of any well defined scheme, and the absolute necessity of being well grounded in general chemistry. There are no tables which can be followed in a mechanical way by the student, but on the contrary he is obliged to exercise his knowledge and judg- ment at every step. For this very reason the introduction of this subject into the list of those now taught is greatly to be desired.” 156 MICRO-CHEMISTRY [CH. VI It will be seen by the last paragraph that in chemistry as in biology, the microscope is only an aid to investigation , it cannot take the place of thorough training in the fundamentals of the subject under investiga- tion. § 229. The following list of substances is suggested by Dr. Chamot for beginning practice as the results given are definite and easily obtained : SUGGESTIONS FOR STUDY ON CRYSTAL SYSTEMS “Isometric. Sodium chlorid, potassium chlorid, potassium iodid. Alums, crystallize in octahedra, cubes or combinations of the two. It is well to recall that the alums have the general formula, M,(SO,); . N.SO,424 H,O, where -M- can be Al, Cr, Mn, Fe, In, Ga, Tl, and -N- Na, K, Rb, Cs, NH, Ag, or Tl. All alums are isomorphous. Tetragonal. Potassium copper chlorid. Ammonium copper chlorid. Nickel sulfate6H,O. This salt is dimorphic, crystallizing also in the mono- clinic system. Nickel sulfate 7H,O is orthorhombic. Orthorhombic. Mercuric chlorid. Silver nitrate. Potassium sulfate. Potassium nitrate. Magnesium sulfate 7H,O. Potassium chromate. Sodium nitrate (also Hexagonal). Monoclinic. Potassium chlorate (sodium chlorate is Isomet. or Tetrag. ) Lead acetate. Copper acetate H,O. Oxalic acid. Ferrous sulfate, this salt forms normally with 7 H,O and is then Mono- clinic, but in presence of zinc sulfate becomes Orthorhombic, and in presence of copper sulfate, Triclinic. Sodium sulfate 10H,O. Triclinic. Copper sulfate 5H,O. Boric acid. Hexagonal. Lead iodid. Sodium nitrate (also Orthorhombic). 4 230. Before performing analytical tests, itis necessary that the student be familiar with the properties of crystals and also thoroughly familiar with the appearance of crystals of the chlorides, nitrates, and sulfates of Sodium, Potassium, and Ammonium ; since some of these salts are sure to appear in almost every test drop examined. For this reason the following experiments should be carefully performed.” CH. VI) MICRO-CHEMISTRY 157 AN EXERCISE FOR PRACTICE 1. ‘Sodium chlorid. Isometric system a. Take a fragment of the salt the size of this period (.) or a little smaller. Dissolve at the corner of a slide in a minute drop of distilled water. Heat over the ‘‘micro’’ flame so as to cause vapid evaporation. When dry examine residue (after cooling) with '% inch obj. and 2 in. eyepiece. Breathe on the preparation several times, allow the moisture to evaporate spontaneously. There should now be obtained well developed crystals instead of a mere crystalline mass. Too rapid crystallization is always to be avoided. The object of the experiment is to emphasize this fact. Select one of the best crystals. Measure its angles. Try its behavior between crossed Nicols. (4% 219.) 6. Place a small drop of water in the usual position add carefully Ferric Chlorid till the drop when held over white paper appears distinctly yellow. Stir. Add two or three tiny fragments of Sodium Acetate at the center of the drop. Place on the stage of the microscope. There is formed Ferric acetate, Sodium chlorid and possibly a double chlorid of iron and sodium. Notice the following points. 1. Tendency toward formation of double salt. 2. That the type crystal of NaClisa cube. 3, Cubes may so grow as to present the appearance of a rectangular prism. 4. In certain positions cubes have the appearance of a pyramid. 5. In other positions they exhibit a hexagonal outline, thus simulating a polyhedron of many faces. 6. There is scarcely any tendency in this case toward the formation of the hoppers observed in the first experiment (a). 7. The crystals often develop fastest along the diagonal planes so that the regular faces are replaced by pyramidal depressions.” Fic. 127. Czapski’s Ocular TLris-diaphragm with cross hairs for examining and accurately determining the axial im- ages of small crystals. The tris diaphragm enables the observer to make the field as large or small as desired. A. Longitudinal section. B. Transection, showing the cross lines and the tris dia- phragm with the projecting part at the left, by which the dia- phragm is opened and closed. (Zeiss? Catalog). For directions and hints in micro-chemical work and crystallography, consult the various volumes of the Journal of the Roy. Micr. Soc. ; Zeitschrift fiir physio- logische Chemie, and other chemical journals; Wormly; Klément & Renard; Carpenter-Dallinger ; Hogg; Behrens, Kossel und Schiefferdecker ; Frey ; Dana, and other works on mineralogy; Davis. Behrens, T. H.—Anleitung zur micro- chemischen Analyse der wichtigsten organischen Verbindungen. Hamburg, 1895-1897. Microchemische Technik, 2d edition, Hamburg, 1900. A manual of microchemical analysis with an jntrodnctory chapter by J. W. Judd, London, 158 TEXTILE FIBERS (CH. VI 1894. Especial attention is also called to the articles by Dr. E. M. Chamot in the Journal of Applied Microscopy beginning with vol. ii. p. 502, and continued in vol. ili and iv. TEXTILE FIBERS, FOOD AND PHARMACOLOGICAL PRODUCTS § 231. The microscope is coming more and more into use for the determination of the character of textile fibers, both in the raw state and after manufacture. As the textile fibers have distinctive char- acters it is not difficult to determine mixtures in fabrics of various kinds. The student is advised to study carefully known fibers, as of cotton, wool, linen, silk, jute, etc., so that he is certain of the appear- ances, and then to determine of what fibers different fabrics are com- posed. He will be astonished at the amount of ‘‘Alabama wool’’ in supposedly all wool goods. For works and articles upon textile fibers see: Herzfeld, J. Trans- lated by Salter. The technical testing of yarns and textile fabrics with reference to official specifications. London, 1898. E. A. Posselt—The structure of fibers, yarns and fabrics. Philadelphia and and London, 1891. Dr. C. Rougher—Des filaments végétaux em- ployés dans l'industrie. Paris, 1873. Wm. P. Wilson and E. Fah- ring—, The conditioning of wool and other fabrics in the technological laboratories of the Philadeiphia Commercial Museum. Journal of Ap- plied Microscopy, Vol. II, (1899) pp. 290-292, 457-460. Bulletin of the National Association of Wool Growers, 1875, p. 470. Proceed- ings of the Amer. Micr. Soc., 1884, pp. 65-68. Besides these referen- ces one is liable to find pictures and discussions of various fibers in general works on the microscope, and in technical and general cyclo- peedias. § 232. From the nature of food and pharmacological products adulterations are in many cases most accurately and easily determined by microscopic examination. The student will find constant reference to the microscopical characters of the genuine and spurious substances in medicines and other pharmacological products in works on pharmacy or pharmacology ; also in pharmacological journals and in druggists reports, e. g. the excellent and well illustrated article by Burt E. Nelson of the chemical laboratory of the Binghamton State Hospital on the Microscopical examination of tea, coffee, spices and condiments. This appeared in Merck’s Report, Oct. 15, Dec. 15, 1898. The micro- scopical Journals also contain occasional articles bearing upon this subject. See also Food Products in bulletins of the U.S. Dep’t Agr. Macé, E.—Les substances alimentaire, etc., Paris, 1891. Schimper, CHN VT | MICRO-METALLOGRA PHY 159 A. F. W. Anleitung, etc. Jena, 1900. Hugh Galt,—The Microscopy of the starches, illustrated by photo-micrographs, London, 1900. THE MICROSCOPE IN METALLOGRAPHY S$ 233. Inthe modern investigation of metals and alloys much light has been thrown upon the structural peculiarities which render some mixtures satisfactory and others unsatisfactory. There are two great methods: First, that of studying fractured surfaces without re- course to any reagents. Second, to polish a metallic surface carefully with emery or carborundum and finally with rouge or diamantine and then etch it with some acid for a longer or shorter time. For either method reflected light must be used. For low powers that obtained at a good window or by alamp ora lamp and bullseye are good. The illum- inating objectives (§ 28), z. e. objectives in which a prism in the side of the objective reflects light down through the lenses which act as a condenser, are preferable for most work and indeed necessary if one uses high powers. For special microscope see Fig. 126 A. Elaborate arrangements have been devised for holding the piece of metal on the stage, but some beeswax, or some clay made plastic with glycerin answers well. For pictures of the appearances seen in studying metallic surfaces, see the journals of engineering and metallurgy, especially the Metallographist, a quarterly publication devoted to the study of metals with special reference to their physics and micro-structure, etc. In twenty-five or more of the great metal manufacturing establishments special laboratories for microscopic examination and investigation have been established. This is an illus- tration of what has frequently occurred—great manufacturing interests have outrun the universities in the appreciation and application of methods of reasearch. Fortunately, however, laboratories are already springing up in connection with the universities, and probably within ten years every great technical school will have its laboratory of micro-metallography where students will have opportunity to perfect themselves in the preparation, photography and microscopic study of the metals and alloys. Beside the sources of information given above, see Dr. H. Ost und Dr. Fr. Kolbeck, Lehrbuch der chemischen Technologie mit einem Schlussabschnitt ‘‘ Metallurgie.’’ Hannover, 1901. Behrens, T. H.— Das mikroskopische Geftige der Matalle, etc. Hamburg, 1894. For an excellent bibliography of 188 titles; see the Metallographist, Vol. I, 1898, and appended to the special papers in all the volumes. Also in Iron Age, Jan. 27, 1898. Carpenter-Dallinger, p. 264. 160 MICRO-METALLOGRAPHY (CH. VI Fic. 126 A. Microscope especially constructed for the study of metals and alloys. (The Boston Testing Laboratories). The stage ts rotary, and may be raised or lowered by rack and pinion. Above the objective ts the arrangement for illumination (see Ch. VIT), CHAPTER VII SLIDES AND COVER-GLASSES ; MOUNTING ; ISOLATION ; SECTIONING BY THE COLLODION AND THE PARAF- FIN METHODS; LABELING AND STORING MICRO- SCOPICAL PREPARATIONS ; REAGENTS SLIDES AND COVER-GLASSES 2234. Slides, Glass Slides or Slips, Microscopic Slides or Slips.—These are strips of clear flat glass upon which microscopic specimens are usually mounted for preservation and ready examination. The size that has been almost universally adopted for ordinary preparations is 25 x 76 millimeters (1 x 3 inches). For rock sections, slides 25 x 45 mm. or 32 x 32 mm. are used; for serial sections, slides 25x 76mm., 50x 76mm. or 37 x 87 mm. are used. For special purposes, slides of the necessary size are employed without regard to any conventional standard. Whatever size of slide is used, it should be made of clear glass and the edges should be ground. It is altogether false economy to mount microscopic objects on slides with unground edges. It is unsafe also as the unground edges are liable to wound the hands. For micro-chemical work Dr. Chamot recommends slides of half the length of those used in ordinary microscopic work. From the rapidity with which they are destroyed, he thinks the ground edges are unnecessarily expensive. He adds further : ‘‘It is a great misfortune that the colorless glass slips used in America and so excellent for ordinary microscopic work should be easily attacked by all liquids ; even water extracts a relatively enormous amount of alkalies and alka- line earths. The slips of greenish glass, while not as neat or desirable for general microscopy, seem to be decidedly more resistant, and are therefore preferable.” Transparent celluloid slides are recommended by Behrens for work where hydro- fluoric acid and its derivatives are to be examined. (Chamot, Jour. Appl. Micr. vol. iii, p. 793). 4235. Cleaning Slides.—For new slides a thorough rinsing in clean water with subsequent wiping with a soft cloth like glass toweling, or thin cotton cloth like bleached cheese cloth (bunting or gauze, or absorbent surgical gauze), usually fits them for ordinary use. If they are not satisfactorily cleaned in this way, soak them a short time in 50% or 75% alcohol, let them drain fora few moments ona clean towel or on blotting paper, and then wipe with a soft cloth. In handling the slides grasp them by their edges to avoid soiling the face of the slide. After the slides are cleaned they should be stored in a place as free as possible from dust. For storing, covered glass dishes are best. Use museum jars or glass boxes (Fig. 150). 162 SLIDES AND COVER-GLASSES [CH VIL Fic. 128. Glass slide or slip of the ordinary size for microscopic work (3.x 71M., 76 x 25mm.). (Cut loaned by the Spencer Lens Company). For old slides, if only water, glycerin or glycerin jelly has been used on them, they may be cleaned with water, or preferably, warm water and then with alcohol if necessary. Where balsam, or any oily or gummy substance has been used upon the slides, they may be freed from the balsam, etc., by soaking them for a week or more in one of the cleaning mixtures for glass. If they are first soaked in xylene, benzin or turpentine to dissolve the balsam, then soaked in the cleaning mixture, the time required will be much shortened (% 242). After all foreign matter is removed the slides should be thoroughly rinsed in water to remove all the cleaning mixture. They may then be treated as directed for new slides. If slides with large covers, as in mounted series, are put into the cleaning mixture, the swelling of the balsam is liable to break the covers. Dissolving away the balsam with turpentine, avoids this, and greatly shortens the time neces- sary for cleaning the old slides and covers. : Another excellent method for balsam mounts is to heat the slides until the balsam is soft and then remove the cover-glasses. The turpentine cleaning mix- ture, etc., can then act on the entire surface. It should be said, however, that at the present price of slides and cover-glasses it costs nearly as much as the slides and covers are worth to clean those that have been used in balsam mounting. 74 236. Cover-Glasses or Covering Glasses.—These are circular or quadr- angular pieces of thin glass used for covering and protecting microscopic objects. They should be very thin, 0.10 to 0.25 millimeter (see table, %29). It is better never to use a cover-glass over 0.20 mm. thick, then the preparation may be studied with a 2 mm. oil immersion as well as with lower objectives. Except for objects wholly unsuited for high powers, it is a great mistake to use cover-glasses thicker than the working distance of a homogeneous objective (% 61). Indeed, if one wishes to employ high powers, the thicker the sections the thinner should be the cover-glass (see % 240). The cover-glass should always be considerably larger than the object over which wt ts placed. 7 237. Cleaning Cover-Glasses.—New cover-glasses should be put into a glass dish of some kind containing one of the cleaning mixtures (7 242) and al- lowed to remain a day or longer. In putting them in, push one in at a time and be sure that each is entirely immersed, otherwise they adhere very closely and the cleaning mixture is unable to act freely. Soiled covers should be left a week or more in the cleaning mixture. An indefinite sojourn in the cleaner does not seem CH. VIT) SLIDES AND COVER-GLASSES 163 to injure the slides or covers. After one day or longer, pour off the cleaning mix- ture into another glass jar, and rinse the cover-glasses, moving them around with a gentle rotary motion. Continue the rinsing until all the cleaning mixture is removed. One may rinse them occasionally, and in the meantime allow a very gentle stream of water to flow on them, or they may be allowed to stand quietly and have the water renewed from time to time. When the cleaning mixture is removed rinse the covers well with distilled water, and then cover them with 50% to 75% alcohol. FIGs. 129-130. Figures of square and of circular cover-glasses. See also Fig. 162 for covers on serial sections. % 238. Wiping the Cover-Glasses.—When ready to wipe the cover-glasses, remove several from the alcohol and put them on a soft, dry cloth, or on some of the lens or filter paper to let them drain. Grasp a cover-glass by its edges, cover the thumb and index finger of the other hand with a soft, clean cloth or some of the the lens paper. The bleached cheese cloth (% 235) is good for wiping covers. Grasp the cover between the thumb and index and rub the surfaces. In doing this it is necessary to keep the thumb and index well opposed on directly opposite faces of the cover so that no strain will come on it, otherwise the cover is liable to be broken. When a cover is well wiped, hold it up and look through it toward some dark object. The cover will be seen partly by transmitted and partly by reflected light, and any cloudiness will be easily detected. If the cover does not look clear, breathe on the faces and wipe again. If it is not possible to get a cover clear in this way it should be put again into the cleaning mixture. As the covers are wiped put them in a clean glass box or Petri dish. Handle them always by their edges, or use fine forceps. Do not put the fingers on the faces of the covers, for that will surely cloud them. Fic. 131. Glass dish for holding covers (Whitall, Tatum & Co.). 4239. Cleaning Large Cover-Glasses.—For serial sections and especially large sections, large quadrangular covers are used (Fig. 162). These are to be put one by one into a cleaning mixture as for the smaller covers and treated in every way the same. In wiping them one may proceed as for the small covers, but special care is necessary to avoid breaking them. It is especially desirable that these large covers should be thin—not over 0.15-0.20 mm. otherwise high ob- jectives cannot be used in studying the preparations. 4240. Measuring the Thickness of Cover-Glasses.—It is of the greatest advantage to know the exact thickness of the cover-glass on an object ; for, (a) 164 SLIDES AND COVER-GLASSES [CH Vil In studying the preparation one would not try to use objectives of a shorter work- ing distance than the thickness of the cover (461); (b) In using adjustable objectives with the collar graduated for different thicknesses of cover, the collar might be set at a favorable point without loss of time; (c) For unadjustable objectives the thickness of cover may be selected corresponding to that for which the objective was corrected (see table, 7 29). Furthermore, if there is a variation from the standard, one may remedy it, in part at least, by lengthening the tube if the cover is thinner, and shortening it if the cover is thicker than the standard (% 102, Fig. 133.) In the so called No. 1 cover-glasses of the dealers in microscopical supplies, the writer has found covers varying from 0.10 mm. to 0.35 mm. To use cover- glasses of so wide a variation in thickness without knowing whether one has a thick or thin one is simply to ignore the fundamental principles by which correct microscopic images are obtained. Brown & Sharpe Mlfg-Co. Prorideace.RLL Fic. 132. Micrometer Calipers (Brown and Sharpe). Pocket Calipers, gradu- ated tn inches or millimeters, and well adapted for measuring cover-glasses. It is then strongly recommended that every preparation shall be covered with a cover-glass whose thickness is known, and that this thickness should be indicated in some way on the preparation. 