THE METRIC SYSTEM \& LIBRARY. ■s, 10 CENTIMETER RULE The upper edge is in millimeters, the lower in centimeters and half centimeters UNITS. The most commonly used divisions and multiples TCTir w PTWB wftu f Centimeter (cm.), o.oi Meter; Millimeter (mm.), o.ooi Meter: THiJf^H < Micron(U), o.ooi Millimeter; theMicron is the unitin Micrometry(§i82). . . ^ Kilometer, iooo Meters; used in measuring roads and other long distances. the gram for f Milligram (mg.), o.ooi Gram. weight . . ( Kilogram, iooo Grams, used for ordinary masses, like groceries, etc. the liter for f Cubic Centimeter (cc), o.ooi loiter. This is more common than the cor- capacity . \ rect form, Milliliter. Divisions of the Units are indicated by the I,atin prefixes ; deci, o.l ; cenii, o.oi ; Milli, o.ooi ; Micro, one millionth (o.oooooi) of any unit. Multiples are designated by the Greek prefixes : deka, lo times ; hecto,ioo times ; kilo, iooo times ; myria, 10,000 times ; Mega, one million (1,000,000) times any unit. TABLE OF METRIC AND ENGLISH MEASURES Meter (Unit of length)=ioo centimeters ; 1 ,000 millimeters ; 1 ,000,000 microtfs (u) 39.3704 inches : 3.2808 feet ; 1.0936 yards. Centimeter (cm.)=io millimeters ; 10,000 microns (fi) 0.01 meter; 0.3937 (|) inch. Millimeter (mm. )=i, 000 microns (u); 0.1 cm.; o.ooimeter; 0.03937 (-^) inch. Micron (/a) (Unit of measure in micrometry (§182)— 0.001 millimeter; one millionth of a meter ; 0.00003937 (^5^5) inch. Yard=3 feet ; 36 inches ; 0.91439 meter ; 91.4399 centimeters. Foot= 12 inches ; 30.4799 centimeters ; 304.799 millimeters. Inch= T V foot ; ^3 yard; 25.3999 millimeters (2.54 centimeters). Liter (Unit of capacity)=i,ooo cubic centimeters (milliliters); (1 quart — . ) Cubic centimeter=o.ooi liter (milliliter); (^ cub. inch.) Fluid ounce (8 fluidrachms) =29.574 cubic centimeters (30 cc. — ). Gram (Unit of weight )=i cc. of water; 15.432 grains. Kilogram=[,ooo grams ; 2.2046 (2A) lbs. avoirdupois. Ounce avoirdupois=437i .grains ; 28. 3 49 grams. \ approx. Ounce Troy or apothecanes=48o grains ; 31.103 grams j J = ' " TEMPERATURE To change Centigrade to Fahrenheit; (C. X|)+3 2 =F. For example, to find the equivalent of 10° Centigrade, C.=io°Xt+32=5°° F. To change Fahrenheit to Cenrigrade ; (F. — 32 ) X§=C. For example, to reduce 50° Fahrenheit to Centigrade, (F.=5o°, and (50 — 32°)Xf=io C. ; or — 40 Fahrenheit to Centigrade, F.= — 40 ( — 40° — 32°)= — 72 , whence — 72°XI= -40 C. Address of American Opticians : For the price of microscopesand microscopical supplies the student is advised to obtain a catalog of one or more of the opticians. Nearly all of them import foreign apparatus. For foreign opticians see the table of tube-length, p. 18. The Bausch & lyOtnb Optical Co , Rochester, New York James T. Dougherty y. 409-411 West 59th St., New York Eimer & Amend 205-211 3d Ave., New York The Gundlach- Manhattan Optical Company _ Rochester, N. Y. E- Leitz 30 East 18th St., New York Edward Pennock 3609 Woodland Ave., Philadelphia, Pa A. B. Porter * 324 Dearborn St., Chicago Queen & Company - _ „__ioio Chestnut St., Philadelphia. Pa. Spencer I^ens Company 367-373 Seventh St., Buffalo, N. Y. Williams, Brown & Earle 918 Chestnut St., Philadelphia, Pa. -- Voigtl&nder und Sohn, A. G 225 Fifth Ave., New YorV^^V^- Joseph Zentmayer .226-228 South 15th St., Philadelphia, "P/fc^ Besides the names here given, nearly every large city has one or more dealers in mij(f scopes and microscope supplies. UBf {<* Cornell University Library QM 551.G131908 The microscope :an introduction to micro 3 1924 001 037 088 Compliments of SIMON HENRY GAGE, Professor of Histology and Embryology, Emeritus, Cornell University, Ithaca, New York, U. S. A. THIS BOOK IS THE GIFT OF iV- 1 - f LIBRARY. THE METRIC SYSTEM \& ^ £■ S, 10 CENTIMETER RULE The upper edge is in millimeters, the lower in centimeters and half centimeters UNITS. The most commonly used divisions and multiples '_ „„___ _„„ ( Centimeter (cm.), o.oi Meter; Millimeter (mm.), o.ooi Meter: isiir™ roR) Micron(U-), o.ooi Millimeter; the Micron is the unit in Micrometry (§182). . . ^ Kilometer, 1000 Meters; used in measuring roads and other long distances. the gram for f Milligram (tag.), 0.001 Gram. weight . . \ Kilogram, 1000 Grams, used for ordinary masses, like groceries, etc. the liter for f Cubic Centimeter (cc), 0.001 loiter. This is more common than the cor- capacity . X rect form, Milliliter. Divisions of the Units are indicated by the I^atin prefixes ; deci, 0.1 ; centi, 0.01 ; Milli^^ 0.001 ; Micro, one millionth (o.oooooil of niw unit -~~- Multiples are de times ; myria, 10,001 TAB Meter (Unit of 1 (#) 39-3704i Centimeter (cm. inch. Millimeter (mm. Micron (/u) (Uni millionth of Yard=3 feet ; 36 Foot=t2 inches ; Inch= T 1 j foot ; 3- 1 Liter (Unit of ca Cubic centimeter Fluid ounce (8 fl Gram (Unit of w Kilogram= i ,000 Ounce avoirdupoi Ounce Troy or ap TEMPERATURE To change Centigrade to Fahrenheit; (C. Xf)+3 2 =F'. For example, to find the equivalent of 10° Centigrade, C.=io°Xf+32=5o° F. To change Fahrenheit to Cenrigrade ; (F. — 32°) Xf=C. For example, to reduce 50 Fahrenheit to Centigrade, (F.=so°, and (50° — 32°)Xf=io C. ; or — 40° Fahrenheit to Centigrade, F.= — 40° ( — 40 — 32°)= — 72°, whence — 7 2°X!= -40 C. Address of American Opticians: For the price of microscopes and microscopical supplies the student is advised to obtain a catalog of one or more of the opticians. Nearly all of them import foreign apparatus. For foreign opticians see the table of tube-length, p. 18. The Bausch & I,omb Optical Co , — - - Rochester, New York James T. Dougherty .*. 409-411 "West 59th St., New York Fyimer & Amend __ 205-211 3d Ave., New York The Gundlach-Manhattan Optical Company Rochester, N. Y. E. Iyeitz__. _ __ 30 East i8th St., New York Edward Pen nock 3609 Woodland Ave., Philadelphia, Pa A. B. Porter 324 Dearborn St., Chicago Queen & Company ,. _-ioio Chestnut St., Philadelphia. Pa. Spencer I Image when field lens is present ; O4 Virtual image ; A Optical tube-length, i. e.: distance from the upper focal plane of the objective (Fi ) to lower focal plane" of eye- piece F2. THE MICROSCOPE MICROSCOPICAL METHODS CHAPTER I THE MICROSCOPE AND ITS PARTS APPARATUS AND MATERIAL FOR THIS CHAPTER A simple microscope (§2, 12); A compound microscope with nose-piece (Figs. 76-95) ; eye-shade (Fig. 67), achromatic ($ 23), apochromatic ({S 25) , dry (^ 20), immersion (§21), unadjustable and adjustable objectives (§26,27); Huygenian or negative (i by an eye at E, and that if a normal or hyperopic eye is at E' a virtual image can be seen -without changing the posi- tion of the simple microscope. The long- sighted eye can see this image best as it naturally focuses converging rays on the retina. The myopic eye either sees no image at all, or a mere blur, depending upon the amount of myopia. A. object; A.' real image above the magnifier; A." virtual image which can be seen below the lens by an eye at E' ; E. eve in position to see a real image ; E.' eve in position to see A" a virtual image ; F. principal focus of the magnifier. Fig. 19. Tripod Magnifier The diagrams, Figs. 17, iS, are introduced to show under what conditions both a virtual and a real image may be seen without changing the position of the magnifier or the object. Simple microscopes are very convenient when only a small magnification (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. 19) is one of the best forms. For many purposes a special mechan- ical mounting is to be preferred. MICROSCOPE AND ACCESSORIES \CH.1 Fig. 20 Lens-holder. Fig. 21. 77;c Hastings Triplet. Fig. 22. Dissecting Microscope. CH. I] MICROSCOPE AND ACCESSORIES >A' J ;Zb A\ &}*> Figs. 23, 24, 25. Diagrams showing the formation of real and of virtual images and of the retinal image in using the simple microscope. See the explanation of Figs. 14, 15, 16. COMPOUND MICROSCOPE I 15. A Compound Microscope. — This enables one to see an enlarged, inverted image. It always consists of two optical parts — an objective, to pro- duce an enlarged, inverted, real image of the object, and an ocular acting in general like a simple microscope to magnif y* this real image (Fig. 26). 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.) § 16. The Mechanical Parts of a laboratory, compound microscope are shown in the frontispiece, and are described in the explanation of that.figure. The student shoula study the figure with a microscope before him and become thoroughly familiar with the names of all the parts. OPTICAL PARTS {S 17. 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, 26) . And as for the formation of real images in all cases, the object must be placed outside the principal focus, in- stead of within it, as for the simple microscope. (See \\ 12, 60, Figs. 16,26.) Modern microscopic objectives usually consist of two or more systems or MICROSCOPE AND ACCESSORIES [CH. I combinations of lenses, the one next the object beirig called the froni combina- tion or lens, the one farthest from the object aBd 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 ac- tion of the system serves to pro- duce an image free from color and from spherical distortion. In the ordinary achromatic objectives of the older period the convex lenses are of crown and the concave lenses of flint glass. In the best modern achromatic objectives the new Jena glass is used for a part or all of the lenses. (Figs. 27, 28.) Fig. 26. Diagram showing the principle of a compound micro- scope with the course of the rays from the object (AB) through the objective to the real image ( B'A') , thence through the ocular and into the eye to the retinal image (A 2 !?'*), and the projection of the retinal image into the field of vision as the virtual image (B^A 3 ) . s A B. The object. A*B*. The retinal image of the inverted real image, (B'A 1 ), formed by the ob- jective. B*As. The inverted vir- tual image, a projection of the retinal image. Axis. The principal optic axis of the microscope and of the eye. 'a Cr. Cornea of the eye. L. Crystalline lens of the eye. R. Single, ideal, refracting 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 ob- jective. C, CH. /] MICROSCOPE AND ACCESSORIES 13 Mirror. The mirror reflecting parallel rays to the object. The light is central. See Ch. II. Pos. Ocular. An ocular in which the real image is formed outside the ocular. Compare the positive ocular with the simple microscope {Fig. 16). NOMENCLATURE OR TERMINOLOGY OF OBJECTIVES I 18. Equivalent Focus. — In America, England, and now 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 T V in. or 2 mm., indicates that the objective produces a real image of the same size as is produced by a simple converging lens whose 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 accordance with the law that the relative size of object and image vary directly as their distance from the center of the lens (Figs. 14, 15, see Ch. IV,) it follows that the less the focal distance of the simple lens or of the equivalent focal distance of the objective, the greater is the size of the real image, as the tube-length remains constant and the image in all cases is formed at 160 or 250 mm. from the objective. I 19. Numbering or Lettering Objectives. — Instead of designating objectives by their equivalent focus, many Continental opticians use letters or figures for this purpose ; in most cases, however, the equivalent focus is also Fig. 27. Section of a dry objective showing working distance and lighting by reflected light. Axis. The principal optic axis of the objective. B C. Back Combination, composed of a plano-concave lens of flint glass {F), and a double couvex lens of crown glass (c). F C. Front Combination. C, 0, si. The cover-glass, object and slide. Mirror. The mirror is represented as above the stage, and as reflecting parallel rays from its plane face upon the object. Stage. Section of the stage of the mi- croscope. • IV. The Working Distance, that is the distance from the front of the objective tojhe object when the objective is in focus. given. 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 arbi- 14 MICROSCOPE AND ACCESSORIES [CH. I trary and does not, like the one above, give direct information concerning the objective. {! 20. Air or Dry Objectives. — These are objectives in which the space between the front of the objective and the object or cover-glass is filled with air (Fig. 27). JMost objectives of low and medium power (i. e., } in. or 3 mm. and lower powers) are dry. §21. 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 ppwer 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 glycerin containing some salt, as stannous chlorid in solution. When oil is used as the immersion fluid the objectives are frequently called oil immersion objectives. The disturbing effect of the cover-glass (Fig. 64) 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 objective ; and when the object is mounted in balsam there is practically no refraction in the ray from the time it leaves ' the balsam till it enters the objective. Fig. 28. Sectional view of an Immersion, Adjustable Objective, and the object lighted with axial or central and with oblique light. Axis. The principal optic axis of the objective. B C, M C, F C. The back, middle and front combination of the objective. In this case the front is not a combination, but a single plano-convex lens. A, B. Parallel rays reflected by the mir- ror axially or centrally upon the object. C. Ray reflected to the object obliquely. I. Immersion fluid between the front of the objective and the cover glass or object ( O) . Mirror. The mirror of the microscope. O. Object. It is represented without a cover-glass. Ordinarily objects are covered whether examined with immersion or -with dry objectives. Stage. Section of the stage of the micro- scope. Non-Achromatic or Chromatic Objectives. — These are objectives in which the chromatic 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. (\\ 8, 9, Figs. 12, 13. ) ' 1* II *t : >s ■< i ' v '- . as. 33, 34, 35 are somewhat modified from Ellenberger, and are introduced to illustrate the relative amount of utilized light, with dry, zvaler immersion and homogeneous immersion objectives of the same equivalent focus. The point from which the rays emanate is in air in each case. If Canada balsam were beneath the cover-glass in place of the air there would be practically no refraction of the rays on entering the cover-glass (§21). Fig. 33. Showing the course of the rays passing through a cover- glass from an axial point of the object, and the num- ber that finally enter the front of a dry objective. ^ f? ISp^ ~-^\\\ i//s^ \\\\1 \ll//cove,v Pig. 34. Rays from the axial point of the object traversing a cover of the same thickness as in Fig. 33, and entering the front lens of a water immersion objective. Fig. 35. Rays from an axial point of the object traversing a cover-glass and entering the front of a homogeneous im- mersion objective. next lower sine whose angle is known. Add this number of minutes to the angle of the next lower sine and the sum will represent the desired angle. Or if the sine whose angle is to be found is nearer in size to the sine just greater, proceed exactly as before, getting the difference in the sines, but subtract the number of minutes of difference and the result will give the angle sought. For example take the case in Section 10S where the sine of the angle of 28° 54' is given as 0.48327. If one consults the table the nearest sines found are 0.48099, the sine of 28° 45', and 0.4848 r, the sine of 29°. Evidently then the angle sought must lie between 28 45', and 29 . If the difference hetween o 48481 and 0.48099 is obtained, 0.48481 — 0.48099—0.00382, and if this increase for 15' be divided by 15 it will give the increase for 1 minute ; 0.00382^-15^0.000254. Now the difference between the sine whose angle is to be found and the next lower sine is 0.48327 — 0.48099=0.00228. If this differ- ence be divided by the amount found necessary for I minute it will give the total minutes above 28 45', 0.00228-^-0.000254=9. That is, the angle sought is •9 minutes greater than 28° 45'=28° 54'. 22 MICROSCOPE AND ACCESSORIES \_CH. I With the water immersion objective the medium in front is water, and its inde'x of refraction is 1.33, whence «= 1.3-3. Half the angular aperture is ^°=47 , and by the table the sine of 47° is found to be 0.731, i. e., sin u= 0.731, whence N. A.=» or 1.33XSH1 « or 0.731=0.972. With the oil immersion in the same way N. A.=w sin u ; n or the index of refraction of the homegeneous fluid in front of the objective is 1.52, and the semi-angle of aperture is \°-°=45°. The sine of 45° is 0.707, whence N. A.=n or i.52Xsin u or 0.707=1.074. By comparing these numerical apertures: Dry 0.799, water 0.972, homo- geneous immersion 1.074, the same idea of the real light efficiency and image power of the different objectives is obtained, as in the graphic representations shown in Figs. 33-35. If one knows the numerical aperture (N. A.) of an objective the angular aperture is readily determined from the formula ; and one can determine the equivalent angles of objectives used in different media {i. e., dry or immer- sion). For example, suppose each of three objectives has a numerical aper- ture (N. A. ) of 0.80, what is the angular aperture of each ? Using the formula of N. A.=ra sinw, one has N. A=o.8o for all the objectives. For the dry objective «=i (Refractive index of air). For the water immersion objective #=1.33 ( Refractive index of water). For the homogeneous immersion objective 72=1.52 (Refractive index of homogeneous liquid). And 2 u is to be found in each case. For the dry objective, substituting the known values the formula becomes 0.80=1 sin u, or sin «=o.8o. By inspecting the table of naturalsines (3d page of cover) it will be found that 0.80 is the sine of 53 degrees and 8 minutes. As this is half the angle the entire angular aperture of the dry objective must be 53 8'X2=io6° 16'. For the water immersion objective, substituting the known values in the 0.80 formula as before : 0.80=1.33 s ' n u % or s ^ n u ~ =0.6015. i-33 Consulting the table of sines as before, it will be found that 0.6015 is the sine 01 36° 59' whence the angular aperture (water angle) is 36 59^X2=73° 58'. For the homogeneous immersion objective, substituting the known values, 0.80 the formula becomes : 0.80=1.52 sin u whence sin «= =0.5263. And by 1-52 consulting the table of sines it will be found that this is the sine of 31° 45^ / whence 2 u or the entire angle (balsam or oil angle) is 63°3i/. That is, three objectives of equal resolving powers, each with a numerical aperture of 0.80 would have an angular aperture of 106° 16' in air, 73° 58' in water and 63 31' in homogeneous immersion "liquid. For the apparatus and method of determining aperture, see Ch. X. I 39. Table of a group of Objectives with the Numerical Aperture (N. A.) and the method of obtaining it. Half the angular aperture is designated by u and the index of refraction of the medium in front of the objective by n. For CH, I] MICROSCOPE AND ACCESSORIES 23 dry objectives this is air and n=/, for water immersions n=/-3j, and for homogeneous immersions n=/.jz. (For a table of natural sines, see third page of cover.) Objective •It; 8 Natural Sine of half the angular aperture (sin u. ) Index of Refraction of the medi- um iu front of the objec- tive («) Numerical Aperture (N. A. )=n sin u 25 mm, Dry. 25 mm. Dry. 12}4 mm. Dry. 12^ mm. Dry. 6 mm. Dry. 6 mm. Dry. 3 mm. Dry. 3 mm. Dry. 2 mm. Water Immersion 2 mm. Homogeneous Immersion 2 mm. Homogeneous Immersion 4o" 42° 75° 136° 1 15° 163 96°I2' 20 Sin =0.1736 2 40 Sin =0.3420 2 42- Sin =0.3584 2 100 ■ Sin =0.7660 2 - 7S Sin =0.6087 2 136 Sin =0.9272 2 115 Sin =0.8434 2 163 Sin =0.9890 2 9 6°I2' Sin =0.7443 iio 38' iio°38 / Sin -=0.8223 2 i34°io' Sin- =0.9211 »=i-33 «=1.52 =1.52 N.A.= iXo- 1 736= 0.173 N.A.= 1X0-3420=0.342 N.A.= 1X0.3583=0.358 N.A.= 1X0.7660=0.766 N.A.= 1X0.6087=0.609 N.A.= 1X0.9272=0.927 N.A.= 1X0.8434=0.843 N.A.= 1X0.9890=0989 N.A.=i.33Xo. 7443=0.99 N.A.=i. 52X0. 8223=1. 25 N.A.= 1. 52X0. 9210=1. 40 § 40. 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 24 MICROSCOPE AND ACCESSORIES, [ CH. 1 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 aperture 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 2A.XN.A. That is twice the num- ber 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 element 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, and 1906, p. 521) it is believed that not more than ^ 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 with a given light the formula would become 2/\xXN.A.=3/2/lN.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 3/2N.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 46,666X3/2=70,000 approximately. If the N.A. is 1, then the objective will resolve or make visible 70,000 lines to the inch, or approximately 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, while here only % of the aperture is assumed to be available. (2) The illuminating power of an objective of a given focus is found to vary directly as the square of the numerical aperture (N.A.) 2 . Thus if two 4 mm. objectives of N.A. 0.20 and N.A. 0.40 were compared as to their illumi- nating power it would be found from the above that they would vary as O.20 2 :o.4o 2 =o.o4oo:o.i6oo or 1 14. That is the objective of 0.20 N.A. would have but % 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, I is found to vary as the reciprocal of the numerical aperture so that in N.A. an objective of a given focus the greater the aperture the less the penetrating power. UH. I] MICROSCOPE AND ACCESSORIES 25 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 it is as with the unaided eye, the telescope and the photo- graphic camera. This is 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 ; Wright, Gordon and Beck). (B) The other view originated with Professor Abbe, and in the words of Carpenter-Dallinger, pp. 62, 43: "What this is becomes explicable by the researches of Abbe. It is demonstrated that microscopic vision is sui generis. There is and can be, no 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 dioptrical 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 alternations of dark and light occur." For a consideration of the aperture question, its history and significance, 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, 4bo; 1883, p. 790; 1884, p. 20; 1896, p. 681 ; 1897, p. 71 ; 1898, pp. 354, 362, 592 ; Mercer, Proceedings Amer. Micr. Soc, 1896, pp. 321-396; Lewis Wright, Philos. Mag., June, 1898, pp. 480-503 ; Carpenter-Dallinger, Chapter II ; Nelson, Jour. Quekett Micr. Club, VI, pp. 14-38 ; Jour. Roy. Micr. Soc, 1906, pp. 521-531 ; A. E. Wright's Prin- ciples of Microscopy ; Conrad Beck, Theory of the Microscope. Gordon, Jour. Roy. Micr. Soc, 1902. THE OCULAR \ 41. A Microscopic Ocular or Eye-Piece consists of one or more con- verging 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. Depending upon the relation and action of the different lenses form- ing oculars, they are divided into two great groups, negative and positive. (i 42. 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. 26). As the field-lens of the ocular aids in the forma- tion 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. 26 MICROSCOPE AND ACCESSORIES \_CH.I I 43. 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 the dia- phragm is below the ocular lenses with the positive ocular and between the lenses in the negative ocular (Figs. 36-37). Fig. 36. Sectional view of a Huygenian ocular to show the formation of the Eye-Point. Axis. Optic axis of the ocular. D. Diaphragm of the ocular. E. L. Eye-Lens. F. L. Field-Lens. E. P. Eye-Point. As seen in section, it appears something like an hour-glass. When seen as looking into the ocular, i. e. , in transection, it appears as a cir- cle of light. It is at the point where the most rays cross. TABLE OF OCULARS \ 44. 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 Ger- man equivalents, and a brief statement of their character. Achromatic Ocular; Fr. Oculaireachromatique; Ger. achromatisches Oku- lar. Oculars in which chromatic aberation is wholly or nearly eliminated. — Aplanatic Ocular; Fr. Oculaire aplanatique; Ger. aplanatisches Okular (see (S 24) . — Binocular, stereoscopic Ocular; Fr. Oculaire binoculaire stereoscopique; Ger. stereoskopisches 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 microscope. By an arrangement of pri9tns the image forming rays are divided, half being sent to each eye. The most satisfactory form was worked out by.Tolles and is constructed on true stereotomic princi- ples, both fields, being equally illuminated. His ocular is also erecting-. — Campani 1 s Ocular (see Huygenian Ocular). — Compound Ocular; Fr. Oculaire compost; Ger. zusammengesetztes Okular. An ocular of two or more lenses, e. g., the Huygenian (see Fig. 36). — Continental Ocular. An ocular mounted in a tube of uniform diameter as in Fig. 37. — Deep Ocular, see high ocular. — Erecting Ocular; Fr. Oculaire redresseur; Ger. bildumkehrendes Okular. An ocular with which an erecting prism is connected so that the image is erect as with the simple microscope. Such oculars are most common on dissecting microscopes. — Filar micrometer Ocular; Screw m. o., Cobweb m. o., Ger. Okular-Schraubenmikrometer. A modification of Ramsden's Telescopic Cob- web micrometer ocular. — Goniometer Ocular; Fr. Oculaire 3, goniometre; Ger. \_CH.I MICROSCOPE AND ACCESSORIES 27 Goniometer-Okular. An ocular with goniometer for measuring the angles of minute crystals. — High Ocular, sometimes called a deep ocular. One that magnifies the real, image considerably, i. e., 10 to 20 fold. — Huygenian Ocular, Huygens' O., Campani's O., Airy's O.; Fr. Oculaire d'Huygens, o. de Cam- pani; Ger. Huygens'sches Okular, Campanisches Okular, see \ 45. — Index Ocular; Ger. Spitzen-O. . An ocular with a minute pointer or two pointers at the level of the real image. The points are movable and serve for indicators and also, although not satisfactorily, for micrometry. — Kellner's Ocular, see orthoscopic ocular — Low ocular, also called shallow ocular. An ocular which magnifies the real image only moderately, i. e., 2 to 8 fold. — Micrometer or micrometric Ocular; Fr. Oculaire micrometrique ou & micrometre; Ger. Mikrometer-Okular, Mess Okular Benches O, Jack- son m. o., see $48. — Microscopic Ocular; Fr. Oculaire microscopique ; Ger. mikroskopisches Okular. An ocular for the microscope instead of one for a telescope. — Negative Ocular, see \ 42. — Nelson's screw- micrometer ocular. A modification of the Ramsden's screw or cob-web micrometer in which positive compensating oculars may be used. — Orthoscopic Oculars; also called Kellner's Ocular; Fr. Oculaire orthoscopique; Ger. Kel- ner'sches oder orthoskopisches Okular. An ocular with an eye-lens like one of the combinations of an objective (Figs. 27, 29) and a 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. — Par-focal Oculars, a series of oculars so arranged that the microscope remains in focus when the oculars are inter- changed (Pennock, Micr. Bulletin, vol. iii, p. 9. 3r, 1886). — Periscopic Ocular; Fr. Oculaire periscopique ; Ger. periskopisches Okular. A positive ocular devised by Gundlach. It consists of a double convex field-lens and a triplet eye-lens. It gives a large, flat field. — Positive Ocular, see \ 43. — Projection Ocular ; Fr. Oculaire de projection ; Ger. Projections-Okular, see \ 47. — Ramsden's Ocular ; Fr. Oculaire de Ramsden ; Ger. Ramsden'sches Okular. A positive ocular devised by Ramsden. It consists of two plano-convex lenses placed close together with the convex surfaces facing each other. Only the central part of the field is clear. Searching Ocular ; Fr. Oculaire d'orienta- tion ; Ger. Sucher-Okular, see $46. Shallow Ocular, see low ocular. — Solid Ocular, holosteric O.; Fr. Oculaire holost£re ; Ger. holosterisches Okular, Vollglass-Okular. A negative eye-piece devised 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 dia- phram, a groove is cut at a proper level and filled with black pigment. It is especially excellent where a high ocular is desired. — Spectral or spectroscopic Ocular ; Fr. Oculaire spectroscopique ; Ger. Spectral-Okular, see Microspec- troscope, Ch. VI. — Stauroscopic Ocular ; Fr. Oculaire Stauroscopique ; Ger. Stauroskop-Okular. An ocular with a Bertrand's quartz plate for mineralog- cal purposes — Working Ocular ; Fr. Oculaire de travail ; Ger. Arbeits- Okular, see \ 46. \ 45. 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. 36. ), aid- 28 MICROSCOPE AND ACCESSORIES CI I. /] ing the objective in forming the real image, and an eye-lens which magnifies the real image. While 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. Ocular lo 2 Fig. 37. Compensating Oculars of Zeiss, with section removed to show the construction. The tine A-A is at the level of the upper end of the tube of the microscope while B-B represents the lower focal points. It will be seen that the mounting is so arranged that the lower focal points in all are in the same plane and therefore the microscope remains in focus upon changing oculars. ( The oculars are par-focal.) The lower oculars 2, 4. and 6 are negative, and the higher ones, 8, 12, /S, are positive. The numbers 2, 4, 6, 8, 12, /S, indicate the magnification of the ocular. From Zeiss' Catalog. ) \ 46. Compensating Oculars.— These are oculars specially constructed for use with the apochromatic objectives. They compensate for aberrations outside the axis which could not be so readily eliminated in the objective it- self. An ocular of this kind, mangifying but twice, is made for use with high powers, for the sake of the large field in finding objects; it is called a search- ing ocular; those ordinarily used for observation are in contradistinction called working oculars. Part of the compensating oculars are positive and part negative. (Fig. 37.) \ 47. Projection Oculars. — These are oculars especially designed for pro- jecting a microscopic image on the screen for class demonstrations, or for photographing with the microscope. While they are specially adapted for use with apochromatic objectives, they may also be used with ordinary ach- romatic objectives of large numerical aperture. The projection oculars (Fig. 38) consist of a collective lens or field lens and of a carefully corrected system for the eye lens. The eye lens is movable so that a sharp image of the diaphragm between the field and eye lens may be projected upon the screen at different screen distances. % 48. Micrometer Ocular. — This is an ocular connected with an ocular [CH. I MICROSCOPE AND ACCESSORIES 29 micrometer. The micrometer may be removable, or it nay be permanently in connection with the ocular, and arranged with spring and screw, by which it may be moved back and forth across the field. (See Ch. IV.) \ 49. Spectral or Spectroscopic Ocular. — (See Micro-Spectroscope, Ch. VI.) DESIGNATION OF OCULARS \ 50. Equivalent Focus. — As with objectives, some opticians designate the oculars by their equivalent focus (<< 15 ). With this method the power of No. 2. Fig. 38. Projection Oculars with section removed to show the construction. Below are shown the upper ends with graduated circle to indicate the amotmt 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. (Zeiss' Catalog.) the ocular, as with objectives, varies inversely as the equivalent focal length, and therefore the greater the equivalent focal length the less the magnifica- tion. This seems as desirable a mode for oculars as for objectives and is com- ing more and more into use by the most progressive opticians. It is the method of designation advocated by Dr. R. H. Ward for many years, and was recommended by the committee of the American Microscopical Society, (Proc. Amer. Micr. Soc, 1SS3, p. 175, 1884, p. 228). \ 51. Numbering and Lettering. — Oculars like objectives may be num- bered 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. £ mm. 25 I2# " Huygenian 45 mm. . 7.0 4.0 37^ mm. 25 12^ " Huygenian 17 mm N. A. =0.25 3-0 2.0 1.6 37^ mm. 25 " 12^ "• Huygenian 5-7 2.8 1.4 0.97 180 mm. 45 15 " 10 Compensation 5 mm. _ . N. A. =0.92 o.54i 0.371 0.290 37^ mm. 25 " I2# " Huygenian 0.850 0.501 0.250 0.173 180 mm. 45 " 15 " 10 Compensation 2 mm N. A.=i.25 0.270 0.186 0.147 37^ mm. 25 I2# " Huygenian ' 0-45O 0.251 0.125 0.088 180 mm. 45 15 10 Compensation 34 MICROSCOPE AND ACCESSORIES [CH. I FUNCTION OF AN OBJECTIVE § 60. Put a 50 mm. objective on the microscope or screw off the front combination of a 16 mm., (^-in), and put the back com- bination on the microscope for a low objective. Place some printed letters or figures under the microscope, and s m ™ . r 17 mm ■8 j m }n ^FiG. 42. Figures showing approximately the actual size of the field with objectives of 85 mm., 4.5 mm. , 17 mm., 5 mm. and 2 mm., equivalent focus, and an ocular of 37 l /i mm. equivalent focus in each case. This figure shows graphically what is also very clearly indicated in the table ($ 59). 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 micro- scopes- Lower the tube of the microscope by means of the coarse ad- justment until the objective is within 2 to 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, i. 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. 26). If the objective is not raised sufficiently, and the head is held too near the microscope, the objective will act as a simple micro- scope. If the letters are erect, and appear to be down in the micro- scope and not on the screen, hold the head farther from it, shade the *\ 61. 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. CH. I] MICROSCOPE AND ACCESSORIES 35 screen, and raise the tube of the microscope until the letters do ap- pear on the ground glass. To demonstrate that the object must be outside the principal focus with the compound microscope, remote the screen and turn the tube of the microscope directly toward the sun. Move the tube of the microscope with the coarse adjustment until the burning or focal point is found (§ 7, 13). Measure the distance from the paper object on the stage to the objective, and it will represent approx- imately the principal focal distance (Figs. 10, n). Replace the screen over the top of the tube, no image can be seen. Slowly raise the tube of the microscope and the image will finally appear. If the distance between the object and the objective is now taken, it will be found considerably greater that the principal focal distance {compare § 12). § 62 Aerial Image. — After seeing the real image on the ground-glass, or paper, use the lens paper over about half of the opening of the tube of the microscope. Hold the eye about 250 mm. from the microscope as before and shade the top of the tube by holding the hand between it and the light, or in some other way. The real image can be seen in part as if on the paper and in part in the air. Move the paper so that the image of half a letter will be on the paper and half in the air. Another striking experiment is to have a small hole in the paper placed over the center of the tube opening, then if a printed word extends entirely across the diameter of the tube its central part may be seen in the air, the lateral parts on the paper. The advantage of the paper over part of the opening is to enable one to accomodate the eyes for the right distance. If the paper is absent the eyes adjust themselves for the light circle at the back of the objective, and the aerial image appears low in the tube. Furthermore it is more difficult to see the aerial image in space than to see the image on the ground-glass or paper, for the eye must be held in the right position to receive the rays projected from the real image, while the granular surface of the glass and the deli- cate 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. § 63 The Function of an Objective, as seen from these ex- periments, is to form an enlarged, inverted, real image of an object, 36 MICROSCOPE AND ACCESSORIES [CH. I this image being formed on the opposite side of the objective from the object (Fig. 26). FUNCTION OF AN OCULAR § 64. Using the same objective as for § 53, get as clear an image of the letters as possible on the lens paper or ground-glass screen. Look at the image with a simple microscope (Fig. 19, 21) 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 (§ 12). Remove the screen and observe the aerial image with the tripod. Put a 50mm. (A, No. 1 or 2 in.), ocular i. e., an ocular of low magnification in position (§ 55). Hold the eye about 10 to 20 millimeters from the eye-lens and look into the microscope. The letters will appear as when the simple microscope was used (see Fig. 43. Diagram, of the simple microscope showing the course of the rays and all the images, and that the eye forms an integral part of it. A x B 1 . The object within the principal focus. A* Z?3. 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. 2? 2 A". Retinal image of the object (A' B 1 ) The virtual image is simply a projection of the retinal image into the field of vision. Axis. The principal optic axis of the micro- scope and of the eye. Cr. Cornea of the eye. L. Crystalline tens of the eye. R. Ideal refracting surface at which all the refractions of the eye may be assumed to take place. above), the image will become more distinct by slightly raising the tube of the microscope with the coarse adjustment. § 65. 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. 26), and that formed by a. simple microscope (Fig. 43). CH. /] MICROSCOPE AND ACCESSORIES 37 It should be borne in mind, however, that the rays from an object as usually examined with a simple microscope, extend from the object in all directions, and no matter at what angle the simple microscope is held, provided it is sufficiently near and points toward the object, an image may be seen. The rays from a real image, however, are continued in certain definite lines and not in all direc- tions; hence, in order to see this aerial image with an ocular or simple microscope, or in order to see the aerial image with the unaided eye, the simple microscope, ocular or eye must be in the path of the rays (Fig. 26). § 66. 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. 36.) Demonstrate this by screwing off the field-lens and using the eye-lens alone as an ocular, refocusing if necessary. Note that the image is bordered by a colored haze (§ 8). 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 con- siderably farther away than when the field-lens is in place. § 67. 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. 