4 241. Cover-Glass Measurers, Testers or Gauges. For the purpose of measuring cover-glasses there are three very excellent pieces of apparatus. The micrometer calipers (Fig. 132) used chiefly in the mechanic arts, are convenient and from their size easily carried in the pocket. The two cover-glass measurers specially designed for the purpose are shown in Figs. 133-134. With either of these the covers may be more rapidly measured than with the calipers. With all of these measures or gauges one should be certain that the index stands at zero when at rest. If the index does not stand at zero it should be adjusted to that point, otherwise the readings will not be correct. As the covers are measured the different thicknesses should be put into different boxes and properly labeled. Unless one is striving for the most accurate possible results, cover-glasses not varying more than 0.06 mm, may be put in the same box. For example, if one takes 0.15 mm. as a standard, covers varying 0.03 mm, on each side may be put into the same box. In this case the box would con- tain covers of 0,12, 0.13, 0.14, 0.15, 0.16, 0.17 and 0.18 mm. CH. VIT) SLIDES AND COVER-GLASSES 165 Fag isl ‘ Fic. 133. Cover-Glass Measurer (Edward Bausch). The cover glass is placed in the notch between the two screws, and the drum ts turned by the milled head at the right till the cover is in contact with the screws. The thickness 1s then indicated by the knife edge on the drum and may be read off directly in 0.001 of a millimeter or inch. In other columns is given the proper tube-length for various unadjustable objectives (4,4, 1, and py in.) (Bausch and Lomb Optical Company). Fic. 134. Zetss Cover-Glass Measurer. With this the knife edge jaws are opened by means of a lever and the cover inserted. The thick- ness may then be read off on the face as the pointer indicates the thick- ness in hundredths millimeter in the outer circle and in thousandths inch on the inner circle. 7242. Cleaning Mixtures for Glass.—The cleaning mixtures used for clean- ing slides and cover-glasses are those commonly used in chemical laboratories: (Dr. G. C. Caldwell’s Laboratory Guide in Chemistry). 166 MOUNTING AND LABELING (CH. VIT (A) Dichromate of Potash and Sulphuric Acid. Dichromate of potash (K,Cr,0O, ) - - - 200 grams Water, distilled or ordinary - - - - 800 ce. Sulphuric acid (H,SO,) - - - - 1200 Cc. Dissolve the dichromate in the water by the aid of heat, using an agate or other metal dish, then pour it into a heavy iron kettle lined with sheet lead (Pr. Trans. Amer. Micr. Soc., 1899, p. 107). Add the sulphuric acid to the dissolved dichromate in the kettle. The purpose of the lead lined kettle is to avoid break- age from the great heat developed upon the addition of the sulphuric acid. The lead is very slightly affected by the acid, iron would be corroded by it. For making this mixture, ordinary water, commercial dichromate and strong commercial sulphuric acid may be used. Itis not necessary to employ chemically pure materials. This is an excellent cleaning mixture and is practically odorless. It is exceed- ingly corrosive and must be kept in glass vessels. It may be used more than once, but when the color changes markedly from that seen in the fresh mixture it should be thrown away. (B) Sulphuric and Nitric Acid Mixture. Nitric acid (HNO,) - - - - - 200 cc. Sulphuric acid (H,SO,) = - - = - 300 cc. The acids should be strong, but they need not be chemically pure. The two acids are mixed slowly, and kept in a glass stoppered bottle. This is a more cor- rosive mixture than (A), and has the undesirable feature of giving off stifling fumes, therefore it must be carefully covered. It may be used several times. It acts more rapidly than the dichromate mixture, but on account of the fumes js not so well adapted for general laboratories. MOUNTING, AND PERMANENT PREPARATION OF MICROSCOPICAL OBJECTS 2243. Mounting a Microscopical Object is so arranging it upon some suit- able support (glass slide) and in some suitable mounting medium that it may be satisfactorily studied with the microscope. The cover-glass on a permanent preparation should always be considerably larger than the object ; and where several objects are put under one cover-glass tt is JSalse economy to crowd them too closely together. 2244. Temporary Mounting.—In a great many cases objects do not need to be preserved ; they are then mounted in any way to enable one best to study them, and after the study the cover glass is removed, the slide cleaned and made ready for future use. In the study of living objects, of course only temporary preparations are possible. With amoebae, white blood corpuscles, and many other objects both animal and vegetable, the living phenomena can best be studied by mounting them in the natural medium. That is, for amoebae, in the water in which they are found ; for the white blood corpuscles, a drop of blood is used and, as the blood soon coagulates, they are in the serum. Sometimes it is not easy or convenient to get the natural medium, then some liquid that has been found to serve in place of the natural medium is used. For many things, water with a CH. VIT) MOUNTING AND LABELING 167 little common salt (water Ioo cc., common salt ;{,ths gram) isemployed. This is the so-called normal salt or saline solution. For the ciliated cells from frogs and other amphibia, nothing has been found so good as human spittle. Whatever is used, the object is put on the middle of the slide and a drop of the mounting medium added, and then the cover-glass. The cover is best put [Stide | on with fine forceps, as shown in Fig. 136. After the cover is in place, if the preparation is to be studied for some time, it is better to avoid currents and evaporation by painting a ring of castor oil around the cover in such a way that part of the ring will be on the slide and part on the cover (Fig. 165.) Cover Fic. 135. Needle Holder (Queen & Co.). By means of the screw clamp or chuck at one end the needle may be quickly changed. sult | 4a o20f Fic. 136. Zo show the method of putting a cover-glass upon a microscopic preparation. The cover 1s grasped by one edge, the opposite edge is then brought down to the slide, and the cover gradually lowered upon the object. FIG. 136. 2 245. Permanent Mounting.—For making permanent microscopical prepara- tions, there are three great methods. Special methods of procedure are necessary to mount objects successfully in each of these ways. The best mounting medium and the best method of mounting in a given case can only be determined by ex- periment. In most cases some previous observer has already made the necessary experiments and furnished the desired information. The three methods are the following : (A) Dry or in air (% 246); (B) Ln some medium :miscible with water, as glycerin or glycerin jelly (4 250); (C) Ln some resinous medium like Canada Balsam (% 255). 7246. Mounting Dry or in Air.—The object should be thoroughly dry. If any moisture remains it is liable to cloud the cover-glass, and the specimen may deteriorate. As the specimen must be sealed, it is necessary to prepare a cell slightly deeper than the object is thick. This is to support the cover-glass, and also to prevent the running in by capillarity of the sealing mixture. 4 246a. Order of Procedure in Mounting Objects Dry or in Air. 1. Acell of some kind is prepared. It should be slightly deeper than the object is thick (7 248). 2. The object is thoroughly dried (dessicated) either in dry air or by the aid of gentle heat. 3. If practicable the object is mounted on the cover-glass ; if not it is placed in the bottom of the cell. 4. The slide is warmed till the cement forming the cell wall is somewhat sticky, or a very thin coat of fresh cement is added ; the cover is warmed and put 168 MOUNTING AND LABELING (CH. VII on the cell and pressed down all around till a shining ring indicates its adherence (4 249). 5. The cover-glass is sealed (% 249). 6. The slide is labeled (7 308). 7. The preparation is cataloged and safely stored (% 309, 311). 2247. Example of Mounting Dry, or in Air.—Prepare a shallow cell and dry it (? 248). Select a clean cover-glass slightly larger than the cell. Pour upon the cover a drop of 10% solution of salycilic acid in 95% alcohol. Let it dry spon- taneously. Warm the slide till the cement ring or cell is somewhat sticky, then warm the cover gently and put it on the cell, crystals down. Press on the cover all around the edge (7% 246); seal, label and catalog (7 253, 308, 309). A preparation of mammalian red blood corpuscles may be satisfactorily made by spreading a very thin layer of fresh blood on a cover with the end of a slide. After it is dry, warm gently to remove the last traces of moisture and mount blood side down, precisely as for the crystals. One can get the blood as directed for the Micro-spectroscopic work (4% 210). Fic. 137. Zurn-Table for sealing cover-glasses and making shallow mount- ing cells. (Queen & Co.) 74248. Preparation of Mouuting Cells.—(A) 7hin Cells. These are most conveniently made of some of the cements used in microscopy. Shellac is one of the best and most generally applicable. To prepare a shellac cell place the slide on a turn-table (Fig. 137) and center it, that is, get the center of the slide over the center of the turn-table. Select a guide ring on the turn-table which is a little smaller than the cover-glass to be used, take the brush from the shellac, being sure that there is not enough cement adhering to it to drop. Whirl the turn-table and hold the brush lightly on the slide just over the guide ring selected. An even ring of the cement should result. If it is uneven, the cement is too thick or too thin, or too much was on the brush. After a ring is thus prepared remove the slide and allow the cement to dry spontaneously, or heat the slide in some way. Before the slide is used for mounting, the cement should be so dry when it is cold that it does not dent when the finger nail is applied to it. A cell of considerable depth may be made with the shellac by adding succes- sive layers as the previous one drys. (B) Deep Cells are sometimes made by building up cement cells, but more frequently, paper, wax, glass, hard rubber, or some metal is used for the main part of the cell. Paper rings, block tin or lead rings are easily cut out with gun punches. These rings are fastened to the slide by using some cement like the shellac. CH. VIT) MOUNTING AND LABELING 169 2 249. Sealing the Cover-Glass for Dry Objects Mounted in Cells.—When an object is mounted in a cell, the slide is warmed until the cement is slightly sticky or a very thin coat of fresh cement is put on. The cover-glass is warmed slightly also, both to make it stick to the cell more easily, and to expel any re- maining moisture from the object. When the cover is put on it is pressed down all around over the cell until a shining ring appears, showing that there is an in- timate contact. In doing this use the convex part of the fine forceps or some other blunt, smooth object ; it is also necessary to avoid pressing on the cover except immediately over the wall of the cellfor fear of breaking the cover. When the cover is in contact with the wall of cement all around, the slide shonld be placed on the turn-table and carefully arranged so that the cover-glass and cell wall will be concentric with the guide rings of the turn-table. Then the turn- table is whirled and a ring of fresh cement it painted, half on the cover and half on the cell wall (Fig. 165.) If the cover-glass is not in contact with the cell wall at any point and the cell is shallow, there will be great dangerof the fresh cement running into the cell and injuring or spoiling the preparation. When the cover- glass is properly sealed, the preparation is put in a safe place for the drying of the cement. It is advisable to add a fresh coat of cement occasionally. Fic. 138. Centering Card. A card with stops for the slide and circles in the position occupied by the center of the slide. If the slide is put upon such a card tt is very easy to arrange the object so that it will be approximately in the center og the slide. The position of the long cover used for serial sections 1s also shown (Fig. 762). (From the Microscope, December, 1886). % 250. Mounting Objects in Media Miscible with Water.—Many objects are so greatly modified by drying that they must be mounted in some medium other than air. In some cases water with something in solution is used. Glycerin of various strengths, and glycerin jelly are also much employed. All these media keep the object moist and therefore in a condition resembling the natural one. The object is usually and properly treated with gradually increasing strengths of glycerin or fixed by some fixing agent before being permanently mounted in strong glycerin or either of the other media. 170 MOUNTING AND LABELING (CH. Vil In all of these different methods, unless glycerin of increasing strengths has been used to prepare the tissue, the fixing agent is washed away with water before the object is finally and permanently mounted in either of the media. For glycerin jelly no cell is necessary unless the object has a considerable thickness. 2251. Order of Procedure in Mounting Objects in Glycerin. 1. A cell must be prepared on the slide if the object is of considerable thick- ness (4% 248, 249). 2. Asuitably prepared object (% 250) is placed on the center of a clean slide, and if no cell is required a centering card is used to facilitate the centering (Fig. 138). 3. A drop of pure glycerin is put upon the object, or if a cell is used, enough to fill the cell. 4. In putting on the cover-glass it is grasped with fine forceps and the under side breathed on to slightly moisten it so that the glycerin will adhere, then one edge of the cover is put on the cell or slide and the cover gradually lowered upon the object (Fig. 136). The cover is then gently pressed down. Ifa cell is used, a a fresh coat of cement is added before mounting (7 249). Fic. 139. Slide and cover-glass showing method of anchoring a cover-glass with a glycerin preparation when | no cell is used. A cover-glass so anchored is not liable to move when the cover ts being sealed (4% 253). | Fic. 140. Glass slide with cover-glass, a drop of re- oe agentand a bit of absorbent paper to show method of irri- gation (2 262, 263). 5. The cover-glass is sealed (% 249). 6. The slide is labeled (4 3038). 7. The preparation is cataloged and safely stored (4% 309, 311). 2 252. Order of Procedure in Mounting Objects in Glycerin Jelly. I. Unless the object is quite thick no cell is necessary with glycerin jelly. 2. A slide is gently warmed and placed on the centering card (Fig. 138) and a drop of warmed glycerin jelly is put on its center. The suitably prepared object is then arranged in the center of the slide. 3. A drop of the warm glycerin jelly is then put on the object, or if a cell is used it is filled with the medium. 4. The cover-glass is grasped with fine forceps, the lower side breathed on and then gradually lowered upon the object (Fig. 136) and gently pressed down. 5. After mounting, the preparation is left flat in some cool place till the glycerin jelly sets, then the superfluous amount is scraped and wiped away and the cover-glass sealed with shellac (% 253). 6. The slide is labeled (% 308). 7. The preparation is cataloged and safely stored (4 309, 311). % 253. Sealing the Cover-Glass when no Cell is used.—(A) For glycerin mounted specimens. The superfluous glycerin is wiped away as carefully as possi- ble with a moist cloth, then four minute drops of cement are placed at the edge of the cover (Fig. 139), and allowed to harden for half an hour or more. These will CH. V1I) MOUNTING AND LABELING 171 anchor the cover-glass, then the preparation may be put on the turn-table and a ring of cement put around the edge while whirling the turn-table. A B C Fic. 141. A—Simple form of moist chamber made with a plate and bowl. B, bowl serving as a belljar, P, plate containing the water and over which the bowl ts inverted ; S, slides on which are mounted preparations which are to be kept moist. These slides are seen endwise and rest upon a bench made by cementing short pieces of large glass tubing to a strip of glass of the desired length and width. B— Two cover-glasses (C) made eccentric, so that they may be more easily sepa- rated by grasping the projecting edge. C—Slide (S) with projecting cover-glass (C). The prozection of the cover en- ables one to grasp and raise it without danger of moving it on the slide and thus Jolding the substance under the cover. (From Proc. Amer. Micr. Soc., 1891). (B) For objects in glycerin jelly, Farrants’ solution or a resinous medium. The mounting medium is first allowed to harden, then the superfluous medium is scraped away as much as possible with a knife, and then removed with a cloth moistened with water for the glycerin jelly and Farrants’ solution or with alcohol, chloroform or turpentine, etc., if aresinous medium is used. Then the slide is put on a turn-table and a ring of the shellaccement added. (C) Balsam preparations may be sealed with shellacas soon as they are prepared, but it is better to allow them to dry for a few days. One should never use a cement for sealing prepara- tions in balsam or other resinous media if the solvent of the cement is a solvent also of the balsam, etc. Otherwise the cement will soften the balsam and finally run in and mix with it, and partly or wholly ruin the preparation. Shellac is an excellent cement for sealing balsam perparations, asit neverrunsin. Balsam preparations are rarely sealed. 2 254. Example of Mounting in Glycerin Jelly.—For this select some stained and isolated muscular fibres or other suitably prepared objects. (See under isola- tion 4 259). Arrange them on the middle of a slide, using the centering card, and mount in glycerin jelly as directed in % 252. Air bubbles are not easily removed from glycerin jelly preparations, so care should be taken to avoid them. 4255. Mounting Objects in Resinous Media.—While the media miscible with water offer many advantages for mounting animal and vegetable tissues the preparations so made are liable to deteriorate. In many cases, also, they do not produce sufficient transparency to enable one to use high enough powers for the demonstration of minute details. 172 MOUNTING AND LABELING [CH VIT By using sufficient care almost any tissue may be mounted in a resinous medium and retain all its details of structure. For the successful mounting of an object in a resinous medium it must in some way be deprived of all water and all liquids not miscible with the resinous mounting medium. There are two methods of bringing this about: (A) By dry- ing or desiccation (2? 256), and (B) by successive displacements (% 258). Fic. 142. Small spirit lamp modified into a balsam bottle, a glycerin or glycerin zjelly bottle, or a bottle Sor homogeneous tmmersion liquid. For all of these purposes it should contain a glass rod as shown in the figure. By adding a small brush, tt answers well for a Shellac bottle also (See Fig. 170). 2 256. Order of Procedure in Mounting Objects in Resinous Media by Desiccation : : 1. The object suitable for the purpose (fly’s wings, etc.) is thoroughly dried in dry air or by gentle heat. 2. The object is arranged as desired in the center of a clean slide on the centering card (Fig. 138). 3. A drop of the mounting medium is put directly upon the object or spread on a cover-glass. 4. The cover-glass is put on the specimen with fine forceps (Fig. 136), but in no case does one breathe on the cover as when media-miscible with water are used. 5. The cover-glass is pressed down gently. 6. The slide is labeled (4 308). 7. The preparation is cataloged and safely stored (4 309, 311). 24257. Example of Mounting in Balsam by Desiccation.—Find a fresh fly, or if in winter, procure a dead one from a window sill or a spider’s web. Remove the fly’s wings, being especially careful to keep them the dorsal side up. With acamel’s hair brush remove any dirt that may be clinging to them. Place a clean slide on the centering card, then with fine forceps put the two wings within one of the guide rings. Leave one dorsal side up, turn the other ventral side up. Spread some Canada balsam on the face of the cover-glass and with the fine forceps place the cover upon the wings (Fig. 136). Probably some air-bubbles will appear in the preparation, but if the slide is put in a warm place these will soon disappear. Label, catalog, etc., (7307-311). 7258. Mounting in Resinous Media by a Series of Displacements.