36), 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 (§60). Light the object brightly, focus the microscope, shade the ocular, then hold some ground-glass or a piece of the lens paper above the ocular and slowly raise and lower it until the smallest circle of light is found. By using different oculars it will be seen that the eye-point is nearer the eye-lens in high than in low oculars, that is the eye-point is nearer the eye-lens for an ocular of small equivalent focus than for one of greater focal length. * The bright circle above the ocular is sometimes called the Ramsden Circle or Disc. See Carpenter-Dallinger, p. 106; Spitta, 114-118; Wright p. 157 ; Beck, p. 14. 38 MIQROSCOPE AND ACCESSORIES [ CH. I 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 micro- scope may be consulted with great advantage for different or more exhaustive treatment. The most satisfactory work in English is Carpenter-Dallinger, 8th 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, 1897-1901+. Carpenter-Dallinger, 8th Ed. Petri, Das Mikroskop. The following special articles in periodicals may be examined with advan- tage: Apochromatic Objectives, etc. Dippel in Zeit. wiss. Mikr., 1886, p. 303; also in the Jour. Roy. Micr. Soc, 1886,'pp. 316, 849, 1110; same, 1890, p. 480, Zeit. f. Instrumentenk., 1890, pp. 1-6; Micr. Built., 1891, pp. 6-7. Tube-length, etc. Gage, Proc. Atner. Soc. Micrs., 1887, pp. 168-172; also in the Microscope, the Jour. Roy. Micr. Soc, and in Zeit, wiss. Mikr., 1887-8. Bausch, Proc. Amer. Soc. Micrs., 1890, pp. 43-49; also in the Microscope, 1890; pp. 289-296. Aperture. J. D. Cox, Presidential Address, Proc. Amer. Soc. Micrs., 1884, PP- 5-39. Jour. Roy. Micr. Soc, 1881, pp. 303, 348, 365, 388; 1882, pp. 300, 460; 1883, p. 790; 1884, p. 20. Czapski, Theorie der optischen Instrumente nach Abbe. Theory of Microscopic vision, Wright, Jour. Roy. Micr. Soc. 1905 p. I, Biography of Abbe, same, p. 156. See also the references to \ 40. 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. 60); Homogeneous immersion liquid, xylene, alcohol, distilled water; Mounted preparation of fly's wing (§ 79); Mounted preparation of Pleuro- sigma (I 88, 89) ; Stage or ocular micrometer (\ 103); Glass slides and cover- glasses (Ch. VII) ; 10 per ct. solution of salicylic acid in 95 per ct. alcohol (§ 103); Preparation of stained bacteria ( g 119); Vial of equal parts olive or cotton seed oil or liquid vaselin and xylene (§123); Eye shade (Fig. 67); Screen for whole microscope (Fig. 66, 68). FOCUSING \ 68. Focusing is mutually arranging an object and the microscope so that a clear image may be seen. . With a simple microscope ($ 12) 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. I 69. Working Distance.— By this is meant the space between the simple microscope and the object, or between the front lens of the compound micro- scope and the object, when the microscope is in focus. This working distance is always considerably less than the equivalent focal length of the objective. For example, the front-lens of a 6 mm. or % in. objective would not be 6. 4 o LIGHTING AND FOCUSING [ CH. II millimeters or % 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 object. (See \ 27, 33.) If too thick covers are used it may be impossible to get an objective near enough an object to get it in focus. For objects that admit of examination with high powers it is always better to use thin covers. I 70. Free Working Distance — In the microscope catalog of Zeiss there is givena table of the size of the field and also of the " free working-distance." This free working-distance is the space between the lower end of the objective and the cover glass of T Y^ mm. thickness, when the objective is in focus on an object immediately under the cover. This is exceedingly practical information for a possessor of a microscope, and it is hoped that the other opticians will adopt the suggestion. Naturally, however, the free working-distance for each optician should be reckoned from the top of the cover for which his unadjus- table objectives are corrected. If, for example, the thickness of cover for which an objective is corrected is fifc mm. then the free working-distance should be that between the top of this and the objective when the objective is in focus on an object under the cover. (See the table of cover-glass thick- ness, I 33). LIGHTING WITH DAYLIGHT \ 71. 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. 66) 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 thor- oughly 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 des- ignated as reflected and transmitted, axial and oblique. \ 72. 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 little used ; but in the study of opaque objects, like whole insects, etc., it is used a great deal. For low powers, ordinary daylight that naturally falls upon the object, or is reflected or condensed upon it with a mirror or condensing lens, answers very well. For high powers and for CH. II] LIGHTING AND FOCUSING special purposes, special illuminating apparatus has been devised also Carpenter-Dallinger, Ch. IV. ) 31). 41 (See \ 73. Transmitted Light.— By this is meant light which passes through an object from the opposite side. The details of a photographic negative Stig 44 Figs. 44-45- For full explanation see Figs. 27 and 28. 45 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 trans- mitted light, and they are in some way rendered transparent or semi-trans- parent. 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 ($ 99), and thence transmitted to the object from below (Figs. 54-57). I 74. — 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 microscope, or a diverging or a converging cone of light whose axial ray is coincident with the optic axis of the microscope. In either case the object is symmetrically illuminated. § 75. Oblique Light. — This is light in which parallel rays from a plane mirror form an angle with the optic axis of the microscope (Fig. 45). Or if a concave mirror or a condenser is used, the light is oblique when the axial ray of the cone of light forms an angle with the optic axis (Fig. 45). 42 LIGHTING AND FOCUSING [ CII. II DIAPHRAGMS I 76. Diaphragms and their Proper Employment. — Diaphragms are opaque disks with openings of various sizes, which are placed between the source of light or mirror and the object. In some cases an iris diaphragm is used, and then the same one is capable of giving a large range of openings. The object of a diaphragm in general, is to cut off all adventitious light and thus enable one to light the object in such a way that the light finally reach- ing 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. ji 77. 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 considerable distance below the object, (1) it allows considerable adventi- tious light to reach the object and thus injures the distinctness of the micro- scope image; (2) it orevents the use of very oblique light unless it swings with the mirror ; (3) it cuts off a part of the illuminating cone from a concave mirror. On the other hand, even with a small diaphragm, the whole field will be lighted. With an illuminator or condenser (Figs. 47, 54), the diaphragm serves to narrow the pencil to be transmitted through the condenser, and thus to limit the aperture (see §95). 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 pro- produced (f! 103; Fig. 57). ARTIFICIAL ILLUMINATION J 78. For evening work and for certain special purposes, artificial illumi- nation 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 (seeCh. 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 EXPERIMENTS § 79. Lighting with a Mirror. — As the following experi- CH. II] LIGHTING AND FOCUSING 43 ments are for mirror lighting only, remove the subtage condenser if present (see § 90, for condenser). Place a mounted fly's wing under the microscope, put the 16 mm. (^in.) or other low objec- tive 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 (§ 57) is illuminated, by looking at the object on the stage, but more satisfactorily by looking into the microscope. It some- times 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 concave mirror con- denses the light, the field will look brighter with it than with the plane mirror. It is especially desirable to remember that the excel- lence of lighting depends in part on the position of the diaphragm (§ 77)- If the greatest illumination is to be obtained from the con- cave mirror, its ppsition 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. § 80. Use of the Plane and of the Concave Mirror. — The mirror should be freely movable, and have a plane and a concave face. The concaved face is used when a large amount of light is needed, the plane face when a moderate amount is needed or when it is necesssay to have parallel rays or to know the direction of the rays. FOCUSING EXPERIMENTS* § 81. Focusing with Low Objectives. — Place a mounted \ 82. *Par-Focal Oculars. — By this is meant oculars of different power in which the microscope remains in focus on changing the oculars. As originally constructed the microscope had to be focused every time the oculars were changed. Mr. Edward Pennock in seeking to overcome this inconvenience wrote to Professor Abbe for advice in 188 r. After successfully producing oculars of different powers for the Acme microscopes of Jas. W. Queen & Co., according to the directions given by Professor Abbe, Mr. Pen- nock as editor of the Microscopical Bulletin and Science News published in Vol. Ill, 1886, pp. 9-10, the following with Professor Abbe's letter : " Chang- ing Eyepieces without altering focus, etc. Some years ago the writer in looking up certain questions in connection with eyepieces took occasion to 44 LIGHTING AND FOCUSING [ CH. II fly's wing under the microscope ; put the 16 mm. {yi in.) objective in position, and also the lowest ocular. Select the proper opening in the diaphragm and light the object well with transmitted light (§ 73. 77)- 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 micro- scope 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 (§ 50). If the oculars are not par-focal it will be necessary to lower the tube somewhat to get the microscope in focus. Pull out the draw-tube 4 to 6 cm. , thus lengthening the body of the microscope ; it will be found necessary to lower the tube of the microscope somewhat. (For reason, see Fig. 65.) § 83. 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 grad- write to Professor Abbe, and his reply, kindly given, is so clear and to the point, and of such interest and value, that we take the liberty of publishing it for the benefit of our readers." "Jena, June 25th, 1881. Dear Sir : The question which you ask admits of a simple answer : In order to change the oculars of a microscope without chang- ing the focus of the objective, neither the diaphragm nor the field lens must come to the same place in the microscope tube, but the anterior (lower) focal points of the ocular systems must do this. In the case of a Huygehenian eyepiece, the said anterior focus 19 a virtual one situated above the field lens at a place D*, which is more distant from the field lens .than the diaphragm D. The level of D* is the place where the virtual image of the diaphragm appears to an observer looking through the field lens. Rays which are required to emerge from the eye lens as parallel rays (or nearly parallel) must of course enter into the ocular converging to the point D*. Consequently if different oculars are inserted successively in such a way that the point D* comes to the same place of the tube always, the conjugate foci of object and image in the objective remain unaltered." CH. //] LIGHTING AND FOCUSING 45 ually 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. § 84. Focusing with High Objectives.— Employ the same object as before, elevate the tube of the microscope and, if no revolv- ing nose-piece is present, remove the 16 mm. (fi in.) objective as indicated. Put a 4 or 3 mm. (\ or \ in.) or a higher objective in place, and use a low ocular. ^22 2" Ejr.-lw Virtual (.TOO^ ^ Hit Jr»|.ura$ru ruu i»o» Fig. 46 "This arrangement and no other one fulfills at the same time the other request that the amplification of the microscope with different oculars should be in exact inverse proportion of the equivalent focal length of the oculars." " The position of the point D* may be easily calculated for every ocular. If A is the distance of the diaphragm from the field lens and X the focal length of that lens, the distance of the focus D* above the diaphragm (z. e. the dis- A= tance from D to D*) will be: fd= . Hoping that these explanations X— A will be found satisfactory for your aim, I remain yours sincerely, DR. E. ABBE." Oa p. 31 of the Bulletin is the following : " Par- focal Eye-pieces. Referring to the article in the April issue of the Bulletin, on changing eye-pieces with- out altering focus, etc., we announce that we are prepared to furnish eye- pieces as there described with our Acme microscopes at a slight additional expense. We have named these eye-pieces Par-Focai,, meaning of equal focus, from the Latin par (equal) and focus.'' 1 46 LIGHTING AND FOCUSING [ CH. II Light well, and employ the proper opening in the diaphragm, etc. (§ 77.) Look between the front of the objective and the" ob- ject as before"(§ 81), 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 prepara- tion 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 colored. With high powers and scattered objects there might be no object in the small field (see § 57 Fig. 42 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 ap- proaching the focal point. It is sometimes desirable to focus on the edge of the cement ring or on the little ring made by the marker (see Figs. 70-75.) Note that this high objective must be brought nearer the ob- ject than the low one, and that by changing to a higher ocular (if the oculars are not par-focal) or lengthening the tube of the micro- scope it will be found necessary to bring the objective still nearer the object, as with the low objective. (For reason see Fig. 65.) § 86. Always Focus Up, as directed above. If one lowers the tube only when looking at the end of the objective as directed \ 85. Par-Focal Objectives. — By this is meant that the objectives are so mounted that when changed on the microscope the object will remain approx- imately in focus for all if it is in focus for any one. The expression is appli- cable especially to a group of objectives on a revolving nose-piece. The tube-length of the microscope must remain constant, for only a slight change in length (10 to 15 mm.) will destroy the parfocalization. In case the objectives on a revolving nose-piece are somewhat out of parfocalizatian one may correct it by getting one in exact focus, and then noting when the others are rotated in place whether the microscope must be focused up or down to bring the objective in focus. If one winds a piece of string around the objective that is up too high it will prevent it entering the nut of the nose-piece so far and hold it down at the right level. It is not known by the writer who first thought of arranging the objectives so that the different powers would be in focus when in position. It is a recent improvement, coming in as a necessary consequence of parfocalizing the oculars. CH. II] LIGHTING AND FOCUSING 47 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 adjustment constantly, and the focus is continually varied. § 87. Determination of Working Distance. As stated in § 69, this is the distance between the front lens of the objective and the object when the objective is in focus. It is always less than the equivelent focal length of the objective. Make a wooden wedge 10 cm. long which shall be exceedingly thin at one end and about 20 mm. thick at the other. Place a slide on the stage and some dust on the slide. Do not use a cover-glass. Focus the dust carefully first with the low then with the high ob- jective. When the objective is in focus push the wedge under the objective on the slide until it touches the objective. Mark the place of contact with a pencil and then measure the thickness of the wedge with a rule opposite the point of contact. This thickness will represent very closely the working distance. For measuring the thickness of the wedge at the point of contact for the high ob- jective use a steel scale ruled in \ mm. and the tripod to see the di- visions. Or one may use a cover-glass measure (Ch. VIII) for de- termining the thickness of the wedge. For the higher powers if one has a microscope in which the fine adjustment is graduated, the working distance may be readily de- termined when the thickness of the cover-glass over the specimen is known, as follows : Get the object in focus, lower the tube of the microscope, until the front of the objective just touches the cover-glass. Note the position of the micrometer screw and slowly focus up with the fine adjustment until the object is in focus. The distance the objective was raised plus the thickness of the cover- glass represents the working distance. For example, a 3 mm. ob- jective after being brought in contact with the cover-glass was raised by the fine adjustment a distance represented by 16 of the divisions on the head of the micrometer screw. Bach division rep- resented 0.01 mm., consequently the objective was raised 0.16 mm. 4 8 LIGHTING AND FOCUSING [ CH. II As the cover- glass on the specimen used was 0.15 mm. the total working distance is 0.16+0.15=0.31 mm. CENTRAL AND OBLIQUE LIGHT WITH A MIRROR § 88. Axial or Central Light (§ 74). — Remove the con- denser or any diaphragm from the substage, then place a preparation containing minute air bubbles under the microscope. The prepara- tion may be easily made by beating a drop of mucilage on the slide and covering it (see Ch. III). Use a 4 or 3 mm., (>fj.in.) or No. 7 objective and a medium ocular. Focus the microscope and select a very small bubble, one whose image appears about 1 mm. in diameter, then arrange the plane mirror so that the light spot in the bubble appears exactly in the "center. Without changing the position of the mirror in the least, replace the air bubble preparation by one of Pleurosigma angulatum or some other finely marked diatom. Study the appearance very carefully. § 89. Oblique Light {§ 75). — Swing the mirror far to one side so that the rays "reaching the object may be very oblique to the optic axis of the microscope. Study carefully the appearance of the diatom with the oblique light. Compare the appearance with that where central light is used. The effect of oblique light is not so striking with histological preparations as with diatoms. It should be especially noted in §§ 88, 89, that one cannot determine the exact direction of the rays by the position of the mir- ror. This is especially true for axial light (§88). To be certain the light is axial some such test as that given in § 88 should be applied. (See also Ch. Ill, under Air-bubbles.) CONDENSERS OR ILLUMINATORS* § 90. These are lenses or lens-systems for the purpose of *No one has stated more clearly, or appreciated more truly the value of correct illumination and the methods of obtaining it than Sir David Brewster, 1820, 1831. He says of illumination in general: "The art of illuminating microscopic objects is not of less importance than that of preparing them for observation." "The eye should be protected from all extraneous light, and should not receive any of the light which proceeds from the illuminating center, excepting that portion of it which is transmitted through or reflected from the object." So likewise the value and character of the substage con- CH. II] LIGHTING AND FOCUSING 49 illuminating with transmitted light the object to be studied with the microscope. For the highest kind of investigation their value cannot be over-estimated. They may be used either with natural or artificial light, and should be of sufficient numerical aperture to satisfy objectives of the widest angle. It is of the greatest advantage to have the sub-stage condenser mounted so that it may be easily moved up or down under the stage. The iris diaphragm is so convenient that it should be furnished in all cases, and there should be marks indicating the N. A. (§36) of the condenser utilized with different openings. Finally the condenser should be supplied with central stops for dark-ground illumination (§ io 3) an d with blue and neutral tint glasses to soften the glare when artificial light is used (§ 100, 104). Condensers or Illuminators fall into two great groups, the Achromatic, giving a large aplanatic cone, and Non-achromatic, giving much light, but a relatively small aplanatic cone of light. § 91. Achromatic Condenser. — It is still believed by all ex- pert microscopists that the contention of Brewster was right, and the condenser to give the greatest aid in elucidating microscopic structure must approach in excellence the best objectives. That is, it should be as free as possible from spherical and chromatic aberra- tion, and therefore would transmit to the object a very large aplan- atic cone of light. Such condensers are especially recommended for photo-micrography by all, and those who believe in getting the best possible image in every case are equally strenuous that achro- matic condensers should be used for all work. Unfortunately good condensers like good objectives are expensive, and student micro- scopes as well as many others are usually supplied with the non- achromatic condensers or with none. Many excellent achromatic condensers have been made, but the denser was thoroughly understood and pointed out by him as follows: "I have no hesitation in saying that the apparatus for illumination requires to be as perfect as the apparatus for vision, and on this account I would recommend that the illuminating lens should be perfectly free of chromatic and spherical aberration, and the greatest care be taken to exclude all extraneous light both from the object and from the eye of the observer." See Sir David Brewster's treatise on the Microscope, 1837, pp. 136, 138, 146, and the Edinburgh Journal of Science, new series, No. 11 (1831) p. 83. 