—For ex- amples of this see the procedure in the paraffin and in the collodion methods CH. Vil] ISOLATION OF ELEMENTS 173 (% 280, 300). The first step in the series is Dehydration, that is, the water is dis- placed by some liquid which is miscible both with the water and the next liquid to be used. Strong alcohol (95% or stronger) is usually employed for this. Plenty of it must be used to displace the last trace of water. The tissue may be soaked in a dish of the alcohol, or alcohol from a pipette may be poured uponit. Dehy- dration usually occurs in the thin objects to be mounted in balsam in 5 to 15 min- utes. Ifa dish of alcohol is used it must not be used too many times, as it loses in strength. The second step is clearing. Thatis, some liquid which is miscible with the alcohol and also with the resinous medium is used. This liquid is highly refrac- tive in most cases, and consequently this step is called clearing and the liquid a clearer. The clearer displaces the alcohol, and renders the object more or less translucent. In case the water was not all removed, a cloudiness will appear in parts or over the whole of the preparation. In this case the preparation must be returned to alcohol to complete the dehydration. One can tell when a specimen is properly cleared by holding it over some dark object. If it is cleared it can be seen only with difficulty, as but little light is reflected from it. If it is held toward the window, however, it will appear translucent. The third and final step is the displacement of the clearer by the resinous mounting medium. The specimen is drained of clearer and allowed to stand for a short time till there appears the first sign of dullness from evaporation of the clearer from the surface. Then a drop of the resinous medium is put on the object, and finally a cover-glass is placed over it, or a drop of the mounting medium is spread on the cover and it is then put on the object. ISOLATION OF HISTOLOGICAL ELEMENTS 2 259. Fora correct conception of the forms of the cells and fibers of the various organs of the body, one must see these elements isolated and thus be able to inspect them from all sides. It frequently occurs also that the isolation is not quite complete, and one can see in the clearest manner the relations of the cells or fibers to one another. The chemical agents or solutions for isolating are, in general, the same as those used for hardening and fixing. But the solutions are only about one-tenth as strong as for fixing, and the action is very much shorter, that is, from one or two hours to as many days. In the weak solution the cell cement or connective tissue is softened so that the cells and fibers may be separated from one another, and at the same time the cells are preserved. In fixing and hardening, on the other hand, the cell cement, like the other parts of the tissue, are made firmer. In preparing the isolating solutions it is better to dilute the fixing agents with normal salt solution (4% 331) than merely with water. 3 260. Isolation by Means of Formaldehyde.—Formaldehyde in normal salt solution is one of the very best dissociating agents for brain tissue and all the forms of epithelium. It is prepared as follows: 2 cc. of formal, (that is, a 40% solution of formaldehyde) are mixed with 1000 cc. of normal salt solution. This acts quickly and preserves delicate structures like the cilia of ordinary epithelia, 174 ISOLATION OF ELEMENTS (CH. VIL and also of the endymal cells of the brain. It is satisfactory for isolating the nerve cells of the brain. For the epithelium of the trachea, intestines, etc., the action is sufficient in two hours; good preparations may also be obtained after two days or more. The action on nerve tissue of the brain and myel or spinal cord is about as rapid. For the stratified epithelia, like those of the skin, mouth, etc., it may require two or three days for the most satisfactory preparations. oO © a OOOO OOO ® o O Oe FIG. 143 A. Fic. 143 B. FIG. 144. Fic. 143. Preparation Vials for Histology and Embryology. This repre- sents the two vials, natural size, that have been found most useful. They are kept in blocks with holes of the proper size. Fic. 144. Block with holes for containing shell vials. 7261. Example of Isolation.—Place a piece of the trachea of a very recently killed animal, or the roof of a frog’s mouth, in the formaldehyde dissociator. After two hours or more, up to two or three days, excellent preparations of ciliated cells may be obtained by scraping the trachea or roof of the mouth and mounting the scrapings on a slide. If one proceeds after two hours, probably most of the cells will cling together, and in the various clumps will appear cells on end show- ing the cilia or the bases of the cells, and other clumps will show the cells in pro- file. By tapping the cover gently with a needle holder or other light object the cells will be more separated from one another, and many fully isolated cells will be seen. 2262. Staining the Cells.—Almost any stain may be used for the formalin dissociated cells. For example, one may use eosin. This may be drawn under the cover of the already mounted preparation (Fig. 140), or a new preparation may be made and the scrapings mixed with a drop of the eosin before putting on the cover-glass. It is an advantage to study unstained preparations, otherwise one may obtain the erroneous opinion that the structure cannot be seen unless it is CAH. VIT] ISOLATION OF ELEMENTS 175 stained. The stain makes the structural features somewhat plainer ; it also accen- tuates some features and does not affect others so markedly. 2 263. Permanent Preparations of Isolated Cells.—If one desires to make a permanent preparation of isolated cells it may be done by placing a drop of glycerin at the edge of the cover and allowing it to diffuse under the cover, or the diffusion may be hurried by using a piece of blotting paper, as shown in Fig. 140. One may also make a new preparation and either with or without staining mix the cells with adrop of glycerin on the slide and then cover, or one may use glycerin jelly (% 254, 326). Fic. 145. Adjustable lens holder with universal joint. This ts especially use- ful for gross dissections, and for dissecting the partly isolated elements with needles (Leitz: Wm. Krafft, N. Y.). 4264. Isolation of Musculer Fibers.—For this the formal dissociator may be used (7% 260, 324), but the nitric acid method is more successful (% 330). The fresh muscle is placed in this in a glass vessel. At the ordinary temperature of a sitting room (20 degrees centigrade) the connective tissue will be so far gelatinized in from one to three days that it is easy to separate the fascicles and fibers either with needles or by shaking in a test tube or reagent vial (Fig. 143) with water. It takes longer for some muscles to dissociate than others, even at the same temper- ture, so one must try occasionally to see if the action is sufficient. When it is, the acid is poured off and the muscles washed gently with water to remove the acid. If one is ready to make the preparations at once they may be isolated and mounted in water. If it is desired to keep the specimen indefinitely or several days, the water should be poured off and a half saturated solution of alum added (4 314). The alum solution is also advantageous if the specimens are to be stained. The 176 COLLODION SECTIONING (CH. VII specimens may be mounted in glycerin, glycerin jelly or balsam. Glycerin jelly is the most satisfactory, however. Fic. 146. Pfeiffer’s preparation microscope with erecting prism between the objective and ocular (Leitz; Wim. Krafft, New York). THE PREPARATION OF SECTIONS OF TISSUES AND ORGANS 7 265. At the present time there are three principal methods of obtaining thin sections of tissues and organs for microscopic study. These methods are: The Collodion Method, the Parafjiin Method, and the Freezing Method. Each of these methods has its special application, although the collodion method is per- haps the most generally applicable, and the freezing method the most restricted, and is used mostly in pathological work where rapid diagnosis is necessary and the finest details of structure are not so important. With the paraffin method the thinnest sections may be made, and in some ways it is the most satisfactory of all. A good microtome is of great aid in sectioning. 7266. The Collodion Method.—In sectioning by this method the tissues are first hardened properly and then entirely infiltrated with collodion, and the collod- ion hardened. It is not removed from the tissue, since on account of its transpar- ency it does no harm, ‘ 7267. Fixing and Hardening the Tissue.—Any of the approved methods of hardening and fixing may be employed. A good general method which is applicable to nearly all of the tissues and organs is that by Picric-Alcohol. For the preparation of the solution see (% 333). A small piece of tissue or organ not CASKIL | COLLODION SECTIONING 177 containing more than two to three cubic centimeters is placed in 4o or 50 cc. of the picric-alcohol and left 6 to 24 hours, when the first picric-aleohol should be thrown away and fresh added. After one or two days more the picric- alcohol should be poured off and 67% alcohol added. Ina day or two this is re- placed by 75% or 82% alcohol ; 82% is on the whole most satisfactory, and thé tissue may be left in this till it is ready for dehydration. % 268. Dehydration before Infiltration.—When one is ready to imbed for sectioning, the tissue must first be dehydrated in plenty of 95% or stronger alcohol. It is better to take only a small piece for this. The smaller the piece the thinner the sections that may be made. The dehydration will usually be completed in 2 to 24 hours. If the alcohol is changed two or three times the dehydration will be hastened. 2269. Saturating with Ether-Alcohol.(% 322).—The next step is to remove the tissue from the alcohol and place it in a vial of ether-alcohol (% 322) for 2 to 24 hours. The dehydration becomes somewhat more complete by this step, and the tissue is more perfectly prepared for the reception of the collodion. If the dehydration is very thorough in the alcohol, this step may be ommitted, however, but one is surer of success if the ether-alcohol is used. %270. Infiltration with Thin Collodion.—The ether-alcohol is poured off, and a mixture of thin collodion is added (7% 319). Two or three hours will suffice for objects two or three millimeters in thickness. A stay of one or more days does no harm. The larger the object the more time is needed. 4271. Infiltration with Thick Collodion.—The thin collodion is poured off and thick collodion (7 319) added. For very small objects, four or five hours will suffice to infiltrate, but for larger objects a longer time is necessary. The tissue does not seem to be injured at allin the thick collodion, anda stay in it of a day or even a week is more certain to insure a perfect infiltration. 2272. Imbedding.—The tissue may be imbedded in a paper box, such as is used for paraffin imbedding, or in any of the other boxes devised for paraffin. It is better, if paper is used, to put asmall amount of oil on the paper to prevent the collodion from sticking to it. Vaselin spread over lightly and then re- moved, so far as possible, with a cloth or with lens paper, gives the right surface. For small objects it is more convenient to imbed immediately on a holder that may be clamped into the microtome. Cylinders or blocks of glass, vulcanite, wood and cork have all been recommended and used. A cork of the proper size is most convenient, and for many purposes answers well. Some collodion is put on the end of the cork and a pin put near one edge. The tissue is transferred from the thick collodion to the cork and leaned against the pin. Drops of the thick collodion are then poured on the tissue, and by moving the cork properly the thick viscid mass may be made to surround and envelop the tissue. Drops of collodion are added at short intervals until the tissue is well surrounded, and then as soon as a slight film hardens on the surface, the cork bearing the tissue is inverted in a wide-mouth vial of considerably larger diameter than the cork (Fig. 143). The vial should contain sufficient chlorform to float the cork. The vial is then tightly corked. In imbedding somewhat larger objects on the end of a cork or other holder, it is frequently advantageous to wind oiled paper around the holder or cork, tie it tightly and have the projecting hollow cylinder sufficiently long to receive the object. The tissue is then put into the cylinder and sufficient 178 COLLODION SECTIONING (CH. VII collodion added to completely immerse it. As soon asa film has formed over the exposed end, the cork may be inverted and immersed in chloroform as described above. For the use of ‘‘deck plugs’’ see % 274. 4273. Hardening and Clarifying the Collodion.—After a few hours the collodion is hardened by the chloroform. If it acts long enough, and if no water is present, the imbedding mass is rendered entirely transparent. Whenever the collodion is hard, whether it is clear or not, the chloroform is poured off and the castor-xylene* clarifier (4317) added. Ina few hours the imbedded mass will be- come as transparent as glass and the tissue will seem to have nothing around it. The tissue may remain for years in the castor-xylene. Sometimes the collodion remains white and opaque for a considerable time. So far as the writer has been able to judge, this is due to moisture. If one breathes on the mass too inuch while imbedding, or if it is very damp in the room, opacity may result. Sometimes, in objects of considerable size, this may remain for a week. Thisis the exception, however, and if the mass seems sufficiently hard and tough, the cutting may pro- ceed even if the clarification is incomplete. t+ In case the imbedding mass will not clarify after a few days the imbedded object may be placed in 95% alcohol for a day for dehydration, and then passed through chloroform and into the clarifier. There is usually no trouble in getting the mass perfectly clear in this way. If one is in a great hurry, the collodion may be hardened in 10 or 15 minutes by heating the bottle containing the chloroform in a water bath. The imbedding block of hardened collodion may then be transferred to the castor-xylene clarifier and kept warm. It will soon clear the collodion. One can then cut the sections. 7 274. Cutting the Sections.—For cutting the sections the collodion block is usually fastened to some form of holder. For small objects cork is fairly good. Blocks of glass, vitrified fiber, etc., have been used. If one uses wood the ‘‘deck plugs” of the shipwright are satisfactory. They are about the right length when one plug is made into two holders by sawing in two(Ewing & Ferguson). To fasten the collodion block to any form of holder, remove it from the castor-xylene, trim as desired, then dry the end on blotting paper, pour some thick collodion on the holder and press the collodion block down into the collodion. The evapora- tion usually fixes it in two or three minutes, when the holder may be clamped in the jaws of the microtome and the cutting proceed. For collodion sectioning a long, drawing cut is necessary in order to obtain thin, perfect sections. The ob- ject is, therefore, put in the jaws of the microtome at the right level, and the knife arranged so that half or more of the blade of the knife is used in cutting the section. It is advantageous also to have the object with its long diameter parallel with the edge of the knife. The surrounding collodion mass should be cut away, as in sharpening a lead pencil, so that there is not more than a thickness of about *The hydrocarbon xylene (C,H,,) is called xylol in German. In English, members of the hydrocarbon series have the termination ‘‘ene,’’ while members of the alcohol series terminate in ‘‘ol.’’ } The imbedded object may remain in the castor-xylene clarifier indefinitely without harm. The collodion grows somewhat tougher by a prolonged stay in it. After cutting all the sections desired at one time, the imbedded tissue is returned to the clarifier for future sections. CH. VILT| COLLODION SECTIONING 179 two millimeters all around the tissue. This is to render the diameter of the end to be cut as small as possible. The smaller the object the thinner can the sections be made. With an object two or three millimeters thick and not over five milli- meters wide, and a good sharp knife, sections 5/ to 6 can be cut without diffi- culty. When knife and tissue are properly arranged the tissue and the knife are flooded with the clarifier. Make the sections with a steady motion of the knife. Then draw the section up toward the back of the knife with an artist’s brush and make the next section. Arrange the sections in serial order on the knife blade till enough are cut to fill the area that the cover-glass willcover. For large objects one can cut thinner sections by a kind of sawing cut. 2275. Transferring the Sections to the Slide.—If the clarifier has evaporated so as to leave the sections somewhat dry on the knife, add asmall amount. Take a piece of thin absorbent, close-meshed paper about twice the size of a slide and place it directly upon the sections. Press the paper down evenly all around and then pull the paper off the edge of the knife. The sections will adhere to the paper. Place the paper, sections down, on a slide, taking care that the sections are in the desired position on the slide. Use some ordinary lens paper or some absorbent paper, and press it down gently upon the transfer paper. This will ab- sorb the oil, and then the transfer paper may be lifted, with a rolling motion, from the slide. The sections will remain on the slide. (See notes p. 180). 4 276. Fastening the Sections to the Slide.—Drop just enough ether- alcohol (equal parts of sulphuric ether and 95% alchol) on the sections to moisten them. This will melt the collodion and fasten the sections to the slide. Allow the slide to remain in the air till the surface begins to look slightly dull or glazed. Sometimes, especially when the air is moist, the sections wrinkle badly when the ether-alcohol is put on to fasten them to the slide. The excessive wrinkling can be avoided by using one part alcohol and two parts ether instead of using equal parts of each. Perhaps also it would be advantageous in this case to use absolute alcohol. Fic. 147. Reagent bottle with combined cork and pipette ( This 7s made by taking a cork of the proper size and making in ita hole | with a cork borer for the glass tube. Lt 1s advantageous to havea <7 string tied tightly around the rubber bulb as shown). TET 4277. Removing the Oil from the Sections.—As soon as the vas eX ether-alcohol has evaporated sufficiently to leave the surface dull, \ place the slide in a jar of ordinary commercial benzin. It may be || | left here a day or more without injury to the sections, but if moved around in the jar the oil will be removed in three to five | minutes. From the benzin transfer to a jar of 95% alcohol to | wash away the benzin. One may use alcohol in the beginning, | but it dissolves the oil far less rapidly than the benzin. The slide | may remain in the alcohol half a day or more if one wishes, but a | stay of five minutes or a thorough rinsing of half a minute or so ne .| by moving the slide around in the alcohol will suffice. Vee Xylene is to be preferred to benzin for removing the oil, but it is more expensive. 180 COLLODION SECTIONING [CH. VII 2 278. Staining the Sections with an Alcoholic Stain.—If an alcoholic stain containing 50% or more alcohol (for example, hydrochloric acid carmine in 70% alcohol) is used, the slide may be removed from the 95% alcohol, drained somewhat and then the stain poured upon the sections, or preferably, the slide immersed in a jar of the stain. The stain is finally washed away with 67% or stronger alcohol, the sections dehydrated in 95% alcohol, cleared and mounted in balsam. 2 279. Staining the Sections with an Aqueous Dye.—In staining with a watery stain, the slide bearing the sections is transferred from the 95% alcohol and plunged into a jar of water, and either allowed to remain a few minutes or moved around in the water a moment. Then it is placed horizontally and some of the stain placed on the sections with a pipette, or preferably, it is immersed in a jar of the stain; in case of immersion the slide should stand vertically or nearly so, then any particles of dust, etc., in the stain will settle to the bottom of the vessel and not settle on the sections. When the sections are stained, usually within five minutes, they are thoroughly washed with water either by the use of a pipette or preferably by immersing in a jar of water. They may then be counter- stained for half a minute with some general dye, like eosin or picric acid, or mounted with but the one stain. { *Various forms of paper have been used to handle the collodion sections. It should be moderately strong, fine meshed and not liable to shed lint, and fairly absorbent. One of the first and most successful papers recommended is ‘‘closet or toilet paper.’’ Cigarette paper is also excellent. In my own work the heavy white tissue paper has been found almost perfect for the purpose. Ordinary leus paper or thin blotting paper for absorbing the oil may be used with it. (4% 275). tIf one is a long time in cutting a series of sections, it sometimes occurs that the xylene evaporates, and while the sections may not look dry, they are practically in castor oil and not easily transferable. In sucha case fresh clarifier or even a little xylene to thin the oil on the sections may be used. If the oil is too thick it is viscid and there is difficulty in handling the sections with the paper as they stick rather firmly to the knife. (4275). {In the past the plan for changing sections from 95% alcohol to water, for ex- ample, has been to run them down gradually, using 75, 50 and 35% alcohol, suc- cessively. Each percentage may vary, but the principle of a gradual passing from strong alcohol to water was advocated. On the other hand I have found that the safest method is to plunge the slide directly into water from the 95% alcohol. The diffusion currents are almost or quite avoided in this way. There is no time for the alcohol and water to mix, the alcohol is washed away almost instantly by the flood of water. So in dehydrating after the use of watery stains, the slide is plunged quickly into a jar of 95% alcohol. The diffusion currents are avoided in the same way, for the water is removed by the flood of alcohol. This plan has been submitted to the severe test of laboratory work, and has proved itself perfectly satisfactory. GEE VIT) COLLODION SECTIONING 181 Fic. 148. Fic. 148. Waste bowl with rack for supporting slides and a small funnel in which the slides stand while draining. This outfit ts easily made by any tinsmith. The rack is composed of two brass rods about 4 mm.in diameter. The bent end pieces are sheet lead. The funnel is made of tin, copper or brass. Either copper or brass ts preferable to tin. A glass dish like that shown in Fig. 160 1s better than a bowl, as it can be more readily and thoroughly cleaned. (Cut loaned by Win. Wood & Co.) Fic. 149. Round glass aquarium. This glass vessel is better than the bowl for all the uses described for the bowl. (Whitall, Tatum & Co.) Fic. 150. Glass box or ointment jar with cover. These boxes may be had of various sizes and can be used advantageously for water, and for cleaning mixture for slides and cover glasses (4 242). (Whitall, Tatum & Co.) Fic. 151. Section lifter. This ts of thin, springy, flexible metal placed in a handle as shown. These are made of various sizes for large or small sections. Such an instrument ts exceedingly helpful in handling loose sections. (Queen & Co.) 182 COLLODION SECTIONING (CH. VIT Fic. 152. Perforated section lifter. This is easily made by soldering a wire to some very thin sheet brass or copper, and then perforating this with a coarse needle or fine awl. Any roughness must be removed by using a fine oil stone. ORDER OF PROCEDURE IN MAKING MICROSCOPICAL PREPARATIONS BY THE COLLODION METHOD 2 280. It will be seen from this table, and sections 267-281, that it requires about five days to get a microscopical preparation if one commences with the fresh tissue. Other methods of hardening might require as many months. It is evident, therefore, that one must exercise foresight in histology or much time will be wasted. I. Fixing and hardening the tissue s| 12, Removing the oil from the sections (% 267), 4 days or more. with benzin and alcohol (§ 277), 3-5 2. Dehydrating the objectto be cut in | Tata eee Wen 95% or stronger alcohol (4 268), 2- 24 hours. 13. Staining the sections with an alco- holic dye (% 278), 2 minutes to 24 3. Saturating the tissue in ether-alcohol hours. | (2 269), 2-24 hours. | 14. Staining the sections with an aque- ; ; : ‘ ous dye (% 279), 2-10 minutes. 4. Infiltrating with thin collodion | y @ 279) (% 270), 2 hours to 2 days. 15. Removing the superfluous dye by washing in water or alcohol (2 278- 279), 2-5 minutes. 5. Infiltrating in thick collodion(? 271), 5 hours to several days. ear : ‘ : | 16. Staining with a general dye (% 279), 6. Imbedding the tissue (7272), 15 to 15-30 seconds. 20 minutes. : : 17. Washing with water or alcohol (% 278-279), 1 to 2 minutes. 7. Hardening the collodion with chlo- roform (% 273), 5-24 hours. 18. Dehydrating the sections in 95% al- 8. Clarifying and further hardening the cohol (4281), § min. to 24 hours. collodion with castor-xylene (7 273), | 19. Clearing the sections (4281), 5 min- 10-36 hours. utes to 24 hours. . . Cutting the sections (7 274), ro min- | 20. Draining the sections, 1-2 minutes. , 274), g utes to 2 hours. 21. Mounting in Canada balsam (? 281), 10, Transferring the sections to a slide | I-2 minutes. with paper (% 275), 1 minute. 22. Labeling the preparation (7 308), 2 11, Fastening the sections to the slide | minutes. with ether-alcohol (4276), 1 or 2 23. Cataloging the preparation (2 309), minutes. 5-10 minutes. CH. Vil] PARAFFIN SECTIONING 183 2 281. Mounting in Balsam.—After the sections are stained they must be dehydrated and cleared before mounting in balsam. For the dehydration the slide is plunged into a jar of 95% alcohol. For clearing after the dehydration the slide is drained of alcohol and put down flat and theclearer poured on, or the whole slide is immersed in a jar of clearer (7 318). Clearing usually is sufficient in a few minutes ; a stay of an hour or even over night does not injure most sections. In mounting in balsam the clearer is drained away by standing the slide nearly vertically on some blotting paper, or by using the waste bowl and standing it up in the little funnel (Fig. 148). Then the balsam is put on the sections or spread on the cover-glass and that placed over the sections. For cataloging and labeling, see % 307-310. Fic. 153. Simall spirit lamp modified into a bal- sam bottle, or a glycerin or glycerin-jelly bottle, or a bottle for homogeneous timmersion liquid. For all of these purposes tt should containa glass rod. See also Fig. 168. , 282. The Collodion Method with Alcohol.—A good method of procedure for making collodion sections is to do exactly as described, including % 272, and then instead of hardening the collodion in chloroform and clarifier, it is hardened in 82% alcohol for a day or two before sectioning. In sectioning the knife and tissue are kept wet with 82% alcohol and the sections are dehydrated with 95% alcohol and then fastened to the slide with ether alone or with ether-alcohol. The staining and mounting (% 278-251) are as described. One may preserve the tissue after imbedding for a long time in the 82% alcohol before sectioning and sections may be made at any time. While this method appears somewhat simpler, the results are not so satisfactory as by the oil method given above. THE PARAFFIN METHOD § 283. As with the collodion method, the tissues are first properly fixed and hardened and then entirely filled with the imbedding mass, but unlike the collo- dion the mass must be entirely removed before the sections are finally mounted. The tissue thus imbedded and infiltrated is like a homogeneous mass and sections may be cut of extreme thinness. 7 284. Harden perfectly fresh tissue in picric-alcohol (% 333) from one to three days. (Any good method for fixing and hardening the elements may be used. One must observe in each case, however, the special conditions necessary 184 PARAFFIN SECTIONING (CAH. VIL for each method. The time might be longer or shorter than for the picric-alcohol. (See Lee, the Microtomists’ Vade-Mecuni. ) If picric-alcohol is used, pour it off after the proper time for fixing has elapsed, and add 67% alcohol. Leave this on the tissue from one to three days, and if it becomes very yellow it is well to change it twoorthreetimes. After two or three days pour off the 67% alcohol and add 82%. The tissue should remain in this one or two days, and it may remain indefinitely. In case the alcohol becomes much yellowed, it should be changed. 7285. Dehydration and Preparation for Imbedding.—From the pieces of tissue fixed and hardened in any approved manner, cut pieces 5 to 10 millimeters long and 2 to 3 millimeters in breadth. Place one or two pieces in a shell vial (Fig. 143.) and add 95% alcohol. Change the alcohol after two or three hours, and within 6 to 24 hours, depending on the size of the piece to be dehydrated, the dehydration will be completed. The secret of success is the use of plenty of alcohol and sufficient time. Absolute alcohol for the second change would act more promptly and efficiently, but if plenty of 95% is used one will succeed, unless the day, or the climate in general, is too damp. Fic. 154. Copper pail with water bottom for melting para- fin. This also serves asa water bath for large bottles in which saturated solutions of dichro- mate and other salts are pre- ie pared, [se (If one is studying organs, then the whole organ may need to be prepared for imbedding, but for minute structure small pieces are preferable, as thinner sections may be made. ) 4 286. Displacing Alcohol and Clearing Tissues with Cedar-wood Oil and Infiltrating with Paraffin.—(Lee, p. 66. Neelson and Schiefferdecker, Arch. fiir Anat. und Physiol., 1882, p. 206.) When the tissue is dehydrated it is removed to a vial of cedar-wood oil. When the alcohol used for dehydration is displaced by the oil, the tissue will look clear and translucent. This requires 2 to 24 hours. CHE VIT| PARAFFIN SECTIONING 155 It is hastened by warmth. It is then removed from the cedar-wood oil, drained, and placed in pure, melted paraffin, and this is then put into a paraffin oven and left from 2 to 24 hours (see Ch. X). It is then imbedded for sectioning. * Paraffin for infiltrating has usually a somewhat lower melting point than that forimbedding. Equal parts of paraffin of 43 C. and 54 C., answer well. For imbedding, the paraffin must be of a melting point which will give good ribbons in the temperature of the room where the sectioning is to be done. In a room of 19 to 20 C. a mixture of 1 part 43 C. paraffin with two parts of 54 C. usually answers well. Fic. 155. Hot filter for paraffin, gelatin, balsam, etc. It is entirely surrounded by a water jacket. The water is heated by placing a burner under the projecting part HT. The wire basket is to hold the strainer and allow a free flow of the filtered substance on all sides. There is a bail for suspending the filter, and the filtered sub- stance runs out through the narrowed part F. @ 287. Imbedding in Paraffin.—Make a small paper box, fill it nearly full of hot paraffin, place the box fora half minute on some cold water to makea thin solid layer on the bottom, then transfer the tissue to the box and arrange near one end so that the sections may be cut in the desired plane. Now place the paper box on as cold water as possible so that the paraffin may cool quickly. It will be more homogeneous if cooled quickly, and will shrink tightly against the tissue and avoid air spaces. In imbedding two main things should be looked after: The paraffin should be hot so that it will thoroughly fuse with the paraffin in the tissue. The tissue should be kept from the bottom of the box, either by holding it up in the middle of the box with warmed forceps or a perforated section lifter while a stratum *Thickened cedar-wood oil like that for oil immersion objectives is recom- mended by Lee for clearing. This is very expensive, and for most work unneces- sary. Any form of cedar-wood oil has been found satisfactory in the writer's laboratory. The great thing is to have the tissue thoroughly dehydrated before putting it into the oil. 186 PARAFFIN SECTIONING (CH. VII cools on the bottom, or a stratum of the paraffin may be cooled on the bottom before putting the tissue in the box. Cool quickly after the tissue is in place. Fic.156. Paraffin re- ceptacle P, with water bath and spout for par- affin imbedding (1883). 2 288. Cutting the Sections.—After the imbedding mass is well cooled, re- move the paper box and trim the end containing the tissue in a pyramidal form. clamp the block of paraffin in the holder of the microtome so that the tissue will be at the proper level for cutting. If a ribbon microtome is used, heat the holder and melt the end of the block upon it. Cool and place the holder in its place in the microtome. Use a very sharp, dry razor for cutting the sections. The sections are made with a rapid, straight cut asin planing. Do not try to section witha drawing cut as in collodion sectioning. If the temperature of the room is right for the paraffin used, the sections will remain flat, and if the opposite sides of the block are parallel, and one edge strikes the knife squarely, the sections will adhere and thus make aribbon. If the room is too cold for the paraffin the sections will roll. If it is too warm the sections will crumple. Remember the sections must be very thin, from 3 to 15 to show fine struc- tural details to good advantage. The secret of making good ribbons of sections is to have the block of parathn containing the tissue cut square and properly arranged in the microtome so that the block strikes the edge of the section knife at right angles with the edge ; and finally the paraffin must be of a proper melting point for the room in which the sections are to be cut. Remember that the larger the object the thicker must be the sections, and the softer the paraffin. Frequently one may modify the tem- perature if too cold by a Bunsen burner flame near the microtome. If it is too warm one may go to a basement room. 4289. Extending Sections with Warm Water.—Paraffin sections are liable to have fine wrinkles or folds in them. These folds are very annoying and often obscure the structure. To get rid of them the sections are extended or stretched upon warm water. One may put a ribbon of sections on warm water and then cut the ribbon into pieces and transfer the pieces to slides. Practically, however, the CH. Vil) PARAFFIN SECTIONING 187 extension is almost always accomplished on the slide itself. A slide is albumen- ized (7 290) and the ribbon cut into short pieces and placed on theslide. Distilled or filtered water is then added with a pipette (Fig. 147) until the sections float. Then the slide is moved back and forth over an alcohol or gas flame to warm the water. Care must be taken to avoid melting the paraffin. As the water warms the paraffin containing the sections will flatten and stretch out. One will be sur- prised at the amount of extension. It is necessary to take pieces considerably shorter than the cover-glass to be used or when extended the sections will not all be covered. After the sections are extended, arrange the ribbons carefully on the slide as shown in Fig. 162 if one is making serial sections. Arrange in the middle of the slide if only one or two sections are on each slide (Fig. 138). Let the ex- cess water drain off. Now let the slide stand several hours for the water to evapor- ate completely. The time will depend on the temperature and the dryness of the atmosphere. If there is plenty of time, leave the slides 24 or 48 hours. If one has a register with hot air intake, the slides may be put in the current of hot air. They willdry out in half an hour or an hour. Sections which have been left for a year have given excellent results. oO @ Fic. 157. Zvrays for slides and for ribbons of sections. The figures show the construction. It is important to have the bordering frame with rounded corners so that the trays muy be easily pulled out of a pile or reinserted. The screw eye shown in A makes tt easy to pull out a single tray. For ribbons of sections a piece of paper is placed in the tray and the ribbons are placed on it. (A) Face view, (B) Sec- tional view of the whole tray, (C) Sectional view of one side to show the construc- tion more clearly. Trays of this kind are so cheap ($15.00 per hundred for those holding 50 to 60 slides), that a laboratory can have a greatnumber, (Trans. Amer. Micr. Soc., 1899, p. 107). The slide trays (Fig. 157) are excellent for drying preparations of all kinds. i290. Fastening the Sections to the Slide.—To fasten the sections firmly to the slide, coat the slide with albumen fixative (7 312) asfollows: Putaminute 18S PARAFFIN SECTIONING (CHE IE drop of the albumen on the center of a slide and with a clean finger spread the albumen over the slide, wiping off all that is possible. Finally beat or tap the slide with the end of the finger. This will make a very thin (it cannot be too thin) and even layer. The sections are extended and dried as describedin 7% 289. When the sections are thoroughly dry they are in optical contact with the slide and have a shining appearance when looking on the back of the slide. When the sections are dry coat them with 34% collodion made as follows. Take 34 gram of soluble cotton, put it into a bottle and add 60 ce. of 95% or absolute alcohol, and 4o cc. of sul- phuric ether. Coat the sections with a soft camel’s hair brush. The collodion should dry in a minute or less. If one uses too much ether in this fixing collo- dion, the paraffin will be partly dissolved and will look moist for a long time. The slight excess of alcohol will obviate any such difficulty. Fic. 158. A slide holder and Fic. 759. Slide holier with the bottle for containing the same (Mix, bail hinged so that it may be turned Journal of Applied Microscopy, vol. aside in inserting or removing the 1, 1898, p. 169.) slides, When the collodion is dry place the slide in benzin or xylene to dissolve the paraffin (see 7 291). If the sections are not extended on water, they may be put directly on the albumenized sides, pressed down with the finger and coated with collodion. This is much more rapid, but does not get rid of the fine folds. (See Dr. Agnes Claypole Trans. Amer. Micro. Soc. 1894, p. 66, 127.) 4 291. Removing the Paraffin.—Immerse the slide ina vessel of xylene or benzin. This will dissolve the paraffin. An hour will usually suffice. One can CH. VII) PARAFFIN SECTIONING 189 hasten the solution of the paraffin by moving the slide in the solvent. In this way it may be dissolved in 5 to 10 minutes, or even less. It will do no harm to leave the slide in the benzin or xylene over night. Two or three days even might not do any harm, but it is usually better to proceed at once to the other operations. Fic. 160. Apparatus and regents with which the slide holders are used. With this apparatus it ts easy to prepare specimens in large numbers very expeditiously. After the sections are fastened to the slide and placed in the holder, the slides need not be touched during all the operations until they are finally ready to be mounted in balsam. Each holder contains from 12 to rg slides. (§ 277-300). The bottles for the reagents are glass stoppered specimen or museum bottles. (Mix, Jour. Ap. Micr. 1898, p. 771.) Compare Fig. 14s. 3.292. Removing the Xylene or Benzin—From the xylene or benzin plunge the slide bearing the sections into a jar of 95% alcohol, and leave it for a few minutes, or move it around in the alcohol for half a minute or so. 2 293. Staining the Sections with an Alcoholic Dye.—With an alcoholic stain like hydrochloric acid carmine, remove the slide from the alcohol, and add the stain directly after draining the slide, Do not allow the stain to become dry, for that would injure the tissue. Wash away the stain with 67% alcohol, then dehydrate with 95% alcohol, clear and mount in balsam as described below. Z 294. Staining with as Aqueous Dye.—Wash away the 95% alcohol from the slide bearing the sections by plunging it into a jar of water and moving it around a moment. Then add the stain to the sections with a pipette, or immerse the slide in a jar of the stain, and allow the stain to act from 5 to Io minutes. Wash thoroughly with water. 7295. Staining with a General Dye—Counterstaining.—If it is desired to give a general stain after the nuclear dye (% 294), carmine stained preparations may be tinted with picric-alcohol for half a minute or more (7 333), and the hem- atoxylin stained specimens with eosin (4 321). It usually takes less than a minute for this. Wash away the counterstain with water. 190 PARAFFIN SECTIONING (CH. VII 2 296. Counterstaining with Picro-fuchsin. —For a general dye to use with hematoxylin, eosin is good, but to differentiate the tissues more completely, especially connective tissue, which is present in practically every section made, it is better to use Van Gieson’s mixture of picric acid and acid fuchsin. (Picric acid, saturated aqueous solution 75 cc., water 25 cc. 1% aqueous solution of acid fuchsin, ro cc.) Sections are first strongly stained with hematoxylin, well washed with water and then stained 3 seconds to 15 minutes in the picro-fuchsin. They are then washed in distilled water ; or in tap water, to which has been added a drop or two of glacial acetic acid to 100 cc. of water. They are then dehydrated, cleared and mounted in acid balsam, that is in balsam which has not been neutral- ized (7 315). If glycerin or glycerin jelly is used as a mounting medium it should be slightly acid. Unless the mounting medium is slightly acid, the red of the acid fuchsin soon fades. In some cases less acid fuchsin should be used, and in some a greater amount. Acid fuchsin alone without the picric acid is also good for a counterstain, The picro-fuchsin is a very valuable differential stain and combined in different proportions with picric acid will give great assistance in al- most every case. It does not seem to be a permanent stain. (See Freeborn, Trans. N. Y. Path, Soc., 1893, p. 73. Also studies from the department of pathol- ogy of the College of Physicians and Surgeons, Columbia University, N. Y., 1894-1895). r6r, Coplin’s staining dish. A. The entire dish, B. the dish in cross section. This is made of glass and is a very neat piece of apparatus, With it ten slides may be stained at once. (Whitall, Tatum & Co.) 4297. Dehydration of the Stained Sec- tions.