5o LIGHTING AND FOCUSING [CM. II most perfect of all seems to be the apochromatic of Powell and Lea- land (Carpenter-Dallinger, p. 302). To attain the best that was possible many workers have adopted the plan of using objectives as condensers. A special substage fitting is provided with the proper screw and the objective is put into position, the front lens being next the object. As will be seen below (§ 94, 95), the full aperture of an objective can rarely be used, and for histological preparations perhaps never, so that an objective of greater equivalent focus, i e., lower power, is used for the condenser than the one on the micro- scope. It is much more convenient, however, to have a special condenser with iris diaphragm or special diaphragms so that one may use any aperture at will, and thus satisfy the conditions neces- sary for lighting different objects for the same objective and for lighting with objectives of different apertures. An excellent con- denser of this form has been produced by Zeiss (Fig. 47). It has a total numerical aperture of 1.00, and an aplanatic aperture of 0.65. Fig. 47. Zeiss' Achromatic Con- denser, c. s. c. s. Centering screws for changing the position of the con- denser and making its axis continuous with that of the microscope. A seg- ment of the condenser is cut away to show the combination of lenses. For very low powers the upper lens is sometimes screwed off. There is an iris diaphragm between the middle and lower combinations. (Zeiss' Catalog. ) § 92. Centering the Condenser.— To get the best possible illumination for bringing out in the clearest manner the minute de- tails of a microscopic object two conditions are necessary, viz.: The principal optic axis of the condenser must be continuous with that of the microscope (see frontispiece) and the object must be in the focus of the condenser, i.e., at the apex of the cone of light given by the condenser. The centering is most conveniently accomplished as follows al- though daylight may be used with almost equal facility. A very small diaphragm is put below the condenser. (If the Zeiss achro- matic condenser is used, the diaphragm of the Abbe illuminator serves for this. If there is no pin-hole diaphragm one can be made CM. II] LIGHTING AND FOCUSING 5i of stiff, black paper. Care must be taken, however, to make the opening exactly central. This is best accomplished by putting the paper disc over the iris or metal diaphragms and then making the hole in the center of the small circle uncovered by the metal diaphragm For the hole a fine needle is best), l^ight well and lower the objec- tive so that it is at about its working-distance from the top of the condenser. If now the condenser is lowered or racked away from the objective the image of the diaphragm will appear. If the open- ing is not central it should be made so by using the centering screws of the condenser. A better plan than to lower the condenser to focus the image of the diaphragm, is to raise the body of the microscope slowly with the coarse adjustment. It is almost impossible to make apparatus so accurate that two parts like the body of the microscope and the substage, each working on different sliding surfaces, shall continue in exactly the same plane. So one will find that if the condenser be accurately centered with the condenser lowered, and then the con- denser be racked up close to the stage and the image of the dia- phragm opening brought again into focus by racking up the body of the microscope, it will not be accurately centered in most cases. For this reason it is advised that the condenser be left in position . close to the stage and the tube of the microscope be used to focus the diaphragm exactly as in ordinary work. Fig. 48. Shows that the optic axis of the condenser does not coin- cide with that of the microscope. (D). Image of the diaphragm of the con- denser shown at one side of the field of view. Fig. 49. Shows the image of the diaphragm (Z>) in the center of the field of the microscope, and thus the coincidence of the axis of the con- denser with that of the microscope. Fig. 49 § 93. Centering the Image of the Source of Illumination. — For the best results it is not only necessary that the condenser be properly centered, but that the object to be studied should be in the image of the source of illumination and that this should also be cen- tered (Figs. 50, .51). After the condenser itself is centered the iris diaphragm is opened to its full extent or the diaphragm carrier 52 LIGHTING AND FOCUSING \_CH. II turned wholly aside. A transparent specimen like the fly's wing is put under the microscope and focused. The condenser is then turned up and down until the image of the flame is apparently on the specimen. If this cannot be Accomplished the relative position of the lamp and condenser is not correct and should be so changed that the image of the edge of the flame is sharply defined. This image must also be centered. This is easily accomplished by manip- ulation of the mirror or, if a lamp is used, by changing the position of the lamp or of the bull's eye (Fig. 60). § 94. Proper Numerical Aperture of the Condenser. — As stated above, the aperture of the condenser should have a range by means of properly selected diaphragms to meet the requirements of all objectives from the lowest to those of the highest aperture. It is found in practice that for diatoms, etc., the best images are obtained when the object is lighted with a cone which fills about three-fourths of the diameter of the back lens of the objective with light, but for histological and other preparations of lower refractive power only one- half or one-third the aperture often gives the most satisfactory images (§ 40). Fig. 50. Shows the image of the flame {Fl.) in the center (C) of the field of the microscope and illuminating the object. Fig. 51. Shows the image of the flame {Fl.) at one side of the center {Exc.) and not properly il- luminating the object. To determine this in any case focus upon some very transparent object, take out the ocular, look down the tube at the back lens. If less than three-fourths of the back lens is lighted, increase the open- ing in the diaphragm — if more than three-fourths diminish it. For some objects it is advantageous to use less than three-fourths of the aperture. Experience will teach the best lighting for special cases. § 95. Aperture of the Illuminating Cone and the Field. — It is to be remarked that with a very small source of light the entire aperture of the objective may be filled if a proper illuminator or condenser is used. The aperture depends on the diaphragm used CH. II] LIGHTING AND FOCUSING 53 with the condenser. And the size of the diaphragm must be directly as the aperture of the objective. That is, it is just the reverse of the rule for diaphragms where no condenser is used (§ 76) ; for there the diaphragm is made large for low powers, and consequently low apertures, while with the condenser the diaphragm is made small for low and large for high powers as the aperture is Ob] Ob] o Fig. 5? Fig. 53 Figs. 52-53. Figures showing the dependence of the objective upon the illuminating cone of the condenser (Nelson) . Fig. 52 (A). The illuminating cone from the condenser (Ilium.). This is seen to be just sufficient to fill the objective (Obj. ). (B.) The back lens of the objective entirely filled with light, shozving that the numerical aperture of the illuminator is equal to that of the objective. Fig. 53 (A). In this figure the illuminating cone from the condenser (Ilium.) is seen to be sufficient lofill the objective (Obj.) . (B.) The back lens of the objective only partly filled with light, due to the restricted aperture of the illuminator, greater in the high powers of a given series of objectives. It is very instructive to demonstrate this by using a 16 mm. objective and opening the diaphragm of the condenser till the back lens is just filled with light. Then if one uses a 3 or 4 mm. objective it will be seen that the back lens of the higher objective is only partly filled with light and to fill it the diaphragm must be much more widely opened. With a condenser, then, the diaphragm has simply to regulate the aperture of the illuminating cone, and has nothing to do with lighting a large or a small field. With the condenser there are two conditions that must be ful- filled, — the proper aperture must be used, and that is determined by the diaphragm, and secondly the whole field must be lighted. The latter is accomplished by using a larger source of light, as the face instead of the edge of a lamp flame, or by lowering or raising S4 LIGHTING AND FOCUSING [ CH. II the condenser so that the object is not in the focus of the condenser, but above or below it, and therefore lighted by a converging or diverging beam where the light is spread over a greater area (Figs. 54-57, § 99)- § 96. Non-Achromatic Condensers. — Of the non-achromatic condensers or illuminators, the Abbe condenser or illuminator is the one most generally used. From its cheapness it is also much more commonly used than the achromatic condenser. It consists of two or three very large lenses and transmits a cone of light of i.^o N.A. to 1.40 N.A., Figs. 58-59, but the aberrations, both spherical and chromatic, are very great in both forms. Indeed, so great are they that in the best form with three lenses and an illuminating cone of 1 . 40 N. A., the aplanatic cone transmitted is only 0.5, and it is the apla- natic cone which is of real use in microscopic illumination where de- tails are to be studied. There is no doubt, however, that the results obtained with a non-achromatic condenser like the Abbe are much more satisfactory than with no condenser. The highest results can- not be attained with it, however. ( Carpenter- Dallinger, p. 309.) § 97. Position of the Condenser. — The proper position of the illuminator for high objectives is one in which the beam of light traversing it is brought to a focus on the object. If parallel rays are reflected from the plane mirror to it, they will be focused only a few millimeters above the upper lens of the condenser ; consequently the illuminator should be about on the level of the top of the stage and therefore almost in contact with the lower surface of the slide. For some purposes when it is desirable to avoid the loss of light by reflection or refraction, a drop of water or homogenous immersion fluid is put between the slide and condenser, forming the so-called immersion illuminator. This is necessary only with objectives of high power and large aperture or for dark-ground illumination. § 98. Centering the Condenser.— The illuminator should be centered to the optic axis of the microscope, that is the optic axis of the condenser and of the microscope should coincide.. Unfortun- ately there is extreme difficulty in determining when the Abbe illuminator is centered. Centering is approximated as follows : Put a pin-hole diaphragm — that is a diaphragm with a small central hole — over the end of the condenser (Fig. 58), the central opening should appear to be in the middle of the field of the microscope. If CH. II] LIGHTING AND FOCUSING 55 it does not the condenser should be moved from side to side by loosening the centering screws until it is in the center of the field. In case no pin-hole diaphragm accompanies the condenser, one may put a very small drop of ink, as from a pen-point, on the center of the upper lens and look at it with a microscope to see if it is in the center of the field. If it is not, the condenser should be adjusted until it is. When the condenser is centered as nearly as possible remove the pin-hole diaphragm or the spot of ink. The microscope and illuminator axes may not be entirely coincident even when the center of the upper lens appears in the center of the field, as there may be some lateral tilting of the condenser, but the above is the best the ordinary worker can do, and unless the mechanical arrange- ments of the illuminator are deficient, it will be very nearly centered. It is to be hoped that the opticians will devise some kind of mounting for this the most commonly used condenser whereby it may be centered as described for the achromatic condenser instead of by the crude methods described above. If the condenser mount- ing regularly possessed centering screws as in the microscope of Watson & Sons and there were a centering diaphragm in the proper position so that its image could be projected into the field of view, the operation would be very simple. If, further, the condensers of Powell and Lealand were selected as models the condensers need not be so bulky, and would still retain all their efficiency. Fortunately the Royal Microscopical Society of London, which has done so much toward standardizing microscopical apparatus, has proposed a standard size for the substage fitting for the condenser of 1.527 in. =38. 786 mm. (see § 53). § 99. Mirror and Light for the Abbe Condenser. — It is best to use light with parallel rays. The rays of daylight are prac- tically parallel; it is best therefore to employ the plane mirror for all but the lowest powers. If low powers are used the whole field might not be illuminated with the plane mirror when the condenser is close to the object ; furthermore, the image of the window frame, objects outside the building, as trees, etc., would appear with un- pleasant distinctness in the field of the microscope. To overcome these defects one can lower the condenser and thus light the object with a diverging cone of light, or use the concave mirror and attain the same end when the condenser is close to the object (Fig. 54). 5 6 LIGHTING AND FOCUSING [ CH. II § ioo. Artificial Light. — If one uses lamp light, it is recom- mended that a large bull's eye be placed in such a position between the light and the mirror that parallel rays fall upon the mirror or in some cases an image of the lamp flame. If one does not have a bull's eye the concave mirror may be used to render the rays less divergent. It may be necessary to lower the condenser somewhat in order to illuminate the object in its focus. ABBE CONDENSER : EXPERIMENTS § ioi. Abbe Condenser, Axial and Oblique Light. — Use a diaphragm a little larger than the front lens of the 3 mm. (}4 in) objective, have the illuminator on the level, or nearly on the level of the upper surface of the stage, and use the plane mirror. Be sure that the diaphragm carrier is in the notch indicating that it is central in position. Use the Pleurosigma as object. Study care- fully the appearance of the diatom with this central light, then make the diaphragm eccentric so as to light with oblique light (§ 89). The differences in appearance will probably be even more striking than with the mirror alone. § 102. Lateral Swaying of the Image. — Frequently in studying an object, especially with a high power, it will appear to sway from side to side in focusing up or down. A glass stage micrometer or fly's wing is an excellent object. Make the light central or axial and focus up and down and notice that the lines simply disappear or grow dim. Now make the light oblique, either by making the diaphragm opening eccentric or if simply a mirror is used, by swinging the mirror sidewise. On focusing up and down, the lines will sway from side to side. What is the direction of apparent movement in focusing down with reference to the illumi- nating ray ? What in focusing up ? If one understands the experi- ment it may sometimes save a great deal of confusion. (See under testing the microscope for swaying with central light § 130.) § 103. Dark-Ground Illumination. — When an object is lighted with rays of a greater obliquity than can get into the front lens of the objective, the field will appear dark (Fig. 57). If now the object is composed of fine particles, or is semi-transparent, it will refract or reflect the light which meets it, in such a way that a ■CII. II] LIGHTING AND FOCUSING 57 part of the very oblique rays will pass into the objective, hence as light reaches the objective only from the object, all the surrounding field will be dark and the object will appear like a self-luminous one on a dark back-ground. This form of illumination is most 54 55 56 57 Figs. 54-57. Sectional views of the Abbe Illuminator of 1 .20 N.A. show- ing various methods of illumination { I 101). Fig. 54, axial light with parallel rays. Fig. 55, oblique light. Fig. 56, axial light with converging beam. Fig. 57, dark-ground illumination with a central stop diaphragm. Axis. The optic axis of the illuminator and of the microscope. The illuminator is centered, that is its optic axis is a prolongation of the optic axis of the microscope. S. Axis. Secondary axis. In oblique light the central ray passes along a secondary axis of the illuminator, and is therefore oblique to the principal axis. D. D. Diaphragms. These are placed in sectional and in face views. The diaphragm is placed between the mirror and the illuminator. In Fig. 55 the opening is eccentric for oblique light, and in Fig. 57 the opening is a nar- row ring, the central part being stopped out, thus giving rise to dark-ground illumination ( \ 103). Obj. Obj. The front of the objective. successful with low powers. It is well to make the illuminator immersion for this experiment, (see § 116). (A) With the Mirror. — Remove all the diaphragms so that 53 LIGHTING AND FOCUSING [CU. II very oblique light may be used, employ a stage micrometer in which the lines have been filled with graphite, use a 16 mm. ( 2 3 in.) objective, and when the light is sufficiently oblique the lines will appear something like streaks of silver on a black back- ground. A specimen like that described below in (B) may also be used. (B) With the Abbe Condenser. — Have the illuminator so that the light is focused on the object (see § 97) and use a diaphragm Fig. 58. Abbe Condenser of r. 20 Fig. 59. Abbe Condenser of 1.40 N.A. in section. N.A. in section. Cuts loaned by Voigtlander &Sohn, A.G. with the annular opening (Fig. 57); employ the same objective as in (A). For object place a drop of 10 % solution of salicylic acid in 95 "0 alcohol on the middle of a slide ; it will crystallize. The crystals will appear brilliantly lighted on a dark back-ground. Put in an ordinary diaphragm and make the light oblique by making the diaphragm eccentric. The same specimen may also be tried with a mirror and oblique light. In order to appreciate the differ- ence between this dark-ground and ordinary transmitted-light illu- mination, use a central diaphragm and observe the crystals. A striking and instructive experiment may be made by adding a very small drop of the solution to the dried preparation, putting it under the microscope quickly, lighting for dark-ground illumination and then watching the crystallization. § 103a. Dark-Ground Illumination for High Powers. — There are two methods for making objects appear as if self lumin- ous in a black field : (1) To light the objects by rays so oblique that none of them will enter the objective unless they are deflected by some object in the field. This method was employed above for low powers. For high powers very wide apertures must be used for the condenser. No rays below 1.00 N. A. can be successfully CH. II} LIGHTING AND FOCUSING 59 utilized. To accomplish this, Siedentopf and Beck employ a para- bolic reflector instead of a condenser of the usual type. Others used condensers specially modified. That of Reichert is conical and silvered on the conical surface ; that of L,eitz makes use of two internal reflections. By all these pieces of apparatus a hollow cone of light of an aperture greater than i.oo N. A. is concentrated upon the field, henee high powers as well as low ones can be used pro- vided a sufficiently brilliant source of light is employed (sunlight, arc lamp, etc.). Ultramicroscopy . — In 1903 Siedentopf and Zsigmondy published a method by which a further evolution of dark-ground illumination was attained according to the general principle just considered. By their method the field is illuminated by a very brilliant cone or wedge of light from the side, i. e. t at right angles to the axis of the microscope. It is evident that none of the rays can enter the micro- scope with even the widest apertured objectives unless the light is de- flected by something in the field. The brilliant light so used renders minute particles luminous something as sunlight entering a small hole in a darkened room renders particles of dust luminous. As this method of lighting rendered particles luminous and therefore visible that were invisible with the microscope as ordinarily used, the use of the microscope with this lighting has come to be called Ultramicroscopy. (2) The second method was used by Toppler, 1867, and has been revived by Gordon, (J. R. M. S. 1906) and others. In this method the object is lighted by a solid cone of light from the con- denser as usual, but the aperture of the condenser must only fill the middle part of the aperture of the objective. In the first method the aperture of the condenser must be great and that of the objective moderate, while in this the reverse is the case, and the objective should have a large aperture and the condenser a moderate aperture. The solid cone of light used for illumination has some of its rays deflected by objects in the field so that they enter the marginal zones of the objective. To secure dark-ground illumination in this manner only these marginal rays are utilized for the image, and the central, solid cone of light entering the objective must be eliminated. This is accomplished by placing a diaphragm or stop on the back lens of the objective of just the right size to cut out the central solid cone and allow the marginal rays to pass on to form the image. 6o LIGHTING AND FOCUSING [ CH. II This gives fairly good results with all powers. The same may also be accomplished, as shown by Gordon, 1906, by using a stop in the eye-point or Ramsden circle (§ 67). For a discussion of dark-ground illumination and ultrami- croscopy see : A. E. Wright, Principles of Microscopy, Ch. XIV ; Siedentopf, Jour. Roy. Micr. Soc. 1903, p. 573, 1907, p. 733 ; Gordon, 1906, p. 167; Beck, 1908, p. 238; Reichert, 1908, p. 374; Leitz, 1905, p. 502 and Catalog No. 42, and special catalog. Top- pier, Poggendorff's Annalen, 1867, p. 33 ; Beck's Cantor Lectures, 1907 ; Zeiss special catalog on Ultramicroscopy and dark-ground illumination, 1907, gives the apparatus needed, the methods and application, also bibliography ; Cotton et Mouton, Les Ultrami- croscopes, Paris, 1906. ARTIFICIAL ILLUMINATION § 104. For evening work and for regions where daylight is not sufficiently brilliant, artificial illumination must be employed. Furthermore, for the the most critical investigation of bodies with fine markings like diatoms, artificial light has been found superior to daylight. A petroleum (kerosene) lamp with flat wick gives a satisfactory light. It is recommended that instead of the ordinary glass chim- ney one made of metal with a slit-opening covered with an oblong cover-glass is more satisfactory, as the source of light is more restricted. Very excellent results may be obtained, however, with the ordinary bed-room lamp furnished with the usual glass chimney. The acetylene light promises to be excellent for microscopic observation and for photo-micrography. (See under photo- micrography.) See also § 103a. Whenever possible the edge of the flame is turned toward the microscope, the advantage of this arrangement is the great bril- liancy, due to the greater thickness of the flame in this direction. § 105. Mutual Arrangement of Lamp, Bull's Eye and Microscope. — To fulfil the conditions given above, namely, that the object be illuminated by the image of the source of illumination the lamp must be in such a position that the condenser projects a sharp image of the flame upon the object (Fig. 60) , and only by trial can this position be determined. In some cases it is found ad- CH. II~\ LIGHTING AND FOCUSING 61 vantageous to discard the mirror and allow the light from the bull's eye to pass directly into the condenser. In most cases no bull's eye need be used. The proper distance of the lamp from the mirror and the proper elevation of the condenser give the required results. The position of lamp and condenser can be determined by trial in each case. § 1 06. Illuminating the Entire Field. — With low objectives and large objects, the entire object might not be illuminated if the Fie 60. 1. Lamp with sHt-opening in metal chimney. 2. Bull's eye on separate stand. 3. Screen showing image of flame. above method were strictly followed ; in this case turn the lamp so that the flame is oblique, or if that is not sufficient, continue to turn the lamp until the full width of the flame is used. If necessary the condenser may be lowered, and the concave mirror used. (See also § 95.) REFRACTION AND COLOR IMAGES \ 107. Refraction Images are those mostly seen in studying microscopic objects. They are the appearances produced by the refraction of the light on entering and on leaving an object. They therefore depend (a) on the form of the object, (b) on the relative refractive powers of object and mounting medium. With such images the diaphragm should not be too large (see \ 94). If the color and refractive index of the object were exactly like the mount- ing medium it could not be seen. In most cases both refractive index and color differ somewhat, there is then a combination of color and refraction 62 LIGHTING AND FOCUSING [ CH. II images which is a great advantage. This combination is generally taken advantage of in histology. 'The air bubble in \ 151 is an example of a purely refractive image. \ 10S. Refraction.— Lying at the basis of microscopical optics is refrac- tion, which is illustrated by the above figures. It means that light passing from one medium to another is bent in its course. Thus in Fig. 61 light pass- ing from air into water does not continue in a straight line but is bent toward the normal N-N', the bending taking place at the point of contact of the air 61. N' 62. N' 63. N' Figs. 61-63. Diagrams illustrating refraction in different media and at plane and curved surfaces. In eaeh case the denser medium is represented by line shading and the perpendicular or normal to the refracting surface is repre- sented by the doited line N-N', the refracted ray by the bent line A C. and water ; that is, the ray of light A B entering the water at B is bent out of its course, extending to C instead of C. Conversely, if the ray of light is passing from water into air, on reaching the air it is bent from the normal, the ray C B passing to A and not in a straight line to C". By comparing Figs. 62-63 in which the denser medium is crown glass instead of water, the bending of the rays is seen to be greater as crown glass is denser than 'water. It has been found by physicists that there is a constant relation between the angle taken by the ray in the rarer medium and that taken by the ray in the denser medium. The relationship is expressed thus : Sine of the angle of incidence divided by tbe sine of the angle of refraction equals the index of re- fraction. In the figures, ^ — t^wit?, = index of refraction. Worked out com- oin Lrffv' pletely in Fig. 61, ABN=4o°, C£N'=2&° 54' and Sln 4 °° = °- 6 4^7 5 * ° 4 Sin 28 54' 0.48327 1.33, i. e., the index of refraction from air to water is 1.33. (See § 39.) In Figs. 62-63, illustrating refraction in crown glass, the angles being given, the problem is easily solved as just illustrated. (For table of natural sines see .third page of cover; for interpolation, J 38. ) \ 109. Absolute Index of Refraction. — This is the index of refraction ob- CH. //] LIGHTING AND FOCUSING 63 tained when the incident ray passes from a vacuum into a given medium. As the index of the vacuum is taken as unity, the absolute index of any substance is always greater than unity. For many purposes, as for the object of this book, air is treated as if it were a vacuum, and its index is called unity, but in reality the index of refraction of air is about 3 ten-thousandths greater than unity. Whenever the refractive index of a substance is given, the absolute index is meant unless otherwise stated. For example, when the index of refraction of water is said to be 1.33, and of crown glass 1.52, etc., these figures represent the absolute index, and the incident ray is supposed to be in a vacuum. \ no. Relative Index of Refraction. — This is the index of refraction be- tween two contiguous media, as for example between glass and diamond, water and glass, etc. It is obtained by dividing the absolute index of refrac- tion of the substance containing the refracted ray, by the absolute index of the substance transmitting the incident ray. For example, the relative index from water to glass is 1.52 divided by 1.33. If the light passed from glass to water it would be, 1.33 divided by 1.52. By a study of the figures showing refraction, it will be seen that the greater the refraction the less the angle and consequently the less the sine of the angle, and as the refraction between two media is the ratio of the sines of the angles of incidence and refraction ( — I , it will be seen that whenever \sm rj the sine of the angle of refraction is increased by being in a less refractive medium, the index of refraction will show a corresponding decrease and vice versa. That is the ratio of the sines of the angles of incidence and refraction of any two contiguous substances is inversely as the refractive indices of those sub- stances. The formula is : (Sine of angle of incident ray \ / Inedx of refraction of refracting medium \ Sine of angle of refracted ray / \ Index of refraction of incident medium / Abbreviated ( — I = I : — ; — ) . By means of this general formula one \sinr/ \ index z / J ° can solve any problem in refraction whenever three factors of the problem are known. The universality of the law may be illustrated by the following cases : (A) Light incident in a vacuum or in air, and entering some denser medium, as water, glass, diamond, etc. V Sine of angle made by the ray in air \ _ / Index of ref.of denser med \ \ Sine of angle made by ray in denser med. / — \ Index of ref . of air ( 1 ) / If the dense substance were glass ( ^-^ ) = ( ^T^ ) ' If the two media were weter and glass, the incident light being in water the formula would be ; / sin t \ _ / 1.52 \ _ jf tlle i nc ident ray were glass and the refracted ray \sin rj \ 1.33 / in water- / sin * \_/JL33_ \ And similarly for any two media ; and as \sin rj \ 1.52 / stated above if any three of the factors are given the fourth may be readily found. 64 LIGHTING AND FOCUSING [ CH. II \ in. Critical Angle and Total Reflection.— In order to understand the Wollaston 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 refractive 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 critical angle, it will be totally reflected at the surface of the denser medium, that surface acting as a perfect mirror. By consulting the figures it will be seen that there is no such thing as a critical angle and total reflection in the rarer of two contiguous media. To find the critical angle in the denser of two contiguous media :— Make the angle of refraction {i. e., the angle in the rarer of the two „ , .- /sin i\ / index r \ T . ,, media) 90 and solve the general equation : ( — I = I ^—5 r \ . I,et the two substances be water and air, then the sine of r ( 90 ) is 1 , and the index of air is 1, that of water 1.33, whence ( ) = ( ) or sin 2=7514-. This is. the sine of 48°-)-, and whenever the ray in the water is at an angle of more than 48° 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 i \_/ index water (1.33) \/ sin z\ / 1.33 \ \ sin r (sin 90° =-i) / "V index glass (1.52) / \ 1 / \ 1.52 / \ whence sin j'=.875 sine of critical angle in glass covered with water. The corresponding angle is approximately 61 °. \ ii2. Color Images. These are images of objects which are strongly colored and lighted with so wide an aperture that the refraction images are drowned in the light. Such images are obtained by removing the diaphragm or by using a larger opening. This method of illumination is especially applicable to the study of deeply stained bacteria. (See below \ 119.) ADJUSTABLE, WATER AND HOMOGENEOUS OBJECTIVES : EXPERIMENTS \ 113. Adjustment for Objectives. As stated above (| 27), the aberration produced by the cover-glass (Fig. 64) , is compensated for by giving the com- binations 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 which the objective is capable by selecting covers of the thick- ness for which the objective was corrected. (See table \ 33.) Adjustment may be made also by increasing the tube-length for covers thinner than the CH. II] LIGHTING AND FOCUSING 65 .3 \ 2 1 Y^ \\ •o» 7 ' N \ • ,' as clearly defined colored ob- jects on a bright field. Fig. 66. Screen for shading the microscope and the face of the observer. This is very readily con- structed as shown in the figure by supporting a wire in a disc of lead, iron, or heavy wood. The screen is then completed by hanging over the bent wire, black ■cloth or paper 30 x 40 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 eyes of the observer. § 120. Shading the Object.— To get the clearest image of an object no light should reach the eye except from the object. A handkerchief or a dark cloth wound around the objective will serve the purpose. Often the proper effect may be obtained by simply shading the top of the stage with the hand or with a piece of bristol board. Unless one has a very favorable light the shading of the object is of the greatest advantage, especially with homogeneous immersion objectives. The screen (Fig. 66) is the most satisfactory means for this purpose, as the entire microscope above the illuminat- ing apparatus is shaded. § 121. 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 (§ 125), then if the fluid is cedar oil, wet one corner of a fresh piece in xylene or chloroform and wipe the front lens with it. Im- mediately afterward wipe with a dry part of the paper. The cover- glass of the preparation can be cleaned in the same way. If the homogeneous liquid is a glycerin mixture proceed as above, but use water to remove the last traces of glycerin. 7 o LIGHTING AND FOCUSING [ CH. II CARE OF THE MICROSCOPE § 122. The microscope should be handled carefully and kept perfectly clean. The oculars and objectives should never be allowed to fall. When not in use keep it in a place as free as possible from dust. All parts of the microscope should be kept free from liquids, especially from acids, alkalies, alcohol, xylene, turpentine and chloroform. § 123. Care of the Mechanical Parts. — To clean the mechan- ical parts put a small quantity of some fine oil (olive oil or liquid vaselin and gasoline or xylene, 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 lubrjcator is rarely needed. When opposed brass surfaces "cut," i. e., when from the introduction of some gritty material, minute grooves are worn in the opposing surfaces, giving a harsh movement, the opposing parts should be separated, carefully cleaned as described above and any ridges or prominences scraped down with a knife. Where the ten- dency 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, xylene 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. § 124. Care of the Optical Parts. — These must be kept scrupulously 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, consequent- ly it is necessary that only soft, clean cloth or paper be used in 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. § 125. Lens Paper. — The so-called Japanese filter paper, which from its use with the microscope, I have designated lens paper, CH. II] LIGHTING AND FOCUSING 71 has been used in the author's laboratory since 1885 for cleaning the lenses of oculars and objectives, and especially for removing the fluid used with immersion objectives. Whenever a piece is used once it is thrown away. It has proved more satisfactory than cloth or chamois, because dust or sand is not present; and from its bib- ulous character it is very efficient in removing liquid or semi-liquid substances. § 126. Removal of Dust. — Dust may be removed with a camel's hair brush, or by wiping with the lens paper. Cloudiness may be removed from the glass surfaces by breathing on them, then wiping quickly with a soft cloth or the lens paper. Cloudiness on the inner surfaces of the ocular lenses may be removed by unscrewing them and wiping as directed above. A high objective should never be taken apart by an inexperienced person . If the cloudiness cannot be removed as directed above, moisten one 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 albuminous 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 moist- ened, (water 100 cc. , ammonia 1 cc). Canada Balsam, damar, paraffin, or any oily substance may be removed with a cloth or paper wet with chloroform, gasoline 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 Cememt may be removed by the paper or a cloth moist- ened 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. 