—Place the slide with the stained sec- lions in a jar of 95% or absolute alcohol and leave it a few minutes, or wave it around in | the alcohol for half a minute or so. Remember that the larger and thicker y yV) the sections the more time it requires to de- a eae hydrate them. In a moist atmosphere, 95% IN POSITION. alcohol is not completely satisfactory, and one must use a stronger alcohol. When it is dry, 95% answers very well. 7298. Clearing the Sections.—Drain off the alcohol, and place the slide in a jar of clearer (7 318, A or B) or put a drop or two of clearer on the sections. The clearing is usually accomplished in two or three minutes. 2299. Mounting in Balsam.—For this the clearer is drained from the slide, and wiped away with blotting paper, cloth, etc. The balsam is then put upon the sections and the cover added, or a cover-glass is spread with the balsam and then put over the sections. (If the sections show a whitish appearance and are opaque they were not sufficiently dehydrated. If natural balsam is used the sections will clear up in time). CH. VIT) SERIAL SECTIONS Ig1 ORDER OF PROCEDURE IN MAKING MICROSCOPICAL PREPARATIONS BY THE PARAFFIN METHOD. 2300. It will be seen from this table and from sections 283 to 299 that it re- quires from 5 to 7 days to get a microscopical preparation by the paraffin method if one starts with a fresh tissue. Depending on the method of fixing and harden- ing, the time may be much greater. Much time will be lost in waiting unless one plans ahead in histological work. 1. Fixing and hardening the tissue or | 11. Washing with water, note, p. 180. organ (% 284), 4 days or more. 12. Staining with an aqueous dye 2. Dehydrating the object to be cut in (2.294), 2 minutes to 24 hours. 95% or stronger alcohol (7% 285), 1 to 24 hours. Displacing the alcohol and clearing ide ; A tissues with cedar-wood oil. (See iq, Stating with a general dye (% 295- @ 286), 2 to 24 hours. 296), 10 seconds to Io minutes. 4. Infiltrating the tissue with paraffin | 15. Washing the sections with water in the paraffin oven (% 286), 2 to (4% 295-296). 24 hours. 13. Washing away the superfluous stain with water (4% 294). ) oo 16. Dehydrating the stained sections in 5. Imbedding in paraffin (7% 287), io 95% alcohol (7% 297), 3 minutes to minutes. . | 24 hours. 6. Cutting the sections(% 2838), 10 min- | 17. Clearing the sections (% 298) 2 min- utes. utes to 24 hours. 7. Extending the sections with warm) 18. Mounting in Balsam (% 299), 1 to 5 Water. (See % 289.) | minutes. 8. Fastening the sections to a slide | 19. Sealing the cover-glass (7% 253), 2 (% 290), 5 minutes to 24 hours. | minutes. g. Removing the paraffin (7291), Io | 20. Labeling the preparation (% 308), 2 minutes to 24 hours. | minutes. é | ; : 5 io. Removing the xylene or benzin | 21. Cataloging the preparation (4 309), (4 292). 5 to Io minntes. SERIAL SECTIONS 2301. In histological studies it is frequently of the greatest advantage to have the sections in serial order, then an obscure feature in one section is fre- quently made clear by the following or preceding sections. While serial sections are very desirable in histological study, they are absolutely necessary for the solu- tion of morphological problems presented in complex organs like the brain, in embryos and in minute animals where gross dissection is impossible. 7302. Arrangement of Tissues for Sections in Histology.—They should be so arranged that the exact relations of each part to the organ can be readily deter- mined. For example, an organ like the intestine, a muscle or a nerve, should be so arranged that exact transections or longisections can be made. Organs like the liver and other glands, the skin, etc., should be so arranged that sections parallel with the surface or at right angles to it, (surface or vertical sections) may be made, Oblique sections are often very puzzling. 192 SERIAL SECTIONS CAL VIL, With cylindrical objects, especially botanical specimens, one may cut tangen- tial sections, 7. ¢., sections at right angles to a radius, or parallel with the radii (radial sections), or transections, 7. é., sections across the long axis. 7303. Arrangement of Serial Sections.—The numerical order may be very conveniently like the words on a printed page, 7. ¢., beginning at the upper left hand corner and extending from left to right. The position of the various aspects of the sections should be in general such that when they are under the compound microscope the rights and lefts will corre- spond with those of the observer. This may be accomplished as follows for sec- tions made in the three cardinal sectional planes, 7vansections, Frontal Sections, Sagittal Sections : (C) Transection, 7. ¢., sections across the long axis of the embryo or animal dividing it into equal or unequal cephalic and caudal parts. (a) In accordance with the generally approved method of numbering serial parts in anatomy, the most cephalic section should be first (Fig. 162). (b) The caudal aspect of the section should face upward toward the cover- glass, the cephalic aspect being next the slide. (c) The dorsal aspect should face toward the upper edge of the slide (Fig. 162). This arrangement may be easily accomplished for the transections as follows : Imbed the embryo with the right side down, taking the precautions against letting the embryo rest against the bottom of the box (% 287). (1) Mount the block of paraffin containing the embryo so that the tail end will be next the microtome holder. The head will then be cut first. (2) Place in the microtome so that the right side of the embryo will meet the edge of the knife. (3) Mount as a printed line and the first or cephalic section will be at the upper left hand corner, and the dorsal aspect of the embryo will be toward the upper edge of the slide. Under the microscope the rights and lefts will appear as in the observers own body, also the dorsal and ventral aspects so that he can easily locate parts by comparing them with his own body. (B) Frontal Sections, 7. ¢., sections lengthwise of the embryo or animal and from right to left (dextral and sinistral), so that it is divided into equal or unequal dorsal and ventral parts. Imbed the embryo with the right side down in the imbedding box as before. (1) Mount the paraffin block so that the ventral side of the embryo is next the microtome holder. The dorsal side will then be cut first. (2) Let the right side of the embryo meet the edge of the knife. (3) Mount the first section on the left end of the slide as before and so that the sections will be crosswise on the slide, the tail toward the upper edge. Under the compound microscope the head will appear toward the upper edge and the rights and lefts will be as in the observer’s own body. (C) Sagittal Sections, that is sections lengthwise of the embryo or animal and from the ventral to the dorsal side, thus dividing the body into equal or unequal right and left parts. For these sections imbed the embryo as in the two preceding cases. CH. VII) SERIAL SECTIONS 193 (1) Put the right side of the embryo next the microtome holder, then the left side will be cut first. (2) Let the caudal end meet the knife edge. (3) Put the first section in the upper left hand part of the slide as in the other cases. The sections will be lengthwise of the slide. This will bring the ventral side up and the head to the right on the slide. Under the microscope the head will appear at the left and the dorsal side toward the upper edge of the slide. 2 304. For serial sections with collodion imbedded objects it is a great advan- tage to have the imbedding mass unsymmetrically trimmed, so that if a section is accidentally turned over it may be easily noticed and rectified. Furthermore it is imperatively necessary that the object be so imbedded that the cardinal aspects, dextral and sinistral, dorsal and ventral, cephalic and caudal, shall be known with certainty. 2305. Thickness of Cover-Glass and of Serial Sections.—It is a great advan- tage to use very thin cover-glasses ( 0.12-0.18 mm.) for serial sections, then the cover will not prevent the use of high powers. When the ordinary slides (25 < 76mm., 1 3 inch) are used cover-glasses 24 >< 50 mm. may be advantageously employed. 79 Homo 5 5 Slide 10 10 86 Sec’s 79 T. 20u 100 9% 1901 Fie, 162; Fic. 162. A slide of serial sections showing the position of the sections, the label and the size of the cover-glass (24 X 50mm.). The numbers next the cover at the left indicate the first section of each row, and illustrate the method of placing the sections as if they were words on a printed page. T. Transections. The combined thickness of the sections on a slide is easily determined by not- ing carefully the position of the microtome screw at the first and last sections and measuring the elevation. With good modern automatic microtomes the successive sections are almost exactly uniform in thickness, hence it is easy to determine the combined thickness of the sections on a slide. % 306. Labeling Serial Sections.—The label of a slide on which serial sections are mounted should contain at least the following : The name of the embryo and the number of the series; the number of the slide of that series ; the thickness of the sections, and the number of the first and last section on the slide; the date. It is also a convenience to have the informa- tion repeated in part on the left end and the number of the first section in each row, as shown in the sample slide of serial sections. (Fig. 162). 194 LABELING AND CATALOGING (CH. VII LABELING, CATALOGING AND STORING MICROSCOPICAL, PREPARATIONS 2307. Every person possessing a microscopical preparation is interested in its proper management ; but it is especially to the teacher and investigator that the labeling, cataloging and storing of microscopical preparations are of import- ance. ‘‘To the investigator, his specimens are the most precious of his possessions, for they contain the facts which he tries to interpret, and they remain the same while his knowledge, and hence his power of interpretation, increase. They thus form the basis of further or more correct knowledge; but in order to be safe guides for the student, teacher, or investigator, it seems to the writer that every preparation should possess two things: viz., a label and a catalog or history. This catalog should indicate all that is known of a specimen at the time of its prepara- tion, and all of the processes by which it is treated. It is only by the possession of such a complete knowledge of the entire history of a preparation that one is able to judge with certainty of the comparative excellence of methods, and thus to discard or improve those which are defective. The teacher, as well as the investigator, should have this information in an accessible form, so that not only he, but his students can obtain at any time, all necessary information concerning the preparations which serve him as illustrations and them as examples.” 2308. Labeling Ordinary Microscopical Preparations.—The label should possess at least the following information (see % 306 for serial sections): EXAMPLE (1) The number of the preparation, the | No. 475 Cs 15 thickness of the cover-glass and | : : Secs. 8 ju of the sections under it. (2) The name and source of the prepara- Striated Muscle; transection of the tion. Sartorius of the Cat. (3) The date of the specimen ( 2 of | catalog.) \October 15, 1894. wo. DO &IS 3/0 HL zy ever a Fic. 163. Laample of a label of an ordinary histologic ¢ specimen. (See also Fig. 162 for serial sections). cate. Oct. 4 /o: Fig. 163. @ 309. Cataloging Preparations.—It is believed from personal experience, and from the experience of others, that‘each preparation (each slide or each series) should be accompanied by a catalog containing at least the information suggested in the following formula. This formula is very flexible, so that the order may be changed, and numbers not applicable in a given case may be omitted. With many objects, especially embryos and small animals, the time of fixing and hardening may be months or even years earlier than the time of imbedding. So, too, an object may be sectioned a long time after it was imbedded, and finally the sections CA. VIT) LABELING AND CATALOGING 195 may not be mounted at the time they are cut. It would be well in such cases to give the date of fixing under 2, and under 5, 6 and 8, the dates at which the oper- ations were performed if they differ from the original date and from one another. In brief, the more that is known about a preparation the greater its value. General Formula for Cataloging Mi- croscopical Preparations : 1. The general name and source. Thickness of cover glass and of section. | 2. The number of the preparation and the date of obtaining and fixing the specimen ; the name of the preparator. 3. The special name of the prepara- tion and the common and scientific name of the object from which it is de- | rived. Purpose of the preparation. 4. The age and condition of the ob- ject from which the preparation is de- rived. Condition of rest or activity; fasting or full fed at the time of death. 5. The chemical treatment, — the | method of fixing, hardening, dissociat- ing, etc., and the time required. 6. The mechanical treatment,—im- bedded, sectioned, dissected with nee- dles, etc. Date at which done. 7. The staining agent or agents and the time required for staining. 8. Dehydrating and clearing agent, | mounting medium, cement used for sealing. g. The objectives and other accesso- ries (micro-spectroscope, polarizer, etc.,) | for studying the preparation. 10. Remarks, including references to | | 10. A Catalog Card Written According to this Formula: Muscular Fibers. Cat. Cras: Fibers 20 to 4o u thick. 2. No. 475. (Drr. IX) Oct. 1, 1891. S. H. G., Preparator. 3. Tendinous and intra-muscular ter- minations of striated muscular fibres from the Sartorius of the cat (Felis do- mestica). 4. Cat eight months old, healthy and well nourished. Fasting and quiet for 12 hours. 5. Muscle pinned on cork with vas- elined pins and placed in 20 per cent. nitric acid immediately after death by chloroform. Left 36 hours in the acid; temperature 20° C. In alum water (% sat. aq. sol.) 1 day. 6. Fibers separated on the slide with needles, Oct. 3. 7. Stained 5 minutes with Delafield’s hematoxylin. 8. Dehydrated with 95% alcohol 5 minutes, cleared 5 minutes with carbol- turpentine, mounted in xylene balsam ; sealed with shellac. g. Usea16 mm. for the general appear- ance of the fibers, then a 2 or 3 mm. ob- jective for the details of structure.’ Try the micro-polariscope (¢ 218). The nuclei or muscle corpuscles are very large and numerous; many of the intra-muscular ends are branched. See S. P. Gage, Proc. Amer. Micr. Sci., 1890, original papers, or to good figures and p. 132; Ref. Hand-book Med., Sci., Vol. descriptions in books. V., p. 59: % 310. General Remarks on Catalogs and Labels.—It is especially desirable that labels and catalogs shall be written with some imperishable ink. Some form of water-proof carbon ink is the most available and satisfactory. The water-proof India ink, or the engrossing carbon ink of Higgins, answers well. As purchased, the last is too thick for ordinary writing and should be diluted with one-third its volume of water and a few drops of strong ammonia added. 196 LABELING AND CATALOGING ( CH. VIL If one has a writing diamond it is a good plan to write a label with it on one end of the slide. It is best to have the paper label also, as it can be more easily read. Fic. 164. Writing diamond for writing numbers and labels on glass slides, cutting cover-glasses, etc. (Queen and Co.) The author has found stiff cards, 12% x 7!4 cm., like those used for cataloging books in public libraries, the most desirable form of catalog. A specimen that is for any cause discarded has its catalog card destroyed. New cards may then be added in alphabetical order as the preparations are made. In fact a catalog on cards has all the flexibility and advantages of the slip system of notes (see Wilder & Gage, p. 45). Some workers prefer a book catalog. Very excellent book catalogs have been devised by Alling and by Ward (Jour. Roy. Micr. Soc., 1887, pp. 173, 348 ; Amer. Monthly Micr. Jour., 1890, p. 91; Amer. Micr. Soc. Proc., 1887, p. 233). The fourth division has been added as there is coming to be a strong belief, practically amounting to a certainty, that there is a different structural appearance in many if not all of the tissue elements depending upon the age of the animal, upon its condition of rest or fatigue ; and for the cells of the digestive organs, whether the animal is fasting or full fed. Indeed as physiological histology is recognized as the only true histology, there will be an effort to determine exact data concerning the animal from which the tissues are derived. (See Minot, Proc. Amer. Assoc. Ady. Science, 1890, pp. 271-289; Hodge, on nerve cells in rest and fatigue, Jour. Morph., vol. VII. (1892), pp. 95-168; Jour. Physiol., vol. XVIL., pp. 129-134; Gage, The processes of life revealed by the microscope ; a plea for physiological histology, Proc. Amer. Micr. Soc., vol. XVII. (1895), pp. 3-293 Science, vol. II., Aug. 23, 1895, pp. 209-218. CABINET FOR MICROSCOPICAL PREPARATIONS 7311. While it is desirable that microscopical preparations should be pro- perly labeled and cataloged, it is equally important that they should be protected from injury. During the last few years several forms of cabinets or slide holders have been devised. Some are very cheap and convenient where one has but a few slides. For a laboratory or for a private collection where the slides are numerous the following characters seem to the writer essential : (1). The cabinet should allow the slides to lie flat, and exclude dust and light. (2). Each slide or pair of slides should be in a separate compartment. At each end of the compartment should be a groove or bevel, so that upon depressing either end of the slide the other may be easily grasped (Fig. 165). It is also desirable to have the floor of the compartment grooved so that the slide rests only on two edges, thus preventing soiling the slide opposite the object. (3). Each compartment or each space sufficient to contain one slide of the standard size should be numbered, preferably at each end. If the compartments are made of sufficient width to receive two slides, then the double slides so fre- quently used in mounting serial sections may be put into the cabinet in any place desired. CH, VIT | LABELING AND CATALOGING Fic. 165. partof a cabinet drawer seen Srom above. In compartment No. 96 ts repre- sented a slide lying flat. The label of the Slide and the number of the compartment are so placed that the number of the compartment may be seen through the slide. The sealing cement ts removed at one place to show that in sealing the cover-glass, the cement is put partly on the cover and partly on the slide (4% 249, 253). B.—This represents a section of the same part of the drawer, (a) Slide resting as in a. No. 96. The preparation ts seen to be above a groove in the floor of the compartment. (b) One end of the slide ts seen to be uplifted by de- pressing the other into the bevel. (4). The drawers of the cabinet should be entirely independent, so that any drawer may be partly or wholly removed without disturb- ing any of the others. (5). On the front of each drawer should be the number of the drawer in Roman numer- als, and the number of the first and last com- partment in the drawer in Arabic numerals (Fig. 166). Fic. 166. Cabinet for M1- 197 | | | _ | 1660) | _ | Aerve fibers|| | Caz — 70 ] FIG. 165. thy \ i croscopical Specimens, show- ing the method of arrange- mént and of numbering the drawers and indicating the number of the first and last compartment in each drawer. Lt is better to have the slides on which the drawers rest somewhat shorter, then the drawer front may be entire and not notched as here shown. (From. Proc. Amer. Micr. Soc., 1883). Ee 337-357 358-378 SEE eq00-42 — 189 : PREPARATION OF REAGENTS (CH. VII Fic. 167. Slide box for 25 spect- mens. These are cheap and conventent and may be stood on end like books, thus placing the slides in a horizontal position. Smaller boxes, 1.e. for 3,6 and 12 slides are also made, and mail- ing boxes for asingle slide (Bausch & Lomb Co.). SOME REAGENTS FOR FIXING, MOUNTING, ETC. 2312. Albumen Fixative (Mayer’s).