72 LIGHTING AND FOCUSING [ CH. II It frequently happens that the upper surface of the back com- bination 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 surfaces carefully wiped. CARB OF THE EYES § 127. Keep both eyes open, using the eye-screen if necessary {Fig. 67); and divide the labor between the two eyes, i. e. use one eye for observing the image awhile and then the other. In the beginning it is not advisable to look into the microscope con- tinuously for more than half an hour at a time. One never should ■work with the microscope after the eyes feel fatigued. After one Fig. 67. Adjusting Eye-Shade. This is prepared by covering a card about 6 x 12 centimeters with black velveteen. A copper wire about 5 mm. (Y% 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. 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 accomodation is required of the eyes, the eyes remaining nearly in a condition of rest as for distant objects. The CH. II] LIGHTING AND FOCUSING 73 fatigue incident upon 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 micro- scope used instead of the muscles of accommodation. With a micro- scope of the best quality, and suitable light — that is light which is steady and not so bright as to dazzle the eyes nor so dim as to strain them in determining details— microscopic work should improve rather than injure the sight. Fig. 68. Laboratory with adjustable stool. Table § 128. Position and Char- acter of the Work Table. — The work-table should be very firm and large (60 x 120 cm.; 24 x 48 in.), so that the necessary apparatus and ma- terial 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 corres- ponding to the necessities 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 here and in Fig. 66 is very useful (see also under lighting, § 71). LABORATORY TABLE. TESTING THE MICROSCOPE § 129. 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 judgement. Those most ready to offer ad- vice should as a rule be avoided, for in most cases they simply "have an ax to grind," and are sure to commend only those instruments that conform to the " fad" of the day. From the writer's experience it seems safe to say that the 74 LIGHTING AND FOCUSING [ CH. II inexperienced can do no better than to state clearly what he wishes to do with a microscope and then trust to the judgement of one of the optical companies. The makers of microscopes and objectives guard with jealous care the excel- lence 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 stand- ard. 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. \ 130. 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 micoscope 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 micro- scope entirely. It has been much improved recently. It should work smoothly and be so balanced that one cannot tell by the feeling when using it whether the screw is going up or down. Then there should be absolutely no motion except in the direction of the optic axis, other- wise the image will appear to sway even with central light. Compare the ap- pearance 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 {\ 88). § 131. 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 advant- age to the student if he could have the help of such a person. In no case is a microscope 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). LABORATORY AND HIGH-SCHOOL COMPOUND MICROSCOPES \ 132. Optical Parts. — A great deal of beginning work with the micro scope in biological laboratories is done with simple and inexpensive apparatus. CH. II] LIGHTING AND FOCUSING 75 Indeed if one contemplates the large classes in the high schools, the universi- ties and medical schools, it can be readily understood that microscopes costing from $25 to 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 ad- vanced microscopical work in general, a microscope should have something like the following character: Its optical outfit should comprise, (a) dry objec- tives of 50 mm. (2 in.), 16-18 mm. (% in.) and 3 mm. (>£in ) equivalent focus. There should be present also a 2 mm. ( r \ in. ) or r.5 mm. (j l s in. ) homogeneous immersion objective. Of oculars there should be several of different power. A centering substage condenser, and an Abbe camera lucida are also neces- sities, 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. Certainly at the present, the apochromatics stand at the head, although the best achromatic objectives approach them very closely. I 133. Mechanical Parts or Stand. — The stand should be low enough so that it can be used in a vertical position on an ordinary table without inconven- ience; it should have a jointed (flexible) pillar for inclination at any angle to the horizontal. The adjustments for focusing should be two, — a coarse ad- justment or rapid movement with rack and pinion, and a fine adjustment by means of a micrometer screw. Both adjustments should move the entire tube of the microscope. The body or tube should be short enough for objectives corrected for the short or 160 millimeter tube-length. 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 examina- tion 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 en- tirely with diaphragms. The condenser mounting should allow up and down motion. \ 134. Quality and Cost. — In order that teachers and students may get a good general idea of the appearance 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 importers. The microscopes are ar- ranged in alphabetical order of the makers. Laboratory microscopes which will answer nearly all the requirements for work in Biology, including Histology, Embryology, Pathology and Bacteri- ology, are listed in the makers catalogs at about $75.00. The less expensive mi- croscopes shown are listed at $25 to $45. 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 76 LIGHTING AND FOCUSING [ CH. II for the best laboratory microscopes. The mechanical work also is of excel- lent quality. During the last few 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) . STANDARD SIZES RECOMMENED BY THE ROYAL MICROSCOPICAL SOCIETY § 135. Society Screw. — Owing to the lack of uniformity in screws for microscope objectives, the Royal Microscopical Society of London, in 1857, made an earnest effort to introduce a standard size. 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 under- took 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. (See Edward Bausch, Trans. Amer. Micr. Soc, 1884, p. 153.) In 1884 the American Microscopical Society appointed Mr. Edward Bausch and Prof. William A. Rogers upon a committee to correspond with the Roya) 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 considered 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 facili- ties 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 Standard, or " Society Screw," see: Trans. Roy. Micr. Soc, 1857, pp. 39-41; 1859, pp. 92-97; i860, pp. 103-104. (All to be found in Quar. Jour. Micr. Sci., o. s., vols. VI, VII, VIII). Proc. Amer. Micr. Soc. 1884, p. 274; 1886, p. 199; 1893, p. 38. Journal of the Royal Microscopical Society, August, 1896. CH. II} LIGHTING AND FOCUSING 77 In this last paper of four pages the matter 19 very carefully gone over and full specifications of the new screw given. It conforms almost exactly with the original standard adopted by the society, but means have been devised by which it may be kept standard. This paper is of so much importance historically and practically that it de- serves to appear in every work on the modern microscope. It is therefore here repeated entire : FROM THE JOURNAL OF THE ROYAL MICROSCOPICAL SCCIETY AUGUST, 1896 " The Royal Microscopical Society's Standard Screw-Thread for Nose- piece and Object-Glasses of Microscopes." " Being the report of a sub-committee of the Council, drawn up by Conrad Beck, F.R.M.S., Secretary to the Sub-Committee. Read June 17th, 1896." "The so-called Standard Screw-Thread of the Royal Microscopical Society has been but an imperfect standard, and has not ensured that interchangea- bility which it originally promised. It has been our duty to investigate the causes of this state of affairs, and to formulate a plan by which such an incon- venience should be remedied in the future." " Without going too closely into the entire history of the subject, we pro- pose to briefly explain the reasons why the original standard was not efficient for practical purposes, and then to state the plan which the Council of the Royal Microscopical Society has now adopted for the future." The specification of the original standard screw was as follows : § 136. Form of Thread. — " Whitworth thread, i. e., a V-shaped thread, sides of thread inclined at an angle of 55° to each other, one-sixth of the V depth of the thread being rounded off at the top of the thread, and one-sixth of the thread being rounded off at the bottom of the thread." " Pitch of Screw, 36 to the inch. Length of Thread on Object-Glass, 0.125 in. Plain Fitting above Thread of Object-Glass 0.15 in. long, to be about the size of the bottom of male thread. Length of Thread of Nose-Piece not less than 0.125 in. Diameter of the Object-Glass Screw at the bottom of the screw, 0.7626 in. Diameter of the Nose-Piece Screw at the bottom of the thread, 0.8 in." "When the exact form of the Whitworth screw-thread is calculated it will be found that this allows a difference between the male and female screw of 0.0018 in., which is in itself quite sufficient margin of looseness to make an easy fit." " The society had two plug and ring gauges, one 0.8 in., and the other o 7626 in., made by Whitworth as standards for the use of the Society, and it has been shown that if an adjustable tap and die (as recommended by the late Mr. Richard Beck in a paper printed in the "Transactions of the Microscopi- cal Society," 1859, p. 92) be made which could be accurately adjusted to these standard sizes so that the tap exactly fitted the 0.8 in. ring size, and the die 7 8 LIGHTING AND FOCUSING \_CH. II exactly fitted the 0.7626 in. plug, the exact standard screw as originally sug- gested could be adhered to. These adjustable taps and dies were not used for cutting the thread, but for passing over each thread after it had been cut to approximately the right size. That this method will work satisfactorily, is evidenced by the fact that in the late Mr. Richard Beck's firm the method has been in successful operation ever since." "The use, however, of such a system involved the necessity of every maker being provided with adjustable tap and die, and also the two pairs of plug and ring Whitworth sizes, together with a means of accurately sharpening the adjustable tap and die. And it was found in practice that microscope makers were not universally prepared to go to such an outlay for a .matter which at that time did not appear to be of such importance as has since proved to be the case." "Therefore the Society issued solid taps, and finding that, as is well known to be the case, a solid tap could not be made to an exactly accurate size "owing to the alteration of the steel during the process of hardening and tempering, they had them made somewhat larger than the standard 0.8 in. gauge. An additional reason for their being larger was to allow for the slight wearing of the tap after prolonged use." " Here, however, there was no record of the amount larger which the taps were made, and although the first set appear to have been carefully manufac- tured, those which were from time to time obtained were less and less like the original, and in this manner a discrepancy arose which the arrangements now adopted by the Council are intended to correct for the future." " Beyond the fact that the Council specify that the diameter of the plain fitting of the object-glass should be as near as possible to, but not exceeding 0.759 * n -> an d that the length of this fitting has been reduced to 0.1 in., the original specification of the standard screw is only altered as to the exact diameters of the screw itself." " The original specification of these diameters allowed only 0.0018 for clearance between the male and female screw." " If absolutely exact sizing taps and dies could be made which should not wear, the original diameters might have been adhered to, but as has been pre- viously pointed out, adjustable dies in connection with gauges, etc., are requisite for this." " The Council has been able to obtain taps and dies which are guaranteed not to vary more than 1/1000 of an inch larger or smaller than the nominal size. And they are therefore having manufactured a series of taps of the nominal diameter on the top of the screw-thread of 0.8015 i n - which will not vary more than from 0.8005 i n - to 0.8025 in. To insure this the Council has ordered a Whitworth plug and ring, size 0.803 i n - "> diameter, and no tap will be allowed to be stamped with the Society's stamp unless it will pass easily through this 0.803 ' n ' ring, and unless it is of such a size that it will not enter the 0.8 in. standard gauge already in the Society's possession." "They are also having made a series of dies of the nominal inside diameter on the top of the thread of 0.7611 in., which will not vary more than from 0.7601 to 0.7621. To test this the Council has ordered a Whitworth plug and CH. II] LIGHTING AND FOCUSING 79 ring, size 0.7596 in. diameter, and no die will be allowed to be stamped with the Society's stamp unless it will pass easily over the 0.7596 in. plug and will not pass over the 0.7626 in. plug." " These taps and dies will be for sale almost immediately, at cost price, il. 15^. for each pair of tap and dies, and it is earnestly requested that every maker of Microscopes will possess himself of a pair of these sizing gauges." " The Council believe that at such time as these sizing taps and dies have come into universal use the standard screw-thread will have been put upon a permanent basis, and complete interchangeability of all object-glasses will have been established." h 137. Screw. New Specification of the Royal Microscopical Society Standard Fig. 69 "Thread. — Whitworth screw, i. e.,a. V-shaped thread, sides of thread inclined at an angle of 55 to each other, one-sixth of the V depth being rounded off at the top and the bottom of the thread. Pitch. — 36 to the inch. Length of Thread on Object-Glass o. 125 in. 80 LIGHTING AND FOCUSING [ CH. II Plain Fitting above Thread of Object-Glass o. I in. long, not to exceed 0.759 in. in diameter. Diameter (C) of Thread on Object-Glass at top of thread not to exceed o 7982 in., or to be less than 0.7952 in. Diameter (D) of Thread on Object-Glass at bottom of thread not to exceed 0.7626 in., or to be less than 0.7596 in. Length of Screw of Nose-Piece to be not less than 0.125 i fl - Diameter of Screw of Nose-Piece (A) at top of thread not to exceed 0.7674 in., or be less than 0.7644 in. Diameter of Screw of Nose-Piece (B) at- bottom of thread not to exceed 0.803 in., or be less than 0.8 in." \ 138. Standard Size Oculars and Substage Condensers. — For a considera- tion of these, with measurements, see \ 53, 98. 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. £ 139. Marker for Preparations. (Figs. 70-72). — This instrument con. sists of an objective-like attachment which may be screwed into the nose-piece of the microscope. It bears on its lower end 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 position. 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.) \ 140. 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. 26, 36, D). For the purposes 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 a hair from a camel's hair or other fine brush to the upper surface of the CH. II '] LIGHTING AND FOCUSING Si diaphragm (Fig. 36D) so that it will project about half way across the opening. If one uses this ocular, the pointer will appear in the field and one can place the specimen so that the pointer indicates it exactly, as in using a pointer on a diagram or on the black-board. It is not known to the author who devised this method. It is certainly of the greatest advantage in demonstrating objects like aruoebas 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. SS 70 71 72 Figs. 70-72. Sectional Views of the two Forms of the Marker. Fig. 70. The simplest form of marker. It consists of the part SS witk the milled edge (M) . This part bears the society or objective screw for attach_ ing the marker to the microscope. R. Rotating part of the marker. This bears the eccentric brush (B) at its lower end. The brush is on the wire ( W)_, This wire is eccenttic, and may be made more or less so by bending the wire. The central dotted line coincides with the axis of the microscope. The revolv- ing part is connected with the " Society Screw " by the small screw (S). Fig. 71. 55, R, and B. All parts same as with Fig. 70, except that the brush is carried by a sliding cylinder the end view being indicated in Fig. 72. J! 141. Mechanical Stage. — For High School and ordinary laboratory work a mechanical stage is not needed; but for much work, especially where high objectives are used a mechanical stage is of great advantage. It is also advantageous if the mechanical stage can be easily removed. 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 micro- scopes, is excellent for high powers and preparations of moderate dimensions, but for the study of serial sectons and large sections or preparations in general^ mechanical stages like those shown in Figs. 79-89 are more useful. This form of mechanical stage has the advantage of giving great lateral and forward and 82 LIGHTING AND FOCUSING [CU. II backward motion. It is a modification 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., Qeeen & Co., and the Spencer Lens Co., in America. — Jour. Roy. Micr. Soc, 1885, p. 122. See also Zeit. Wiss. Mikro- skopie (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 excel- lent feature that with them one can explore an entire slide full of- serial sec- tions, as the sections are ordinarily mounted, i.e. , under a cover-glass 24X50 mm. 73 74 75 Figs. 73-75. Specimens Showing the Use of the Marker. In Fig. 7_j a section of a series is marked to indicate that this section shows something especially well. In Fig. 7./ some blood corpuscles showing ingested carbon very satisfactorily are surrounded by a minute ring, and in Fig. 75 the lines of a micrometer are ringed to facilitate finding the lines. CM. II] LABORA TORY MICROSCOPES 83 Fig. 76. The Bausch d~ Lomb Optical Co' s Detachable Mechanical Stage. Fig. 77. The Detachable Mechani- cal Stage of Leitz, Fig. 78. 77/i? Spencer Lens Co' s Detachable Mechanical Slan-e of Great Range. 8 4 LABOR A TOR Y MICROSCOPES [ CH. If Fig. 79. The Bauuh & Lamb Optical CaLs. bUm Mudd. nr>H Mivo*rnbr. CH. II ] LA MORATORY MICROSCOPES 35 Fig. So. The Bausch & Lomb Optical Co's Microscope BH. Handle Type 86 LAB( >RA TORY MICFH >SC< )PES [ CH. // Fig. Si. The Bausch & Lomb Optical Co's microscope /• without Handle. CH. W] LABi IRA TORY . I//CR0SO >PES 87 Fig. 82. The Bausch & Lomb Optical Co's Model A H Microscope, Handle Type. The coarse adjustment is by a sliding lithe, and the pillar is not jointed. See also Fig. 140 on p. ijS. 88 LA P,0 R A TORY MICROSCOPES [ CI I. II Fig. 83. Heck's London Microscope, Regents Model, with Handle and New Fine Adjustment, See a/so Fig; /J5. CH. IF] LABOR A TORY MICROSCOPES 89 Fig. 84. Leitz Universal Microscope, Stand A with Large Tube and Special Fine Adjustment. See also Figs. 142, 150. go L .//.'( )RA '/'< )RY i MICROSCOPES [ CI I. II Figs. 85, 86. Quei ^..een & Co's Continental Mia proof, triple nose-piece. I ■oscope, i\'o. II. Dust- n this and the he difference between this and the ordinary form can he seen by comparing with Fig. 87. This form of revolving nose-piece has been made for many years by Winkel of Goeltmgen. See legend of Fig. 40. CH. //] LABORATORY MICROSCOPES 9 1 Fig. 88. Re i chert's Laboratory Microscope with Handle. This handle is so attached that it docs not preclude the ordinary means for fine adjustment. 9 2 LABOR A TOR ) ' MICROSCOPES [ CH. II Fig. 89. The Spencer Ton l o's New Model No. 10 Microscope especially for Photo-Micrography. CH. //] LAB OR A TOR J ' MICROSCOPES 93 Fig. 90. The Spencer Lens Co's Microscope No. 40 ivith curved Spring inside the Arm and Pillar so that they may he safely used as a Handle. 94 LABOR A TORY MICROSCOPES L CH. II '' . (5- Fig. 91. The Spencer Lens Co's Microscope No. 36 with an extra large Stage. CH. II] LABOR A TOR ) ' MICROSCOPES 95 Fig. 92. The Spencer Lens Co's Microscope No. jo with double Nose-Piece and no Condenser. 9 6 LABORATORY MICROSCOPES [ CH. 11 Fig. 93. Voigtlander & Sohn's Laboratory Microscope No. IV. For their large Stum/, see Fig; /, 104 INTERPRETATION OF APPEARANCES [CI/. Ill Search the preparation until an air bubble is found appearing about i 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, cir- cular 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 (§ 74). Focus both up and down, noting that, in focusing up, the cen- tral 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. § 151. Air Bubbles with Oblique Illumination. — Remove the sub-stage of the microscope and all the diaphragms. Swing the mirror so that the rays may be sent very obliquely upon the object (Fig. 28, C). The bright spot will appear no longer in the center but on the side away from the mirror (Fig. 98, A). § 152. Oil Globules. — Prepare these by beating a small drop of clove oil with mucilage on a slide and covering as directed for air bubbles (§ 150), or use a drop of milk. § 153. Oil Globules with Central Illumination. — Use the same diaphragm and light as above (§ 150). Find an oil globule appearing about 1 mm. in diameter. If the light is central a bright spot will appear in the center as with air. Focus up and down as with air, and note that the bright center of the oil globules is clear- est last in focusing up. A w Fig. 9S. Very small Globules of Oil (O) and an Air Bnb- » > bles (A) seen by Oblique Light Surface view. The arrow d$\/ K^fek. indicates the direction of the light rays. O § 154. Oil Globules with Oblique Illumination. — Remove the sub-stage, etc., as above, and swing the mirror to one side and CH. Ill] INTERPRETATION OF APPEARANCES 105 light with oblique light. The bright spot will be eccentric, and will appear to be on the same side as the mirror (Fig. 98,0). § 155. Oil and Air Together. — Make a preparation exactly as described for air bubbles (§ 149), and add at one edge a little of the mixture of oil and mucilage (§ 152); 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 ap- pearing 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 toward it. See Fig. 91.* § 156. Air and Oil by Reflected Light. — Cover the dia- phragm or mirror so that no transmitted light (§ 73) can reach the preparation, using the same preparation as in § 155. The oil and air will appear like globules of silver on a dark ground. The part that was darkest in each with transmitted light will be lighted, and the bright central spot will be somewhat dark.f § 157. Distinctness 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 refrac- tive than the mucilage and the air less. (Figs. 61-63.) Place a fragment of a cover- glass on a clean slide, and cover it * It should be remefnbered that the image in the compound microscope is inverted (Fig. 26) , hence the bright spot really moves toward the mirror for air, and away from it for oil. t 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 ob- servation. If a 16 mm. ( % in. } is used instead of a 3 mm. ( yb in. ) objective, the appearances will vary considerably from that given above for the higher power. It is well to use a low as well as a high power. Marked differences will also be seen in the appearances with objectives of small and of large aperture. ^ 1 06 INTERPRETATION OF APPEARANCES [CH. Ill (see under mounting). The outline will be distinct with the un- aided eye. Use it as object and employ the 16 mm. iffi in.) objec- tive 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 and immerse the fragment. The outline will remain distinct, but the dark band will be somewhat narrower. Remove the cover-glass, wipe it dry, and wipe the frag- ment 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 narrower than before. Fig. 99. Section of an air bub- ble and an oil globule in water (H,0). The air bubble although spherical in form gives only a virtual focus, indi- cated by the dotted lines below the bub- ble. As it is surrounded by a denser ■medium it acts like a concave lens in air {Fig. 10). The focus of the oil globule is real as it is denser than the surrounding medium. Axis, — the principal axis. F, principal focus . It is virtual and below for the air bubble ; real and above for the oil globule. U 2 0. Water or a mixture of water and gum arabic serving as a mounting medium ($ 140). Draw a solid glass rod out to a fine thread. Mount one piece in air, and the other in 50% glycerin. Put a cover-glass on each. Employ the same optical arrangement as before. Examine the one in air first. There will be seen a narrow, bright band, with a wide, dark band on each side (Fig. 100, a;. Fig. 100. Solid glass rod showing the appearance when viewed with transmitted , central light, and with an objective of medium ape7'ture. a. Mounted in air. b. Mounted in 50 per cent glycerin. The one in glycerin will show a much wider bright central band, with the dark borders correspondingly narrow (Fig. 100, 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 CH. Ill] INTERPRETATION OF APPEARANCES 107 band simply represents an area in which the rays are so greatly bent or refracted (Figs. 61-63) that they cannot enter the objective and contribute to the formation of the image ; the edges are dark sim- ply because no light from them reaches the observer. 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, as it is also at the colored zone, but at other levels it can hardly be seen in the liquid. § 158. Highly Refractive. — This expression is often used in describing microscopic objects, (mednllated nerve fibers, for ex- ample), and means that object will appear to be bordered by a wide, dark margin when it is viewed by transmitted light. And from the above (§ 157), it would be known that the refractive power of the object, and the medium in which it was mounted must differ con- siderably. Fig. 10 r. Solid glass rod coated with collodion to show a double con- tour. Toward one end the collodion had gathered in a fusiform drop. § 159. Doubly Contoured.— This means that the object is bounded by two, usually parallel dark lines with a lighter band be- tween them. In other words, the object is bordered by (1) a dark line, (2) a light band, and (3) a second dark line (Fig. 101). This may be demonstrated by coating a fine glass rod (§ 157) 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 * 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. 108 INTERPRETA TWN OF APPEARANCES [ CH. Ill 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. 101). § 160. Optional 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 stud)'ing mammalian red blood-corpuscles on edge. The experiments with the solid glass rods (Fig. ioo)furnish excellent and striking examples of optical sections. § 161. Currents in Liquids. — Employ the 16 mm. (/lin.) objective, 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. § 162. Velocity Under the Microscope. — In studying cur- rents 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. 42 it will be seen that the actual size of the field of the micro- scope 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 * The collodion used is a 6% solution of gun cotton in equal parts of sul- phuric ether and 95% alcohol. It is well to dip the rod two or three times in the collodion 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 membran- ous covering (Fig. 101). CH. 1111 INTERPRETATION OF APPEARANCES 109 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 studying the circulation of the blood. The truth of what has just been said can be easily demon- strated in studying the circulation in the gills of Necturus, or in the frog's foot, by using first a low power in which the field is actually of considerable diameter (Fig. 42, Table, § 58) 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. § 163. Pedesis or Brownian Movement. — Employ the same object as above, but a 3 mm. (}i 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 indfinite, dancing or oscillating motion. This indefinite but continuous motion of small particles in a liquid is called Pe-de' sis or Brownian move?nent. Also, but im- properly, molecular 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. Car- mine 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 divided and in a suitable liquid. In the minds of most, no adequate explanation has yet been offered. Compare the pedetic motion with that of a current by slightly inclining the tube of the microscope. The small particles will con- tinue their independent leaping movements while they are carried along by the current. The pedetic motion makes it difficult to ob- tain good photographs of milk gobules 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). no INTERPRETATION OF APPEARANCES [Of. Ill § 164. Demonstration of Pedesis with the Polarizing Microscope. — (Ch. VI.) The following demonstration shows con- clusively that the pedetic riiotion is real and not illusive. (Ranvier, P- I73-) 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 power then a high power (3 mm. or }& in.). In the field will be seen multitudes of crystals of carbonate of lime; the larger crys- tals 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 (§ 120) and also by shading the eye. Focus sharply and observe the pedetic motion of the small particles, then cross the polarizer and analyzer, that is, turn one or the other until the field is dark. Part of the large motionless crystals will shine continuously and a part will re- main 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. § 165. Muscae Volitantes. — These specks or filaments in the eyes due to minute shreds or opacities of the vitreous sometimes ap- pear as part of the object as they are projected into the field ol vision. They may be seen by looking into the well lighted micro- scope when there is no object under the microscope. They may also be seen by looking at brightly illuminated snow or other white sur- face. By studying them carefully it will be seen that they are some- what movable and float across the field of vision, and thus do not remain in one position as do the objects under observation. Further- CH. III~\ INTERPRETATION OF APPEARANCES in more, 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. § 1 66. Miscellaneous Observations. — 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. Por- tions 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 liable to be present in histological preparations, and unless their character is understood there is chance for much confusion and erroneous interpretation. The scales of butterflies' and moths, es- pecially the common clothes moths. 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. If it is necessary to see all sides of an ordinary gross object, and to observe it with varying illumination and under various con- ditions of temperature, moisture, etc., in order to obtain a fairly ac- curate and satisfactory knowledge of it, so much the more is it necessary not to be satisfied it 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. § 167. Summary for Proper Interpretation. — To summar- ize 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. ii2 INTERPRETATION OF APPEARANCES \_CH. Ill 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. § 168. Zeiss-Greenough Binocular, Erecting Micro- scope. — As shown in figure 102 this consists of a microscope stage with two tubes mounted side by side and moving on the same rack and pinion. Either tube can be used without the other. The ocu- lars are capable of greater or less separation to suit the eyes of different observers. In the large cylinder near the top is placed a Porro prism which erects the image. This microscope gives most perfect stereoscopic images and also erect ones, and therefore is es- pecially adapted for dissection and for studying objects of consider- able thickness, like injected preparations etc. It is interesting to note that the binocular microscope constructed by Cherubin D'Orleans, 1677, was composed in like manner of two microscopes side by side. It of course had no erecting prisms (For statement and figure of this early binocular, see Mayall, p. 17, 18). § 169. Wenham's Binocular Microscope. — This is illus- trated in Figs. 103-104. There is but a single objective. The light from this is divided by a prism, a part of it passing to the right and a part to the left eye. That to the right eye passes directly, that to the left is twice internally reflected by the prism to give it the right inclination. In order to get the stereoscopic effect with the binocular there must be an image in both eyes, and to ensure this the oculars must be separable so that the eye- points are the same width as the pupils of the eyes of the observer. One can tell whether there is binocular vision in his first trials by closing first one eye and then the other. If an image is seen without moving the head whichever eye is closed then of course both eyes are seeing an image and one should get the appearance of relief characteristic of stereoscopic images. If one does not see with both eyes the eye-points are too close or too far seperated for his pupils. The tubes should be seperated or approximated until each CM. Ill] INTERPRETATION OF APPEARANCES 113 eye sees the image. After one is used to the stereoscopic appear- ance when seeing with both eyes he can tell instantly whether the Fig. 102. Greenough's Erecting Binocular Microscope. This consists of two microscope tubes mounted side by side. The oculars may be approximated or separated for the eyes of different observers. The images are erected by the Porro prisms in the large rounded part of the lube. {Zeiss' Catalog.) ii4 INTERPRETATION OF APPEARANCES [CH. Ill binocular is properly adjusted for his eyes. (See Carpenter- Dallinger for fuller discussion of Binoculars. ) 103 104 Fig. 103. Sectional View of Wenham's Binocular Microscope, a. Tin- prism which e a- lends partly across the field and directs about half of the light to the left eye (L). A part of the light extends directly to the right eye (R). c, b. Field lenses of the right and the left oculars. Fig. 104. An enlargement of the Prism used in the H'enham Binocular Microscope, a, b, e, d Represent the course of the ray for the left eye. It is internally reflected at the points />, c, and given the proper direction to enter the left eye. REFERENCES FOR CHAPTER III. For general discussions : Carpenter-Dallinger, A. E. Wright, Principles of Microscopy, Ch. V.; Beale ; Spitta, Microscopy, Ch. xviii.; Beck's Cantor Lectures, lect. IV. For pedesis see Carpenter-Dallinger, p. 431 ; Beale, p. 195 ; Jevons in Quart, four. Science, n. s., Vol. YIII (1S78), p. 167. For the original 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. Sigtu. Schultze, " Mikroskopische Untersuchungen CI I. Ill ] IN TERPRE T ATI ON OF A PRE. I RA NCES "5 iiber des Herren Robert Brown Entdeckuug lebender, selbst im Feuer unzer- stiirbarer Theilchen in alien Korpern." From "Die Gesellschaft fur Befiir- derung der Xaturwissenchaften zu Frieburg." (1828.) For overcoming pedesis for photography see Gage, The use of a solution of gelatin to obviate pedesis in photographing milk globules and other minute objects in water. Transactions Amer. Micr. Soc. , Vol. XXIV., 1902, p. 21. For figures (photo-micrographs, etc. ) of the various forms of starch, see Bulletin Xo. 13 of the Chemical Division of the U. S. Department of Agri- culture. For Hair and Wool, see Bulletin of the National Association of Wool Growers, 1S75, p. 470, Proc. Amer. Micr. Soc, 1884, pp. 65-68. Herzfeld, translated by Salter. — The technical testing of yarns and textile fabrics, Lon- don, 1898. See also the Bibliography at the end for works relating to adulter- ation of foods, etc., for further discussions of the elements used in foods and drugs. For different appearances due to the illuminator, see Nelson, in Jour. Roy. Micr. Soc, 1891, pp. 90-105 ; and for the illusory appearances due to diffrac- tion phenomena, see Carpenter-Dallinger, p. 434. Mercer. Trans. Amer. Micr. Soc, pp. 321-396. Also, A. E. Wright's Principles of Microscopy; Conrad Beck. For the Binocular see Carpenter-Dallinger ; Mayall ; Spitta. 3- 4-5. 6. 7- S. 9- 10. 13- 14- 15- 16. Positive ocular. Draw-tube. Main tube or bod}-. Society screws in the draw-tube and bod}'. Objective in position. Stage. Spring for holding slides. Sub-stage condenser. Iris diaphragm. Plane and concave mirror. Horse-shoe base. Rack and pinion for condenser. Flexible pilar. Spiral spring of fine adjustment. Fine adjustment. Coarse adjustment. THE niCROSCOPE IN SECTION CHAPTER IV MAGNIFICATION AND MICROMETRY APPARATUS AND MATERIAL FOR THIS CHAPTER Simple and compound microscope (fj 172, 174); Steel scale or rule divided to millimeters and \\ Block for magnifier and compound microscope (§ 172, 176); Dividers (§ 172, 176); Stage micrometer (§ 175); Wollaston camera lucida (jS 176); Ocular screw-micrometers (Figs. 118-120); Abbe camera lucida (Fig. 114). Necturus red blood corpuscles (g 184). Eikonometer (J 195). § 170. 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 appar- ent size is obtained by measuring the virtual image (Figs. 26, 43). For determining magnification the object must be of known length and is designated a micrometer (§ 175). In practice a virtual image is measured by the aid of some form of camera lucida (Figs. 108, 114), or by double vision (§ 172). 