—This consists of equal parts of well- beaten white of egg and glycerin. To each 50 cc. of this 1 gram of salicylate of soda is added to prevent putrefactive changes. This must be carefully filtered. For method of use see ? 290. 2313. Alcohol (Ethylic).—Ethyl alcohol is mostly used for histological pur- poses. (A) absolute alcohol (7. ¢., alcohol of 745%) is recommended for many purposes, but if plenty of 95% alcohol is used it answers every purpose in histology, in a dry climate or in a warm, dry room. When it is damp dehydration is greatly facilitated by the use of absolute alcohol. (B) 82% alcohol made by mixing 5 parts of 95% alcohol with 1 part of water. (C) 67% alcohol made by mixing 2 parts of 95% alcohol with 1 part of water. Grades of Alcohol. It has been found by careful tests that quite accurate percentages of alcohol may be obtained by mixing water and alcohol as follows : Pour alcohol into a graduate until the volume of alcohol corresponds to the de- sired percentage. Add water until the volume in cubic centimeters corresponds to the original percentage of the alcohol used. For example, to get 67% from 95% alcohol, pour 67 cc. of 95% into a graduate, and add sufficient water to bring the volume up to 95 cc. Far 50% alcohol from 75%, put 50 cc. of 75% alcohol in a graduate, add sufficient water to make the volume 75 cc. From the change in volume it does not answer to mix given volumes of water and alcohol in these cases. In the first case, if one mixed 75 cc. of 95% alcohol and 20 cc. of water the resulting mixture would be over 75% ; but if sufficient water is added to bring the volume back to the original percentage more than 20 cc. of water is added, that is enough more to compenate for the shrinkage, and the result is approximately accurate. Methyl Alcohol is much cheaper than ethyl, and answers well in most micro- scopical processes. It has recently been so carefully refined that the disagreeable odor is very little noticeable. Synthol. This is said to be a synthetic form of alcohol. It seems to serve the purpose of alcohol in histology. Absolute synthol is far cheaper for the private worker than absolute ethyl alcohol. 2314. Alum Solution.—For muscle dissociated in nitric acid use a satu- rated solution (2. é., a solution in which the water holds all the alum it can. If one adds an excess so that there will always be some undissolved alum in the ves- sel he can be sure the solution is saturated after it has stood a few days. An easy way to get a saturated solution is to take 500 cc. of water and add 100 gramis of alum and heat the water in an agate dish. All the alum will be melted, but on CH. VII) PREPARATION OF REAGENTS 199 cooling a part will crystallize out, leaving a cold saturated solution). The satu- rated solution may be used but, if a half saturated solution is employed, it will answer all the purposes. Fora half saturated solution take 100 cc. of water, and 1oo cc. of saturated alum water and mix the two. In case preparations are to be kept some time in alum water, 2% of chloral hydrate should be added to prevent mold. 2315. Balsam, Canada Balsam, Balsam of Fir; Xylene Balsam.—This is one of the oldest and most satisfactory of the resinous media used for mounting microscopical preparations. The natural balsam is most often used, but within the last ten years the belief has arisen that it is better to evaporate the balsam and then dissolve it in xylene or benzole. It certainly dries out more rapidly if so treated. Natural balsam has the advantage of being able to take up a small amount of water so that if sections are not quite dehydrated they will clear up after atime. If xylene balsam is used the dehydration must be almost complete or the preparation will look cloudy. Filtering Balsam. Balsam is now furnished already filtered through filter paper. If one wishes to filter it himself a hot filter like that shown in Fig. 155 is good. If xylene balsam is used it may be made thin and filtered without heat. For filtering balsam and all resinous and gummy materials, the writer has found a paper funnel the most satisfactory. It can be used once and then thrown away. Such a funnel may be easily made by rolling a sheet of thick writing paper in the form of a cone and cementing the paper where it overlaps, or winding a string several times around the lower part. Such a funnel is best used in one of the rings for holding funnels. Fic. 168. Vessel for homogeneous immersion liquid (thick cedar-wood oil). This is filled only a little above the lower end of the inner tube. The oil will not then run outif the vessel is tipped over. Lor applying the oil there isa wire loop attached to the upper cork. The side cork is for the pur- pose of emplying the bottle, and also for the escape of the air when filling it. (The Spencer Lens Co.; see also Zeit. wiss. Mikr., 1897, p. 348, and Jour. Roy. Micr. Soc., 7898, Dp. 238). Natural Balsam. All the samples of balsam tested by the author have been found slightly acid. This is an advantage for carmine, and acid fuchsin stains or any other acid stain.. Also for preparations injected with carmine or Berlin blue. In these cases the color would fade or diffuse if the medium were not slightly acid. For hematoxylin the acid is detrimental. For example, the slight amount of acid in the balsam will cause the delicate stain in the finest fibers of Weigert preparations to fade. Also the fuchsin and other stains which are faded by acids. To neutralize the balsam add some pure sodium carbonate, set the balsam in a warm place and shake it occassionally. After a month or so the soda will settle and the clear supernatant balsam will be found very slightly alkaline. Use this whenever an acid medium would fade the stain in the specimen. 200 PREPARATION OF REAGENTS (CH. VII 2316. Cedar-Wood Oil.—This is used for oil immersion objectives. It is best kept in a bottle like that shown in Fig. 168. This oil is quite thick. For penetrating tissues and preparing them for infiltration with paraffin, thick oil is recommended by Lee. The writer has found, however, that any good cedar wood oil gives excellent results in ordinary histological and embryological work. That known as Cedar Wood Oil (Florida) is excellent, also that known as cedar wood oil (true Lebanon). These forms are far less expensive than that used for immersion objectives. The tissues should be thoroughly dehydrated before put- ting them into cedar-wood oil, and they should remain until they are transparent (4 286). 4317. Clarifier, Castor-Xylene Clarifier.—This is composed of castor oil 1 part and xylene* 3 parts. (Trans. Amer. Micr. Soc., 1595, p. 361). % 318. Clearing Mixture (7281, 298).—(A). One of the most satisfactory and generally applicable clearers is carbol turpentine, made by mixing carbolic acid crystals (Acidum carbolicum. A. phenicum crystallizatum) 40 cc. with rectified oil of turpentine (Oleum terebinthinae rectificatum) 60 cc. If the carbolic acid does not dissolve in the turpentine add 5 cc. of 95% alcohol, or increase the tur- pentine, thus: carbolic acid 30 cc., turpentine 70 cc. (B). Carbol-Xylene Clearer.—Vasale recommends as a clearer, xylene 75 cc., carbolic acid (melted crystals) 25 cc. It is used in the same way as the preceding. % 319. Collodion.—This is a solution of soluble cottont or other form of pyroxy- lin in equal parts of sulphuric ether and 95% alcohol. Two solutions are used: *The hydrocarbon xylene (C,H,,) is called xylol in German. In English members of the hydrocarbon series have the termination ‘‘ene’’ while members of the alcohol series terminate in ‘‘ol.”’ {The substance used in preparing collodion goes by various names, soluble cot- ton or collodion cotton is perhaps best. This is cellulose nitrate, and consists of a mixture of cellulose tetranitrate C,,H;,(NO,),O,, and cellulose pentanitrate, C,.H,;(NO;),0,;. Besides the names soluble and collodion cotton, it is called gun cotton and pyroxylin. Pyroxylin is the more general term and includes several of the cellulose nitrates. Celloidin is a patent preparation of pyroxylin, more ex- pensive than soluble cotton, but in no way superior to it for imbedding. Soluble cotton should be kept in the dark to avoid decomposition. After it is in solution this decomposition is not so liable to occur. The decomposition of the dry cotton gives rise to nitrous acid, and hence it is best to keep it in a box loosely covered so that the nitrous acid may escape. Cellulose nitrate is explosive under concussion and when heated to 150° centi- grade. In the air, the loose soluble cotton burns without explosion. It is said not to injure the hand if held upon it during ignition and that it does not fire gun- powder if burned upon it. So far as known to the writer, no accident has ever occurred from the use of soluble cotton for microscopical purposes. I wish to ex- press my thanks to Professor W. R. Orndorff, organic chemist in Cornell Univer- sity, for the above information. Proc. Amer. Micr. Soc., vol. XVII (1895), pp. 361-370. CA VI) PREPARATION OF REAGENTS 201 (A). 6% to 8% or thick collodion. It is made by mixing 50 cc. of sulphuric ether and 50 cc. of 95% alcohol and adding 6 or 8 grams of soluble cotton. If this is shaken repeatedly the solution will be complete in a day or two. (B). 114% or ¢hin collodion. To prepare this 1% grams of soluble cotton are added to 100 ce. of ether-alcohol (7 322). 2320. 34% collodion or cementing collodion. To, prepare it 3{ths of a gram of soluble cotton is added to alcohol, 60 cc., ether, 4o cc. The excess of alcohol prevents the mixture from softening the paraffin (7 290). 7321. Eosin.—This is used mostly as a contrast stain with hematoxylin, which is an almost purely nuclear stain. It serves to stain the cell-body, ground sub- stance, etc., which would be too transparent and invisible with hematoxylin alone. If eosin is used alone it gives a decided color to the tissue and thus aids in its study (% 144). Eosin is used in alcoholic and in aqueous solutions. A very satis- factory stain is made as follows: 5o cc. of water:and 50 cc. of 95% alcohol are mixed and r-Ioth of a gram of dry eosin added. The eosin is used after the hematoxylin in most cases (7 280), and, as it is in alcoholic solution, it may be washed off with 95% alcohol if the object is to be mounted in balsam. If it is to be mounted in glycerin or glycerin jelly, the excess of eosin should be washed away with distilled water. 4322. Ether, Ether-Alcohol.—Sulphuric ether is meant when ether is men- tioned in this book. For the ether-alcohol mentioned in 7 269, 319, etc., a mixture of equal volumes of sulphuric ether and 95% alcohol is meant. 4323. Farrants’ Solution.—Take 25 grams of clean, dry, gum arabic; 25 cc. of a saturated aqueous solution of arsenious acid; 25 cc. of glycerin. The gum arabic is soaked for several days in the arsenic water, then the glycerin is added and carefully mixed with the dissolved or softened gum arabic. This medium retains air bubbles with great tenacity. It is much easier to avoid than to get rid of them in mounting. 4324. Formaldehyde Dissociator.—This is composed of 2 cc. of a 40% solu- tion of formaldehyde in 1000 cc. of water, to which 6 grams of common table salt (sodium chlorid) have been added. Formaldehyde as bought in the market is a 40% solution in water, and is called formol, formalin, formalose and formal, the last name being the preferable one. For its use in isolating cells see % 260. (Micr. Bulletin and Sci. News, vol. XII. (1895), pp. 4-5). 3325. Glycerin—(A). One should procure pure glycerin for a mounting medium. It needs no preparation, except in some cases it should be filtered through filter paper or absorbent cotton to remove dust, etc. For preparing objects for final mounting, glycerin 50 cc., water 50 cc., forms a good mixture. For many purposes the final mounting in glycerin is made in an acid medium, viz., Glycerin 99 cc., Glacial acetic or formic acid, I cc. By extreme care in mounting and by occasionally adding a fresh coat to the sealing of the cover-glass, glycerin preparations last along time. They are liable to be disappointing, however. In mounting in glycerin care should be taken to avoid air-bubbles, as they are difficult to get rid of. A specimen need not be discarded, however, unless the air-bubbles are large and numerous. 2326. Glycerin Jelly.—Soak 25 grams of the best dry gelatin in cold water in asinall agate-ware dish. Allow the water to remain until the gelatin is softened. It usually takes about half an hour. When the gelatin is softened, as may be 202 PREPARATION OF REAGENTS [CH. Vil readily determined by taking a little in the fingers, pour off the superfluous water and drain well to get rid of all the water that has not been imbibed by the gelatin. Warm the softened gelatin over a water bath and it will melt in the water it has absorbed. Add to the melted gelatin about 5 cc. of egg albumen, white of egg ; stir it well and then heat the gelatin in the water bath for about half an hour. Do not heat above 75° or 80° C., for if the gelatin is heated too hot it will be trans- formed into meta-gelatin and will not set when cold. The heat will coagulate the albumen and form a kind of floculent precipitate which seems to gather all fine particles of dust, etc., leaving the gelatin perfectly clear. After the gelatin is clarified it should be filtered through a hot flannel filter and mixed with an equal volume of glycerin and 5 grams of chloral hydrate and shaken thoroughly. If it is allowed to remain in a warm place (7. ¢,, in a place where the gelatin remains melted) the air-bubbles will rise and disappear. In case the glycerin jelly remains fluid or semi-fluid at the ordinary temper- ature (18°-20° C), the gelatin has either been transformed into meta-gelatin by too high a temperature or it contains too much water. The amount of water may be lessened by heating at a moderate temperature over a waterbath in an open vessel. This is a very excellent mounting medium. Air-bubbles should be avoided in mounting as they do not disappear. 4327. Chloral Hematoxylin.—Hematoxylin is one of the most useful stains employed in histology. An excellent solution for ordinary section staining may be made as follows: Distilled water 200 cc., and potash alum 7} grams, are boiled together for 5 minutes, in an agate-ware or glass vessel, and sufficient boiled water added to bring the water back to 200 ce. After the mixture is cool, 4 grams of chloral hydrate, and #,ths grams of hematoxylin crystals, previously dissolved in 20 cc. of 95% alcohol, are added. The boiling seems to destroy any fungi pres- ent in the alum or water, and the chloral prevents the development of any that may get in afterward, and this solution therefore is quite permanent. At first the color will be rather faint, but after a week or two it will become a a deep purple. The deepening of the color is more rapid if the bottle is left un- corked in the light and is shaken occasionally. It may be prepared for work at once by the addition of a small amount of hydrogen dioxid (H,O,). If the stain is too concentrated it may be diluted with freshly distilled water or with a mixture of water, alum and chloral. If the stain is not sufficiently con- centrated, more hematoxylin may be added. With hematoxylin of the strength given in the formula, sections are usually sufficiently stained in from one to five minutes. As may be inferred from what was said above, the boiling is to destroy any living ferments present in the water or alum, and the chloral hydrate is to prevent the development of germs which accidentally reach the solution after it is made. No precaution is necessary in using this stain for sections, except that appli- cable to all hematoxylin solutions, viz: It must be filtered occasionally and after staining the surplus stain must be very thoroughly washed away with water ; otherwise black granules or needles will appear in or upon the sections. If granules appear in the preparations in spite of the washing, it will be well to boil the solu- tion three to five minutes and filter through paper or absorbent cotton. The addi- tion of one or two per cent. of chloral after the boiling is also advantageous. This stain has not been tried for dyeing in bulk. Other substances than chloral CH. V/T) PREPARATION OF REAGENTS 203 were tried, but not with so good success. (Proc. Amer. Micr. Soc., 1892, pp. 125-127). 2328. Hematein. This is used instead of hematoxylin, as it is believed to give more satisfactory results. Prepare as follows: Prepare a 5% solution of pot- ash alum in distilled water and boil or leave in a steam steralizer an hour or two. While this alum solution is warm add 1 per cent. of hematein dissolved in a small quantity of alcohol. After the fluid has cooled add 2 grams of chloral for each 100 ce. of solution. (Freeborn, Jour. Ap. Micr., 1900, p. 1056. ) 2329. Liquid Gelatin.—Gelatin or clear glue, 75 to 100 grams. Commercial acetic acid (No. 8) too cc., water 100 cc., or glacial acetic acid do cc. and water 160 cc., 95% alcohol Ioo cc., glycerin 15 to 30 cc. Crush the glue and put it into a bottle with the acid, and set in a warm place, and shake occasionally. After three or more days add the other ingredients. This solution is excellent for fastening paper to glass, wood or paper. The brush must be mounted in a quill or wooden handle. For labels, it is best to use linen paper of moderate thickness. This should be coated with the liquid gelatin and allowed to dry. The labels may be cut of any desired size and attached by simply moistening them, as in using postage stamps. Very excellent blank labels are now furnished by dealers in microscopical supplies, so that it is unnecessary to prepare them one’s self, except for special purposes Those like that shown in Fig. 163 may be had for about $4 for 10,000. 7330. Nitric Acid Dissociator.—This is prepared by mixing So cc. of water with 20 cc. of strong nitric acid. It is used mostly in dissolving the connective tissue of muscle and thus making it possible to separate the fibers. Alum water is used as a restrainer (% 314 and 264). (Proc. Amer. Micr. Soc., Vol. XI,( 1889), Pp. 34-45). 4331. Normal Salt Solution or Saline Solution.—Pure water from its differ- ing density from the natural lymph acts injuriously on the tissues. The addition of a little table salt, however, prevents this deleterious action, or greatly lessens it, hence the name of xormal salt solution. It is a 745% solution of table salt (sodium chlorid) in water; water 1000 cc., salt 6 grams, or water 100 cc., salt ;°; gram. 2 332. Paraffin. Paraffin is of various melting points, hence at the ordinary temperature of a laboratory, that melting at the lowest temperature will be moder- ately soft, hence soft paraffin, while that melting at a higher temperature will be hard. For the best results one usually has to mix hard and soft paraffins. The larger the object to be cut and the thicker the sections the softer should be the paraffin. % 333. Picric-Alcohol.—This is an excellent hardener and fixer for almost all tissues and organs. It is composed of 500 cc. of water and 500 cc. of 95% alcohol, to which 2 grams of picric acid have been added. (It is a 4% solution of picric acid in 50% alcohol). It acts quickly, in from one to three days. (% 267, 254). (Proc. Amer. Micr. Soc., Vol. XII (1890), pp. 120-122). 2 334. Shellac Cement.—Shellac cement for sealing preparations and for mak- ing shallow cells (% 248) is prepared by adding scale or bleached shellac to 95% alcohol. The bottle should be filled about half full of the solid shellac then enough 95% alcohol added to fill the bottle nearly full. The bottle is shaken occasionally and then allowed to stand until a clear stratum of liquid appearsonthetop. This clear, supernatant liquid is then filtered through absorbent cotton, using a paper 204 PREPARATION OF REAGENTS (CH. VIL funnel (% 315), into an open dish or a wide-mouth bottle. To every 100 cc. of this filtered shellac, 2 cc. of caStor oil and 2 cc. of Venetian turpentine are added to render the shellac less brittle. The filtered shellac will be too thin, and must be allowed to evaporate till it is of the consistency of thin syrup. It is then put into a capped bottle, and for use, into asmall spirit lamp (Fig, 153). In case the cement gets too thick add a small amount of 95% alcohol or some thin shellac. The solution of shellac almost always remains muddy, and in most cases it takes a very long time for the flocculent substance to settle. One can very quickly obtain a clear solution as follows: When the shellac has had time to thoroughly dissolve, 7. €., in a week or two in a warm place, or in less time if the bottle is frequently shaken, a part of the dissolved shellac is poured into a bottle and about one-fourth as much gasolin or benzin added and the two well shaken. After twenty-four hours or so the flocculent, undissolved substance will separate from the shellac so- lution and rise with the gasolinto the top. The clear solution may then be siphoned off or drawn off from the bottom if one has an aspirating bottle. (R. Hitchcock, Amer. Monthly Micr. Jour, July, 1884, p. 131). If one desires to color the shellac, the addition of a strong alcoholic solution of some of the coal tar colors is good, but it is liable to dissolve in the mounting medium when shellac is used for sealing. A small amount of lampblack well rubbed up in very thin shellac and filtered, is good to darken the shellac. ARRANGING AND MOUNTING MINUTE OBJECTS % 335. Minute objects like diatoms or the scales of insects may be arranged in geometrical figures or in some fanciful way, either for ornament or more satisfac- tory study. To do this the cover-glass is placed over the guide. This guide for geometrical figures may be a net-micrometer or a series of concentric circles. In order that the objects may remain in place, however, they must be fastened to the cover-glass. As an adhesive substance, mucilage or liquid gelatin (% 329) thinned with an equal volume of 50% acetic acid answers well. A very thin coating of thisis spread on the cover with a needle, or in some other way and allowed to dry. The ob- jects are then placed on the gelatinized side of the cover and carefully got into posi- tion with a mechanical finger, made by fastening a cat’s whisker in a needle holder. For most of these objects a simple microscope with stand (Figs. 20, 145, 146) will be found of great advantage. After the objects are arranged, one breathes very gently on the cover-glass to soften the mucilage or gelatin. It is then allowed to dry and if a suitable amount of gelatin has been used, and it has been properly moistened, the objects will be found firmly anchored. In mounting one may use Canada bal- sam or mount dry on a cell (% 247, 255). See Newcomer, Amer. Micr. Soc.’s Proc:, 1886, p. 128; see also E. H. Griffith and H. L. Smith, Amer. Jour. of Micros., iv, 102, v, 87; Amer. Monthly Micr. Jour., i, 66. 107, 113. Cunningham, The Micro- scope, viii, 1888, p. 237. For additional apparatus for this chapter, see Ch. X. CHAPTER Vill PHOTOGRAPHING OBJECTS WITH A VERTICAL CAMERA ; PHOTOGRAPHING LARGE TRANSPARENT OBJECTS; PHOTOGRAPHING WITH A MICROSCOPE: (A) TRANS- PARENT OBJECTS; (B) OPAQUE OBJECTS AND THE SURFACES OF METALS AND ALLOYS* APPARATUS AND MATERIAL FOR THIS CHAPTER Vertical camera with photographic objectives (Fig. 169), small vertical camera with special microscope stand for embryos, etc. (Figs. 183-184); arrangement of camera for large transparent objects (Fig. 181); photo-micrographic cameras ( Figs. 183-184, 192); photographic objectives for gross and microscopic work (Figs. 170- 171, 176-180); microscope, microscopic objectives and projection oculars (Figs. 185, 189, 193); color screens, lamps, dry plates and the chemicals and apparatus neces- sary for developing, printing, etc. 4336. Nothing would seem more natural than that the camera, armed with a photographic objective or with a microscopic objective, should be called into the service of science to delineate with all their complexity of detail, the myriads of forms studied. For photographing many objects in nature the camera remains horizontal or approximately so, but for a great many of those studied in botany, zoology, miner- alogy and in anatomy the specimens cannot be put in a vertical position necessary for a horizontal camera. This difficulty has been overcome by using a mirror or a 45-degree prism. These are practically alike and have the defect of producing a picture with the inversion of a plane mirror. VERTICAL CAMERA 2 337. To meet all the difficulties the object may be left in a horizontal posi- tion and the camera made vertical (fig. 169). For the last twenty-five years such a camera has been in use in the Anatomical Department of Cornell University for photographing all kinds of specimens; *Papers on this subject were given by the writer at the meeting of the Amer- ican Association for the Advancement of Science in 1879, and at the meeting of the Society of Naturalists of the eastern United States in 1883; and in Science Vol. III, pp. 443, 444. 206 PHOTO-MICROGRAPHY (CH. VIII among these, fresh brains, and hardened brains have been photographed without the slightest injury to them. Furthermore, as many specimens are so delicate that they will not support their own weight, they may be photographed under alcohol or water with a vertical camera and the result will be satisfactory as a photograph and harmless to the specimen. A great field is also open for obtaining life-like portraits of water animals. Freshly killed or etherized animals are put into a vessel of water with a contrast- ing back ground and arranged as desired, then photographed. The fins have something of their natural appearance and the gills of branchiate salamanders float out in the water in a natural way. In case the fish tends to float in the water a little mercury injected into the abdomen or intestine will serve as ballast. The photographs obtainable in water are almost if not quite as sharp as those made in air. Even the corrugations on the scales of such fishes as the sucker (Catostomus teres) show with great clearness. Indeed so good are the results that excellent half tone plates may be produced from the pictures thus made, also excellent photogravures. In those cases, as in anatomical preparations, where the photograph rarely answers the requirements of a scientific figure, still a photo- graph serves as a most admirable basis for such a figure. The photograph is made of the desired size and all the parts are in correct proportion and in the correct relative position. From this photographic picture may be traced all the outlines upon the drawing paper, and the artist can devote his whole time and energy to giving the proper expression without the tedious labor of making measurements. “While the use of photography for outlines as bases for figures diminishes the labor of artists about one-half it increases that of the preparator ; and herein lies one of its chief merits. The photographs being exact images of the preparations, the tendency will be to make them with greater care and delicacy, and the result will be less imagination and more reality in published scientific figures ; and the objects prepared with such care will be preserved for future reference.”’ “In the use of photography for figures several considerations arise: (1) The avoidance of distortion ; (2) The adjustment of the camera to obtain an image of the desired size ; (3) Focusing ; (4) Lighting and centering the object. (1). While the camera delineates rapidly, the image is liable to distortion. I believe opticians are agreed, that, in order to obtain correct photographic images, the objective must be properly made, and the plane of the object must be parallel to the plane of the ground glass. Furthermore, as most of the objects in natural history have not plane surfaces, but are situated in several planes at different levels, the whole object may be made distinct by using in the objective a dia- phragm with a small opening. % 338. Scale of Sizes and Focusing.—(2). By placing the camera on a long table, and a scale of some kind against the wall, the exact position of the ground glass for various sizes may be determined once for all, and these positions noted in some way. Fic. 169. Vertical Camera for photographing objects in a horizontal position. The camera is attached to a double frame connected by bent metal pieces Sastened to the lower and sliding in a groove in the upper frame. The two frames can then slide over each other without separating. For moving the outer frame a rack work ts put on the lower or tnner frame and a pinion with a toothed wheel on the outer one. This is turned by the wheel shown. To prevent the camera run- CH, VIIT) PHOTO-MICROGRA PAY 207 ning down in the vertical position a pawl is held in place by a spring. This may be released by a smaller wheel than that serving to move the pinion. This rack and pinion are fine enough for focusing with the photographic objectives employed. i FIG. 169. The camera bed is graduated in centimeters so that the exact extent of the bellows can be determined by inspection. The support on which the specimen rests is of heavy glass on vertical vods about ro cenlimers long. The background ts placed on the table top about ro cm. below. This arrangement of support and background serves to avoid the dense shadows which make it difficull to determine exactly the limits of the specimen. To make the apparatus steady the right hand end of the table is heavily weighted. The tables have leveling screws in the legs. 208 PHOTO-MICROGRA PHY (CH. VIIT Fic. 170. Yurner-Reich anastigmat objective for pho- tography, (Gundlach Opt. Co.) mil a il I | | In the camera here fig- ured, the camera bed is ruled | Wil: in centimeters so that the Ml gouge IT Tr l position of the ground glass 22 30 can be determined with ac- ; curacy and noted. It takes but a moment to set the ground glass or focusing screen at the right level to ‘I ay give any desired size. In practice it is convenient to have attached to the camera a table giving the position of the ground glass for various sizes, and also the distance of the objective from the object in each case. By having this information it takes but a moment to set the camera \, | | and to place it so that it m will be approximately in Hic. a SI oO. Fic. 171. Zeiss anastigimat objective for photography, (Bausch & Lomb Opti- cal Co.) focus. The final focusing is then accom- plished by the use of the rack and pinion movement. It is an advantage to usea focusing glass and a clear focusing screen or the transparent part of the ordinary screen (Fig. 174), for the final focusing. As many objects have not sharp details which one can focus on, it is helpful to place some printed letters on the part to be brought out with the greatest sharp- ness. Of course these are removed before the exposure is made. , 7339. In lighting the object one should take pains to so arrange it with ref- erence to the light that the details will show with the greatest clearness. Naturally for the vertical camera the light will come from the side and not from a skylight, although good results are obtained with a skylight if one so Sah the camera that it does not cast objectionable shadows. CH VITL | PHOTO-MICROGRAPHY Fic. 172. Tripod magnifier as focusing glass. This ts carefully focused on a scratch or pencil mark on the lower or ground surface of the focusing screen. Then whenever the object is sharply focused the focal plane will be at the level of sensitive surface. As shown in Fig. 169, the object is placed upon a glass support and the background is quite a distance below the support. For a dark object the background should be hght, and for a light one dark. Black velveteen is excellent for a back-ground. The advan- tage of the glass support is that the shadows in the FIG. 172. background which often make it difficult to tell just where the specimen ends and the background begins, is wholly done away with, and that too without at all affecting the proper light and shade of the object itself. Rumsey, Canadian Entomologist 1896, p. $4). Fic. 173. Focusing Glass. “It ts achromatic, consisting of a double convex crown lens and a nega- tive meniscus flint lens cemented together.’ Tt screws into the brass tube and ts thus adjustable, enabling one to focus the pencil mark in the clear area of the focus- ing screen (Fig. 174) with great accuracy. It also serves to focus the image with ease and accuracy. The eye must not be too close to the upper end of the focus- ing glass or the field will be restricted. (Gundlach Opt. Co.) (Method of W. E. FIG. 173. FIG. 174. Ground-glass focusing screen with central transparent area for exact Jocusing with a focusing glass when one does not possess a clear focusing screen. (TL) The ground surface ; (2) Central part with oblong cover-glass and Canada balsam on the ground surface to render it transparent. X, The central point in the entire focusing screen. Itis made with a black lead pencil on the ground surface. The focusing glass 1s focused on this cross, then when the image isin focus it will be at the level of the sen- sitive coating of the plate. Fic. 175. CA. VIIT) PHOTO-MICROGRA PHY 211 Fic. 175. Vertical Camera and special microscope stand for photographing embryos and other small specimens in liquids and for photographing large sections. The camera rests on a low table and the operator can stand on the floor while per- Sorming all the operations. The stage of the microscope is attached to the arm in the place of the tube. This stage has two stories. The specimen is shown on the upper and the back- ground on the lower story. In focusing, the coarse and fine adjustment of the special microscope stand are used. The large mirror is to illuminate embryo chicks mounted entire, and other large transparent preparations. Trans. Amer. Micr. roor. 2340. Prints.—If the photographic prints are to be used solely for outlines, the well-known blue prints so much used in engineering and architecture may be made. If, however, light and shade and fine details are to be brought out with great distinctness, either an aristotype, platinotype or a bromide print is preferable. § 341. Recording, Storing and Labeling Negatives.—In or- der to get the greatest benefit from past experience it is necessary to make the results available by means of a careful record. For this pur- pose the table (§ 360) has been prepared. If one gives the information called for in this table, whether the result is successful or not, one can after a time work with great exactness, for the elements of success and failure will stand out clearly in the table. §$ 342. Labeling the Negatives.—After the negative is dry the labeling can be done on the gelatin side with carbon ink. Enough data should be given to enable the certain identification of the negative at any future time. § 343. Storing Negatives.—This is satisfactorily done by put- ting each into an envelope and writing a duplicate label on the upper edge, and then the negatives may be placed in drawers in alphabetical order as are the catalog cards of books in a library. One can then find any negative with the same facility that the title of a book can be found in a card catalog. PHOTOGRAPHING EMBRYOS For photographing embryos and many other small specimens it is more convenient to use a smaller apparatus than the vertical camera just described. It is necessary also to have a more delicate method of focusing. § 344. Camera for Embryos.—This is a vertical camera for photographing with the microscope, with the photographic objective in the end of the camera as for an ordinary camera. ‘This is readily ac- complished by having a society screw adapter, and also adapters for the micro-planars or other objectives which one desires to use. The 212 PHOTO-MICROGRAPHY ( CH. Vill magnification usually required varies from natural size (X1) to five times natural size (x5) up to x20. As with the large camera the position of the ground glass for each magnification and for each objec- tive is determined once for all by using a scale in millimeters. The various positions are accurately noted, then one can set the camera almost instantly for the desired magnification. The supporting rod is divided to half centimeters and therefore the exact position is easily recorded (Fig. 183). Fic. 176. Zeiss Micro-Planar for photographing with low magnification and for projection (see Ch. 12.6), These are made from 20 to 100 mm, equivalent focus, those of 20 and 35 mm. equivalent focus have the standard, Royal Society Screw, the others have a larger screw in order thal the image may not be restricted. (Cut loaned by Bausch & Lomb Optical. Co.) $ 345: Special Microscope Stand.—For the accurate focusing necessary for embryos one should possess a special microscope stand with the stage in two or three stories and attached to the arm in place of the tube of the microscope. The stage proper is absent. This arrangement of the stage permits the use of the coarse and fine adjustment of the microscope to be used for focusing. The position of the camera on a low table (45 to 50 cm. high) makes it possible for the operator to stand on the floor while making all the adjustments of the the embryo and for focusing; and all the parts are within reach (Fig. 175). § 346. Arranging the Embryos.— Micro-chemistry, 155-157 ; slides for, 161. Micro-metallography, objects for, 238. Micrometer, 103; calipers, 164; cob-web, | 117; filarm. ocular, 117, 118; filling | lines of, 106; lines, arrangement of | ocular and stage, 120; lines, finding, 106; net, 128; object or objective, 105; ocular or eye-piece, 114-120; ocular, micrometry with, 116; ocular, ratio, 119; ocular, valuation of, ri, 114; ocular, varying valuation of, | 118; for photo-micrography, 233 ;, negatives, | Microscope Micro-polarizer, 150. 249-267 ; apparatus, 256-257 ; carbons for, 250-254 ; cir- culation of blood with, 257, 264 ; con- denser, 254, 256; current, 254; dem- onstration with, 265 ; magnification, 262; masks for specimens, 264; me- chanical stages, 258-259 ; microscope for, 249; objectives and oculars, 259; pointer for, 265; preparations, 263 ; screen and screen distance, 261; specimen cooler, 257; stage, 255; stains for, 263; water-bath, 255. Microscope, definition, 1; amplification of, 103 ; clinical, 243 ; demonstration, 243; dissecting, 8, 33, 175, 176 228; care of, 59; eye and, 1, 6, 9; field of, 28, 29 ; focusing, 34 ; magnification, 103; for metallography, 159-160 ; for mi- crochemical analysis, 153; for opaque objects, 235-237; for photo-micro- graphy, 225, 231, 237; polarizing, 150; preparation, with erecting prism 176; projection, 249-250; price of, 64,71; putting an object under, 27; sun or solar, 249; screen, 59; stand for large, transparent objects, 215, 216; stand, for embryos, 212; traveling, 245-246. compound, definition, 8; drawing with, 122; figures, frontis- piece, 9, 71-89, 102, 153, 160, 237, 244-246 ; focusing, 38-39; for High schools, 64, 71; for laboratory, 64, 71-89; lamp for, 50; magnification or magnifying power, 105 ; magnifi- cation and size of drawing with Abbe camera lucida, 131; mechani- cal parts, 64; micrometry with, 112; optic axis of, 9, 10; optical parts of, 9g, 64; varying magnification, I09 ; working distance of, 39; testing, 63. screw ocular, 117; stage, 105, 106; Microscope, simple, definition, 1; exper- table of magnification, IIT. Micrometry, definition, 112, 114; with) adjustable objectives, 118 ; compari- | son of methods, 119-120; with com- | iments with, 6; figures, 6-S, 33, 104, 175, 209, 228, 243; focusing with, 34; magnification of, Io4; micrometry with, 112; working distance of, 34; pound microscope, 112; and juris-, Microscopic, objective, 9 ;obj ective low, prudence, 121 ; limit of accuracy in, | 120; with ocular micrometer, 116 | with simple microscope, 112; remarks | on, 115; unit of measure in, 112. Micro-millimeter, 113. Micron, 113; for measuring wave length | of light, 143. Micro-photograph, 220. Micro-photography, distinguished from photo-micrography, 220. attached to camera, 214; objects, drawing, 122; ocular, 22; slides or slips, 161; tube-length, 13, 14. | Microscopical preparations, cabinet for, 197 ; cataloging, 194 ; labeling, 194; mounting, 166-193. | Microtome, hand, 274-275 ; Minot’s ay 275- 276 ; razor support for, 276-277 ; slid- ing, 278. INDEX Micro-spectroscope, 134-150; adjusting, 139; experiments, 145; focusing, 144 ; focusing the slit, 139; lighting for, 144 ; objectives to use with, 144; reversal, apparent, of colors in, 134; slit, mechanism of, 135, 139. Micrum, IT3. Mikron, 113. Milk globules, to overcome pedesis of, 100 Minerals, absorption spectra of, 149. Minot’s microtomes, 275-276. Minute objects, arrangement of 204. Mirror, 9-11; for Abbe illuminator, 48; of camera lucida, arrangement for drawing, 126; concave, use of, 37; dark ground illumination, 50; light) with, central and oblique, 40, 41 ;) _ lighting with, 37; plane, use of, 37. Mixture, clearing, 200. Models, wax, 274. | Moist, chamber, 171. Molecular movement, 99. Monazite sand, spectrum of, 149. Monnting cells, preparation of, 168; me- dia and preparation of, 198 203; ob- jects for polariscope, 151; perma- nent, 167 ; temporary, 166. Mounting objects, dry in air, order of procedure, 167-168 ; in glycerin, or- der of procedure, 170; in glycerin jelly, order of procedure, 170; in media miscible with water, 169; mi-) nute objects, 204; opaque objects, | 239; permanent, 167; in resinous media. by drying or desiccation. or- | der of procedure, 172; in resinous | media, by successive displacements, | order of procedure, 172; temporary, | 166. Movement, Brownian, or molecular, 99. Muscae volitantes, 100. Muscular fibers, isolation of, 175. N Natural balsam, 199. Needle-holder, 167. Negatives, labeling, 211, 214; oculars, 22;rack for drying, 240; record of, 214; storing, 211, 214. Net micrometer, 128, Neutral balsatn, 199. Nicol prism, 150. Nitric acid, dissociator, 203. Nomenclature of objectives, Non-achromatic condenser, ives, II. Non-adjustable objectives, 12; of cover glass for, table, 14. 10. 46; object- thickness 295 Normal salt solution, 203. Nose-piece, 27, 38, 80; marking object- ives on, 7I. Numerical aperture, of condenser, 44; of objectives, 16, 270; table of, 19. O Object, determination of form, 93; hav- ing plane or irregular outlines, rela- tive position in a microscopical prep- ration, 92; and image, size of, Io, 108 ; marking parts of, 65-66 ; mark- ing position of, 248 ; micrometer, 105; mounting, 166; putting under micro- scope, 27; shading, 59; suitable for photo-micrography, 227; transparent with curved outlines, relative posi- tion in microscopic preparations, 94. Objective, 9-13, 121; achromatic, 11; adjustable, 11, 12, 54; adjustable, micrometry with, 118; adjustable, photo-micrography with, 234 ; adjust- ment for, 54; aerial image of, 30; anastigmat, 208; aperture of, 15-22, 270; aplanatic, 11; apochromiatic, 12, 224; back combination of, 10, I1; carrier, 259; cleaning back lens of, 61; collar, graduated for adjustment, 56; cloudiness or dust, how to deter- mine, 92; designation of, 10; dry, IO, 17-19, 121; equivalent focus of, 10, 25, 29, 272; field of, 28-29 ; focus- ing for micro-spectroscope, 144 ; front combination of, 10, 11; function of, 29-31; glass for, 11-13, 71; high, focusing with, 38 ; homogeneous 1m- mersion, I7-I9, 121; homogeneous immersion, cleaning, 59; homogene- ous immersion, experiments, 58; il- luminating, 13, 233; image, power of, 18; immersion, If, 121; index of refraction of medium in front of, 17, 19; initial or independent magnifica- tion of, 273; inverted, real image of, 30; for laboratory microscope, 64; lettering, 10; light utilized with, 17; low, focusing with, 38; magnifica- tion of, 272; marking, by Krauss’ method, 71 ; for micrometallography, 13, 159; micro-planar, 212, 260; to use with micro-polariscope, 151 ; mi- croscopic, 9; to use with wmicro- spectroscope, 144; for micro-spectro- scope, focusing, 144; nomenclature of, 10; non-achromatic, II; non- adjustable, 12; non-adjustable, thick- ness of cover-glass for, table, 14; with nose piece, 38; numbering, 10; numerical aperture, 16-22, 270; oil 96 immersion, 11 ; panto-chromatic, 13; para-chromatic, 13 ; for photography, 208, 212, 214, 224; for photo-microg- raphy, 224; projection, 13, 212, 214, 259; putting in position and remov- ing, 26 ; semi-apochromatic, 13; table of field, 29; terminology of, Io; un- adjustable, 12; variable, 13; visual and actinic foci of, in photo-microg- raphy, 226: water immersion, 17-19, 56; working distance of, 39-40. Oblique light, 36, 41; with Abbe illumi- nator, 48; with a mirror, 41, 50. Ocular, various forms, 22-25 ; cloudiness, how to determine and remove, 60, 92; equivalent focus of, 25, 29, 273; eye- point of, 32; field-lens, 32; filar or screw micrometer, 26, 117; focus, equivalent of, 25, 273; function of, 31-33; indicator, 67, 247; iris dia- phragm for, 157; lettering and num- bering, 26; micrometer, micrometry with, 114-121 ; parfocal, 24, 38; for photo-micrography, 224, 232, 235; pointer, 247; projection, 260; spec- troscopic,134 ; standard size for, 26; table. effect on field, 29. and air, appearances and distinguish- ing optically, 95 ; removal, 61; re- moval from sections, 179. Oil-globules, with central and oblique illuminations, 95. Oil immersion objectives, Ir. Oil, Opaque objects, lighting, 144, 238 ; pho- | tography of, with microscope, 235- 240; projection of, 267. Opera glasses, 262. Optic axis, 2,; of condenser or illumi- nator, 47 ; of microscope, Io. Optical, bench, 237 ; center, 2; focus, 12 ; parts of compound microscope, 9, 64; parts of microscope, care of, and test- ing, 60, 63; section, 98. Order of procedure in mounting ob- jects dry or in air, 167; in glycerin, 170; in glycerin jelly, 170: in resin-_ nous media by desiccation, 172; in resinous media by successive dis- placement, 172. Ordinary ray, with polarizer, 150. Orthochromatic plates, 217. Orthoscopic ocular, field with, 25. Outline distinctness of, 96. Oven paraffin, 279. Over-correction, 5. Oxy-hemoglobin, spectrum of, 138, 147. INDEX P Paper, bibulous, filter, lens, or Japanese for cleaning oculars and objectives, 60, 180. Paraffin, 185, 203; filtering, 185; infil- trating with, 185; dish for infiltrat- ing, 279; imbedding in, 185, 186; method, 183; oven, 279; pail for melting, 184; removal from lenses, 61; removing from sections, 188. Parfocal oculars, 24, 35. Parts, optical and mechanical of micro- scope, 8, 64; testing, 63. Pedesis, 99 ; compared with currents, 99 ; to overcome, 100; with polarizing microscope, 100; proof of reality of, 100, Penetrating power, 21. Penetration of objective, 21. Permanent mounting, 167 ; preparations of isolated cells, 175. Permanganate of potash, absorption spec- trum of, 136, 146. Petri dish, photographing bacterial cul- tures in, 241. Petroleum light, 37, 229; as color screen, 220. Pharmacological products, examination of, 158. Photo-engraving, drawing for, 273; let- tering for, 274. Photographic, camera, 207; negatives, labeling, 211, 214; objectives, 208 ; prints, 211. | Photography, back-ground for, 209; of bacterial cultures, 241-242; color- correct, 217; of colored objects, 215 ; compared with photo-micrography, | 222; of embryos, 211-214; focusing and exposure, 206, 213 ; indebtedness | to photo-micrography, 220; of large transparent objects, 214-216; hghting for, 205, 216; metallic objects, 235- 240 ; objectives for, 208, 212, 214; of objects in alcohol cr water, 206; opaque objects, 235-240; plates for, 217; stage for, 211; with vertical camera, 205-209, 225. | Photo-micrograph, 220; determination of magnification for, 233 ; at 5-20 di- ameters, 212; 20-50 diameters, 230; 100-2500 diameters, 233 ; of metallic surfaces, 235-240; objects suitable for, 227 ; of opaque objects, 235-240; prints of, 211; plates for, 217; repro- ductions of, 232; with and without an ocular, 230-234. Photo-micrographic, ca 236; outfit, 236-237 Bs mera, » stand, 22 2 231. 5s INDEX Photo-micrography, 220-240 ; cover-glass correction, 234; apparatus for, 223; compared with ordinary photogra- phy, 222 ; condenser for, 42, 226, 227; distinguished from micro-photogra- phy, 220; experiments, 229; expos- ure for, 213, 220, 232, 235, 240; focus- ing for, 213, 216 ; focusing screen for, 209 ; lighting, 229, 230, 233, 239; mi- crometer for, 233 ; objectives and oc- ulars for, 13, 224, 232,235 ; vertical camera with, 211, 222, 225; actinic foci in, 226 ; with and without ocular, 230,232, 234 ; record table for, 219. Physiological histology, 196. Picrie-alcohol, 203. Picro-fuchsin, 190. Pillar of microscope, Frontispiece. Pin-hole diaphragm, 47. Pippett, 179; egg, 28o. Plane mirror, use of, 37. Plates, color-correct, 217; exposure of, 213, 220, 232, 235, 240 ; isochromatic, or orthochromiatic, 217 ; size of, 224. Pleochroism, 152. Pleurosigma angulatum, 41. Point, axial, 15 ; burning, 7. Pointer ocular, 247. Polariscope, 140, 150. Polarized light, extraordinary and ordi- nary ray of, 150. Polarizer and analyzer, 140, 151. Polarizing microscope, pedesis with, 100. Position of objects or parts of same ob- ject, 92; marking p, 248. Positive oculars, 30, 22. Power, of microscope, 103 ; illuminating, penetrating, resolving, of objective, 19-21 ; of ocular, 25. Preparation of Canada balsam, Farrant’s solution, glycerin, glycerin jelly,etc., 198-204. Preparation, of clearing mixture, liquid gelatin and shellac cement, 198-204 ; of ground glass, 29; of metallic sur- faces, 239; vials, 174. Preparations, cataloging, 194-196; cabi- net for, 196-197 ; labeling, 194; for. microprojection, 263; permanent, of isolated cells, 175. Price of American and foreign micro- scope, 7I. Principal, focus, 3, 5; focal distances, 3, 30; Optic axis, 2,.'5- Prism of Abbe camera lucida, 124, 127; Amici, 140; comparison, 141; dis- persing, 141; erecting, 176; Nicol, 150; and slit of micro-spectroscope, mutual arrangement, 139; of Wollas- ton’s camera lucida, 125. 297 | Prints, photographic, art. Projection, apparatus, 249, 263-297 ; mi- croscope, 249-266 ; see micro-projec- tion ; objective, 13, 214, 259; ocular, 24, 25, 232, 260; opaque objects, 267 ; in photo-micrography, 232, 234. Putting, on cover-glass, 167; an object under microscope, 27; an objective and ocular in position, 26, 27. Pyroxylin, 200. QO—R Quadrant for camera lucida, 127, 128. Radiant, centering, 255. Ratio, ocular micrometer, 119. | Razor and support, 276-277. Reagent, bottle, 179; for fixing, 198; irri- gation with, 170; for mounting, 198. Real image, 5, 8, 9, 30; magnification, 103, 262. Record, of negatives, 214; table for photo- micrography, 219. Reflected light. 35. Reflection, total, 54. Refraction, 52; images, 52,58; index of, 53; of medium in front of objective, 19. Refractive, doubly, 152; singly, 152. Relative position of objects, 92. Resinous media, mounting objects in, order of procedure, by drying or desiccation, 172; by a series of dis- placements, 172. Resolution and numerical aperature, 20. Resolving power, 20. Retinal image, 6, 9. Revolving nose-piece, marking objectives on, 7I. Ribbon sections, 186-187 ; tray for, 187. S) Sagittal sections, 192. Salicylic acid, crystallization, 50. Salt solution, normal, 203. Scale, of drawing, 131 ; of sizes for pho- tographing, 206; of wave lengths, 142. Screen, color, 218-219; focusing s. for photo-micrography, 209; of ground glass, 29; for micro-projection, 261 ; for microscope, 59. Screw, society, 64; micrometer, 26, 117. Sealing cover-glass, 169, 170. Searching ocular, 24. Secondary axis, 3. Section, lifter, 181-182 ; optical, 98. | Sections, arrangement of tissue for, I9I ; clearing, 190; cutting, 178, 186; de- highly, 97; -° 98 hydration of, 190; extending with water, 186; fastening to slide, 179, 187; frontal, 192; mounting, 183, | Igo ; removing benzine, oil and para- fin from, 179, 188; ribbon, 186; sagittal, 192; serial, 191-193; stain- ing, 180, 159; transferring, 179; thickness for micro-projection, 263. Selenite plate for polariscope, 154. Semi-apochromatic objective, 13. Serial sections, 191-193; arranging and labeling, 192, 193 ; stage for, 67, 69 ; thickness of cover-glass for, 193. Shell vials, 174. Shellac cement, preparation of, 203; re-| moval from lenses, 61. Significance of aperture, 20. Simple microscope, see under microscope. Sines, table of, 3d page of cover. Slides, 161; box for 198; cleaning, I6r ; holder for, 188-189 ; lantern, 241 ; for | micro-chemistry, 161 ; tray ion 187. Sliding microtome, 278. Slips, 161. Shit mechanism of micro-spectroscope, 135, 139. Society screw, 64. Sodium, lines and spectrum, 136-137. Solar spectrum or s. of sunlight, 136-137. Soluble cotton, 200, Solution, alum, 198; Farrants’, 201. Specimen cooler, 257. Spectral, colors, 135; ocular, 134, 139. Spectroscope, direct vision, 134, 145. Spectroscopic, examination of color- screens, 220 ; ocular, 134. Spectrum, 136-150; absorption, 137; amount of material necessary and its proper manipulation, 145; analysis, 150; Angstrom and Stokes’ law of, 138 ; banded, not given by all colored objects, 145 ; of blood, 146 ; of carbon monoxide hemaglobin, 147 ; of car- mine solution, 148 ; of minerals, 149; of colorless bodies, 148 ; comparison, 142; complementary, 139; continu- ous, 137 ; double, 142 ; incandescence; 137 ; line, 137 ; met-hemaglobin, 147, monazite sand, 149 ; oxy-hemoglobin, | 138, 147; permanganate of potash, 136, 146; single-banded of hemaglobin, 135, 147; sodium, 136, 137; solar, 136, 137; two-banded of oxy-hema- globin, 147. Spherical aberration, 4, 5; test for, 268. Stage, Frontispiece, 65; mechanical, 65, 67-70, 259; micrometer, 105; for mi- cro-projection, 258 ; for photograph- ing, 211. INDEX Stain, alcoholic, 180, 189 ; aqueous, 180, 189 ; for micro-projection, 263. | Staining, cells, 174; dish, 190; sections, | 180, 189. Stand, of microscope, 65; photo-micro- graphic, 231 ; special for embryos, 212; special for large transparent ob- jects, 215, 216. Standard, distance (250 mim.) at which the virtual image is measured, 109 ; screw, 64; size for condenser, 47; size for oculars, 26. Stokes and Angstrom’s law of absorption spectra, 138. Storing negatives, 211 ; preparations, 196. Substage, $6, Frontispiece. Substances for crystallography, Sulphonal with polarizer, 154. Sulphuric ether, 201. | Support for Knife of microtome, 276-277. 156. Swaying of image 48. EY nthol, 198. System, back, front, intermediate of | lenses, I0, II ; crystal, 156; metric, | cover Ist p., 133. T Table, for immersion fluid, 272; of mag- nification and valuation of ocular mi- crometer, Ill; magnification with projection microscope, 262; of tube- length and thickness of cover-glasses, 14; natural sines, third page of cover; of numerical aperture, 19; record, for photo-micrography, 219; size of | fields, 29; testing homogeneous liq- | uids, 272; of valuations of ocular micrometer, {II ; weights and meas- ures, 2d page of cover. Temporary mounting, 166, Terminology of objectives, 10. Test of chromatic and spherical aberra- tion, 268-270, | Tester, cover-glass, 164-165 ; geneous liquids, 58, 271. | Testing a camera, 223 ; a microscope and its parts, 63. | Test-plate, Abbe’s, method of using, 268. Textile fibers, examination of, 1or, 158. Thickness, of cover-glass for non- adjust- able Objectives, table, 14; of serial sections, 193. Tissues, arranging for sections, 191; fix- ing or hardening, 176, 183 « washing apparatus for, 28o. Tolles-Mayall mechanical stage, 67. Transections, 192. Transferring sections, 179. Transmitted light, 36. for homo- INDEX Tray for slides, 187. Triplet, aplanatic, 7. Tripod, 7, 104; base for microscope, 86 ; as focusing glass, 209. Tube of microscope, Frontispiece. Tube-length, 13, 14, 56, for cover-glass adjustment, 56, 57; importance of, 56; microscopical, 13, 14; of various opticians, table, 14 ; and optical com- binations, 118. Turn-table, 168. U—V—W—X Unadjustable objectives, 12. Under-correction, 5. Unit of measures, in micrometry, 112; of wave length, 143. Valuation of ocular micrometer, II4-116; table, 11. Variable objective, 13. Varying magnification of compound mi- croscope, 109. Varying ocular micrometer valuation, 118, Velocity under microscope, 98. Vertical, camera, 205-210, 225: illumina- tor, 13, 238-239. 299 | Vials, preparation, 174; blocks for, 174. Virtual image, 5, 6, 9, 32; standard dis- tance at which measured, Io9. Visibility with objectives, 20. Vision, double, 103, 105 ; microscopic, 21. Washing apparatus for tissues, 280. Waste bowl, 181. Water immersion objective, 16-19, 56; light utilized, 17; numerical aper- ture, 19 Water, bath, 235, 255; for immersion ob- jectives and removal of, 56, 58. Wave length, designation of, 143; scale of, 142. Wax models, 274. Weights and measures, see 2d page of cover. Welsbach light, 37, 229. Wollaston’s camera lucida, 107, 125. Work-room for photo-micrography, 224. Work-table, position, etc., 62. Working distance of microscope or ob- jective, II, 34, 39-40. Writing diamond, 196. Xylene, 178, 200; balsam, 199; for re- moving oil, 179; removing from slides, 189. Xylol, German form of xylene, 178, 200. TABLE OF NATURAL SINES Compiled from Prof. G. W. Jones’ Logarithmic Tables MINUTES. DEGREES AND QUARTER DEGREES UP TO 90°. 14 MI ANP WN Ne) 0.00029 0.00058 0.00087, 1,30 O.0O1I6 1,45 0.00145 2 0.00175 2,15 0.00204 2,30 0.00233 2,45 0.00262 3 0.00291 3,15 0.90320! 3,30 0.00349) 3,45 0.00378 4 0.00407 4,15 0.00436) 4,30 0.00465 4,45 0.00495, 5 0.00524 5,15 0.00553) 5,30 0.00582) 5,45 0.00611 6 0.00640 6,15 0.00669 6,30 0.00698 6,45 0.00727) 7 0.00756 7,15 0.00785, 7,30 0.00814) 7,45 0.00844) 8 0.00873 0.00902] 8.30 0.00931| 8,45 0.00960] 9 0.00989} 9,15 0.01018] 9,30 0.01047) 9,45 0.01076) 10 0.01105} 10,15 0.01134|10,30 0.0T164|10,45 O.O1193|11 0.01222/11 ,15 0.01251|11,30 0.01280/11,45 0.01309|12 0.01338/12,15 0.01367|12,30 0.01396|12,45 0.01425|13 0.01454|13,15 0.01483/13,30 0.01513/13,45 0.01542|14 0.01571|14,15 0.01609} 14,30 0.01629/14,45 0.01658|15 0.0168, 15,15 0.01716/15,30 0.01745 |15545 8,15 0.03054) 16, 45 0.03490 17 0.03926/17,15 0.04362/17,30 0.04798 )17,45 0.05234)18 0.05669/18,15 0.06105 18,30 0.06540) 18,45 0.06976/19 0,07411/19,15 0.07846) 19,30 0.08281/19,45 0.08716|20 0.09150) 20, 15 0.09585|20, 30 0. 10019|20, 45 0, 10453|21 0,10887|21,15 0,11320/21,30 0.1£754|21,45 0.12187|22 0.12620/22,15 0.13053|22,30 0.13485 22,45 0.13917|23 0.14349/23,15 oO. 14781 23,30 0.15212|23,45 0.15643|24 0.16074 24,15 0. 16505)24, 30 0.16935 |24,45 0.17365)25 0.17794)25,15 0.18224 25,30 0. 18652|25,45 0.19081/26 0.19509 26,15 0.19937|26,30 0.20364|26,45 0.20791|27 0,21218)27,15 0.21644 27,30 0.22070/27,45 0.22495|28 0.22920 /28,15 0.23345|28,30 0.23769 28,45 0.24192|29 0.24615 /29, 15 0.25038 29, 30 0.25460/29, 45 0.25882|30 0.26303 |30, 15 0.26724!30, 30 0,27144)30, 45 0.28820 31,45 0.29237 /32 0.29654 32,15 0.30071 32,30 0.30486 32,45 0.30902 |33 0.31316 33,15 0.31730 33,30 0.32144/33,45 0.32557 34 0.32969 34,15 0.33381 34,30 0.33792 34,45 0.34202|35 0.34612 35,15 0.35021 35,30 0.35429 35,45 0.35837 36 0.36244/36,15 0.36650 36,30 0.37056 36,45 0.37461|37 0.37865/37,15 0.38268) 37,30 0.38671/37,45 0.39073 38 0.39474/38, 15 0.39875 38,30 0.40275 /38, 45 0.40674'39 0.41072 39,15 0.41469 39,30 0.41866 39,45 0.42262 40 0.42657 40,15 0.43051)/40,30 0.43445 40,45 0.43837 41 0.44229 41,15 0.44620 | 41,30 0.45010 41,45 0.45399 42 0.45787 42, 15 0.46175 42,30 0.46561)42,45 0.46947 43 0.47332 43,15 0.47716 43,30 0.48099 43,45 0,48481)44 0.48862! 44,15 0.49242 44,30 0.49622/44,45 0.50000 45 0.50377 45515 0.50754/45,30 0.51129 45,45 0.52621146, 45 0.52992'47 0.53361 47,15 0.53730/47,30 0.54097 47,45 0.54464 48 0.54829 48, 15 0.55194|48, 30 0.55557/48,45 0.55919 49 0.56280]49, 15 0.56641 49,30 0.57000|49,45 0.57358|50 0.57715 50,15 oO. 58070/50, 30 0.58425'50,45 0.58779 51 0.59131 51,15 0.59482'51,30 0.59832 51,45 0.60182)/52 0.60529'52,15 0.60876'52,30 0.61222,52,45 0.61566/53, 0.61909/53,15 0 62251/53,30 0.62592/53,45 0.62932/54 0.63271 54,15 0,63608 54,30 0.63944 54,45 0.64279 55 0.64612)55,15 0.64945)55,30 0.65276 55,45 0.65606 56 0.65935 56,15 0.66262'56, 30 0.66588 56,45 0.66913/57 0.67237/57,15 0.67559|57,30 0.67880 57,45 0.68200|58 0.68518/58, 15 0.68835|58,30 0.69151/58,45 0.69466|59 0.69779 59,15 0.7009T|59, 30 0.70401/59,45, 0.70711/60 0.71019|60, 15 0.71325|60,30 0.71630160, 45 | Hl | 1°, 0.01745 16°, 0.2756431°, 0.51504'46°, 0.71934 61°, 0.87462)76°, 1°, 15/0.02181 16°, 15’ 0.27983 31°, 1§/0.51877|46°, 15’ 0.72236 Gis uel aya7a 0.02618 16,30 0.28402:31,30 0.52250 0.72837 61,45 0.73135 62 0.73432 62,15 0.73728 62,30 0.74022 62,45 0.74314|63 0.74606 63,15 0.74896 63,30 0.75184 63,45 0.75471 64 0.75756)64,15 0.76041 64,30 0.76323 64,45 0.76604/65 0.76884 65,15 0.77162,65,30 0.77439 65,45 0.77715 66 0.77988 66, 15 0.78261 66,30 0.78532 66,45 0. 78801 67 0.79069 67,15 0.79335 67,30 0.79600 67,45 0.79864 68 0.80125 68, 15 0.80386 68, 30 0,80644)68, 45 0,80902|69 0.81157 69,15 0.81412 69,30 0.81664 /69,45 0.81915|70 0,82165/70,15 0.82413 70,30 0.82659|70,45 0.82904|71 0.83147|71,15 0.83389 /71,30 0.83629/71,45 0.83867/72 0.84104|72,15 0.84339 72,30 0.84573|/72,45 0,84805/73 0.85035|73,15 0.85264)73,30 0.85491/73,45 0.85717|74 0.85941/74,15 0.86163/74,30 0.86384|74,45 0.86603/75 0.86820)75,15 0.87036)75,30 0.87250)75,45 0.88089/76, 45 0.88295 77 0.88499)77, 15 0.88701/77,30 0.88902 )77,45 0.89101|78 0.89298 78, 15 0.89493 78,30 0.89687)78, 45 0.89879 79 0.90070 79,15 0.90259 79,30 0.90446 79,45, 0.90631 [80 0.90814 80,15 0.90996 80, 30 0.91 176180, 45 0.91355 81 0.91531/81,15 0.91706/81, 30 0.91879 81,45 0,92050/82 0,92220/82,15 0.92388 82,30 0.92554/82,45 0.92718|83 0.92881/83, 15 0.93042/83,30 0.93201 |83, 45 0.93358 84 0.93514)84, £5 0.93667|84, 30 0.93819|84,45 0.93969)85 0.94118/85,15 0.94264|85,30 0.94409|85,45 0.94552|86 0.94693|86, 15 0.94832|86, 30 0.94970|86, 45 0.95 106|87 0.95240|87,15 0.95372/87,30 0.95502|87,45 0.95630/88 0.95757|88, 15, 0.95882/88, 30 0.96005|88, 45 0.96126|89 0.96246/89, 15, 0.96363|89, 30 0.96479|89, 45, 0.96593|90 0.96705} . 0.96815 0.96923 0.97030 76°, 15/ 0.97134 46,30 0.72537 61,30 0.87882)/76,30 0.97237 0.97338 0.97437 0.97534 0.97630 0.97723 0.97815 9.97905 0.97992 0.98079 0.98163, 0.98245 0.98325 0.98404 0.98481 0.98556 0.98629 0.98700 0.98769 0.98836 0.98902 0.98965 0.99027 0.99087 0.99144 0.99200 0.99255 0.99307. 9.99357 0.99406 0.99452 0.99497 0.99540 0.99580 0.99619 0.99657 0.99692 0.99725 0.99756 0.99786 0.99813 0.99839 0.99863, 0.99885 9.99905 0.99923 0.99939 0.99953 0.99966 0.99976 0.99985 0.99991 0.99996 0.99999 1.00000. : re i bestia nal ‘ Hearn aA L mee a i a h 7 ee aiid