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 40 mm. and the object mag- nified, 2 mm., the amplification is 40-^2=20, that is, the appar- ent 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 a scale at which a micro- scopical or histological drawing is made, the word magnification is frequently indicated by the sign of multiplication thus : X 450, upon a drawing means that the figure or drawing is 450 times as large as the object. § 171. Magnification of Real Images.— In this case the magnification 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 CH. IV] MA CNIFICA TION AND MICRON E TRY 117 (§ 60 s ), it will be remembered that it forms a real image on the ground glass placed on the top of the tube, and that this real image could be looked at with the eye or measurered 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 is 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 magnification is designated in the same way by dividing the size of the real image measured on the ground glass by the size of the object. For example, if the ob- ject is 400 millimeters long and its image on the ground glass is 25 mm. long the ratio is 25-=-400= Trr . That is, the image is yg- 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 is X T \r. In photog- raphy ( Ch. VIII) and when using the magic lantern and the pro- jection microscope the images are real, and may be measured on the screen as if real pictures. MAGNIFICATION OF A SIMPLE MICROSCOPE § 172. The Magnification of a Simple Microscope is the ratio between the object magnified (Fig. 16, A'B 1 ), and the virtual Fig. 105. Tripod Magnifier image (A S B S ). To obtain the size of this virtual image place the tripod magnifier near the edge of a support of such a height that the distance from the upper surface of the magnifier to the table is 250 millimeters. I lS MAGNIFICATION AND MICROMETRY [CM. IV 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. Fig. 106. Ten Centimeter Rule. The upper edge is divided into milli- meters, the lower into centimeters at the left and half centimeters at 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 (§ 12). 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 others, the two images appearing to be fused in a single visual field. § 173. Measuring the Spread of Dividers. — Thisshould be done on a steel scale divided to millimeters and i. As \ mm. cannot be seen plainly by the unaided eye, place one arm of the dividers at a centimeter line, and with the tripod magni- fier 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 3 B 3 ), 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 mil- limeters, then the magnification is 15-5-2=7^, that is the image is f 1 /, times as long or wide as the object. In this case the image is said to be magnified 7^ diameters, or 7^ times linear. CH. IV] MAGNIFICATION AND MICROMETRY 119 The magnification of any simple magnifier may be determined experimentally in the way described for the tripod ; but this methcd is of course only possible when the observer has two good eyes. If he has but one eye then the magnification m^ be determined by the aid of a camera lucida (§ 176) or the eikonometer (§ 196). MAGNIFICATION OF A COMPOUND MICROSCOPE § 174. The Magnification of a Compound Microscope is the ratio between the final or virtual image (Fig. 26, B 8 A 3 ), and the object magnified (A B). The determination of the magnification of a compound micro- scope may be made as with a simple microscope (§ 172), but this is fatiguing and unsatisfactory. § 175. Stage, Object or Objective Micrometer.— For de- termining 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. Such a finely divided scale is called a micrometer, and for ordinary work one mounted on a glass slide (1 X 3 in., 25 x 76mm.) is most convenient. The spaces between the lines should be y 1 ^ and y^ ¥ mm. (or if in inches, T ^ and TTi Vo i n -)- Micrometers are sometimes ruled on the slide, but more satisfactorily on a cover-glass of known thick- ness, 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 shel- lac 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 sharpness of the micrometer is lost. If the lines are on the slide and uncovered one cannot use the micrometer with an oil im- mersion, as the oil obliterates the lines. Cleaning the slide makes the lines less sharp as stated. If the lines are coarse, it is an advantage to fill them with plumbago. This may be done with some very fine plumbago on the end of a soft cork, or by using a soft lead pencil. Lines properly filled may be covered with balsam and a cover-glass as in ordinary balsam mounting (Ch. VII). § 176. 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 120 MAGNIFICATION AND MICROMETRY [CH. IV used for all powers, and the determination of the standard distance of 250 millimeters at which to measure the images is readily deter- mined (Fig. 108, § 178). Employ the 16 mm. (fi in.) objective and a 37 mm. (orX8 ocular with a stage micrometer as object. For this power the T V mm. spaces of the micrometer should be used as object. Focus sharply. Fig. 107. Abbe's Test Plate to show the enclosure of the micrometer lines by small rings. See also Fig. 75. It is somewhat difficult to find the micrometer lines. To avoid this it is well to have a small ring enclosing some of the micrometer lines (Fig. 107). The light' must also be carefully regulated. If too much light is used, i. e., 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- not afford to break them. As suggested in § 83, 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 position make the tube of the microscope horizontal, by bending the flexible pillar, being careful not to bring any strain upon the fine adjustment (frontispiece).. Put a Wollaston camera lucida (Fig. 108 and Ch. V) in posi- tion, and turn the ocular around if necessary so that the broad flat surface may face directly upward, as shown in the figure. Elevate the microscope by putting a block under the base, so that the per- pendicular distance from the upper surface of the camera lucida to the table is 250 mm. (§ 178). 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 CH. IV] MAGNIFICATION AND MICROMETRY appear to be on the table. It may be necessary to readjust the focus after the camera lucida is in position. If there is difficulty in seeing dividers and image consult Ch. V. Measure the image with dividers and obtain the power exactly as above (§ 172-173). Fig. 108. Wollasion's Camera Lucida, showing the rays from the microscope and from the draw- ing surface, also the position of the pupil of the eye. Axis, Axis. 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 the drawing surface. C. D. Rays from the drawing surface to the eye. A. D A' D' . Overlapping portions of the two fields, where both the ■microscopic image and the drawing surface, pencil, etc., can be seen. It is rep- resented by the shaded part of the overlapping circles at the right. Ocular. The ocular of the microscope. P. The drawing pencil. Its point is shown in the overlapping fields. 10S Thus: Suppose two of the y 1 ^ 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 gf millimeters. If the ob- ject is T 2 7 of a millimeter and the magnified image is of millimeters, the magnification (which is the ratio between size of object and image) is of -7-^—47. That is, the magnificatfon 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 denomination, thus: If the size of the image is found to be of mm. this number may be reduced to tenths mm., so it will be of the same denomina- tion as the object. In 9 mm. there are 90 tenths, and in f there are 4 tenths, then the whole length of the image is 90-1-4=94 tenths MA GNIFICA TION AND MICROME TR V [ CH. 1 V of a millimeter. The object is 2 tenths of a millimeter, then there must have been a magnification of 94-7-2=47 diameters in order to produce an image 94 tenths of a millimeter long. Image Object- Objeetivi Object-b Object-a FIG. 109 Fig. no Fig. 100-110. Figures showing that the size of object and image vary directly as their distance from the center of the lens. In Fig. no one can also see why.it is necessary to focus down, i. e. bring the object and objectives nearer together when the tube is lengthened. See also Fig. 66. 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. 26, B s A 3 ) seen with the CH. IV~\ MAGNIFICATION AND MICROMETRY 123 high ocular is larger than the one seen with the low one. The real image (Fig. 26 A 1 B 1 ) remains nearly the same, and would be just the same if positive, par-focal oculars (§ 43, 82, 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 is greater than with the shorter tube. This is because the real image (Fig. no) 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 lens. The truth of this statement is illustrated by Figs. 109 and no. § 177. Varying the Magnification of a Compound Micro- scope. — It is seen from the above experiments (§ 176) that in- dependently of the distance at which the microscopic image is measured (§ 178), there are three ways of varying the power of a compound microscope. These are named below in the order of desirability. (1) By rising a higher or lower objective. ( 2 ) By using a higher or lower ocular. (3) By lengthening or shortening the tube of the microscope (Fig. no).* § 178. 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 a distance of 250 millimeters. This is not 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 com- mon standard will be seen at a glance in Fig. 111, where is repre- *Amplifier. — In addition to the methods of varying the magnification given in \ 177, the magnification is sometimes increased by the use of an amplifier, that is a diverging lens or combination placed between the objec- tive 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. 124 MAGNIFICATION AND MICROMETRY I CI I. IV sented graphically the fact that the size of the virtual image de- pends directly on the distance at which it is projected, and this size is directly proportional to the vertical distance from the apex of the Fig. hi. Figure shock- ing the position of the mi- croscope, the camera lucida, the eye, and the difference in size of the image depending upon the distance at which it is projected from from the eye. (a) The size at 25 cm.; (6) at 35 cm., (I 17S). Fig. 112. Wollaston's camera lu- cida in position on the tipper end of the tube of the microscope. (Cut loaned by the Spencer Lens Co.) Fig. 113. Simple microscope mechanically supported by a lens holder. One may obtain the magnifying power of a simple microscope by the use of a camera lucida as with the compound microscope. (Cut loaned by the Spen- cer Lens ( 'o. ) CH. 1 V\ JfAGNIFICATION AND MICROMETRY 125 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. 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 milli- meters. Fig. 114. Sectional view of the Abbe Cam- eya Lucida to show that in measuring the stand- ard distance of 250 mill- imeters, one must meas- ure along the axis from the point P, at the left of the prism, to the mir- ror, and from the mir- ror to the drawing sur- face. For a full ex- planation of this camera lucida, see next chapter. In preparing drawings it is often of great convenience to make them at a distance somewhat less or somewhat greater than the standard. In such a case the magnification must be determined for the special distance. (See the next chapter, § 207. ) 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; 1904, pp. 261, 279; A. E. Wright, Practical Microscopy, pp. 129, 145, 163. § 179. Table of Magnification and of the Valuations of 126 MAGNIFICATION AND MICROMETRY [CH. IV the Ocular Micrometer. — The table should be filled out by each student. In using it for Micrometry and Drawing it is necessary to keep clearly in mind the exact conditions under which the determinations were made, and also the ways in which variations in magnification and the valuatio?i of the ocular micrometer may be produced (§ 177, 178, 188, 195- OCULAR OCULAR 37 or 5° mm- 25 mm Objective. Tube in Tube out MM. Tube in Tube out MM. Ocular Micrometer Valuation, tube in. out mm. X X X X X X X X X X X X X X X X X X X X X X X X Simple Microscope. X Fig. 115 micrometry § 180. Micrometry is the determination of the size of objects by the aid of a microscope. MICROMETRY WITH THE SIMPLE MICROSCOPE § 181. With a simple microscope (A), the easiest and best way is to use dividers and then with the simple microscope deter- CH. IV] MAGNIFICATION AND MICROMETRY 127 mine when the points of the dividers exactly include the object. The spread of the dividers is then obtained as above (§ 173) . 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 (§ 172), measure the image at the standard distance. If the size of the image so measured is divided by the magnificatibn of the simple microscope, the quotient give the actual size of the object. One might use the eikonometer also (§ 196). 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 MICROSCPE There are several ways of varying excellence for obtaining the size of objects with the compound microscope, the method with the ocular micrometer (§ 189-193) being most accurate. § 182. Unit of Measure in Micrometry. — As most of the objects measured with the compound microscope are smaller than any of the originally named divisions of the meter, and the common or decimal fractions necessary to express the size are liable to be unnecessarily cumbersome, Harting, in his work on the microscope (1859), proposed the one thousandth of a millimeter (t-jVt mm - or 0.001 mm.) or one millionth of a meter (niwiinr or 0.00000 1 meter) as the unit. He named this unit micro-millimeter and designated it mmm. In 1869, Listing (Carl's Repetorium fur Ex- perimentai-Physik, Bd, X, p. 5) favored the thousandth of a milli- meter as unit and introduced the name Mikron or micrum. In English it is most often written Micron (plural micro, or microns, pronunciation Mik'rSn or Mik'rbn). By universal consent the sign or abbreviation used to designate it is the Greek /x. Adopting this unit and sign, one would express five thousandths of a millimeter (nnnr or °'°°5 mm -> thus > SM-* * The term micromillimeter, abbreviation mmm., is very cumbersome, and besides is entirely inappropriate since the adoption of the definite mean- 128 MAGNIFICATION AND MICROMETRY \_CH. IV | 183. Micrometry by the use of a stage micrometer on which to mount the object. — 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. § 184. Micrometry by dividing tlie size of the image by the magnification of the microscope. — For example, employ the 3 mm. (}4 in.) objective, 25 mm. (1 in.) ocular, anda 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 (§ 176). Divide the size of the image in each case by the magnifi- cation, and the result gives 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 microscope 400 diameters (§ 170), then the actual length of this long axis of the corpuscle is 18 mm. -r- 400 =0.045 mm - or 45/* (§ 182). Fig. 116. Preparation of blood with a ring around a group of blood cor- puscles. As the same three blood- corpuscles are to be measured in three ways, it is an advantage to put a delicate ring around a group of three or more corpuscles, and make a sketch of the whole enclosed group, marking on the sketch the corpuscles measured (Figs. 70, 75). The different corpuscles vary considerably in size, so that accurate comparison of different methods of measurement can only ings 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 inter- national commission on weights and measures, and by original investigators, and is, in the opinion of the writer, the best term to employ. Jour. Roy. Micr. Soc, 1888, p. 502 ; Nature, Vol. XXXVII (1888), p. 388. CH. IV] MAGNIFICATION AND MICROMETRY 129 be made when the same corpuscles are measured in each of the ways. § 185. Micrometry by 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- corpuscles. Measure the same three that were measured in § 184. Remove "the object, place the stage micrometer under the microscope, focus well, and draw the lines of the stage micrometer so as to include the dots representing the limits of the part of the image to be measured. As the value of the spaces on the stage micrometer is known, the size of the object is determined by the number of spaces of the micrometer required to include it. This simply enables one to put the image of a fine rule on the image of a microscopic object. It is theoretically an excellent method, and nearly the same as measuring the spread of the dividers with a simple microscope (§ 173, 197). OCULAR MICROMETER § 186. Ocular Micrometer, Eye-Piece Micrometer. — This, as the name implies, is a 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 equi- distant and T V or ^ mm. apart, every fifth line should be longer and heavier to facilitate counting. If the micrometer is ruled in squares {net micrometer) it will be very convenient for many pur- poses. The ocular micrometer is placed in the ocular, no matter what the form of the ocular (i. e., whether positive or negative) at the level at which the real image is formed by the objective, and the image appears to be immediately upon or under the ocular microme- ter, 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. 13° MA GN1FICA TION AND MIC ROME TR Y [CH. IV § 187. Valuation of the Ocular Micrometer. — This is the value of the divisions of the ocular micrometer for the purposes of micrometry, and is entirely relative, depending on the magnifica- tion 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 (z. e. , objective and ocular), and for every change in the length of the tube of the microscope. That is, it is necessary to determine the ocular micrometer valuation for every condition modifying the real image of the microscope (§ 177). Any Huygenian ocular (Fig. 117) may, however, be used as a micrometer ocular by placing the ocular micrometer at the level of the ocular diaphragm, where the real image is formed. 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 at 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. ■I 1 )4q / - / \ /I « \ ° K L.\ \ : \^>T ) lu. I Fig. 117. Sectional view of a Huygenian ocular. Axis. Optic axis of the ocular, the ocular. E. L. Eye-Lens. F. L. D. Diaphragm of Field-Lens. E. P. Eye-point. In micrometry the ocular microm- eter zvith a Huygenian ocular must be placed at the level of the diaphragm where the real image is formed. In a positive ocular it would be placed below the ocular lenses. § 188. Obtaining the Ocular Micrometer Valuation for an Ocular Micrometer with fixed Lines. — Use the stage micrometer as object. Light the field well and look into the micro- scope. 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 magnifier. When the lines of the ocular micrometer are distinct, focus the microscope (§ 81, 84) for the stage micrometer. The image of the stage micrometer appears to be directly under or upon the ocular micrometer. Make the lines of the two micrometers parallel by rotating the CH. IV] MAGNIFICATION AND MICROMETRY 131 ocular or changing the position of the stage micrometer or both if necessary, 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 micrometer 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 milli- meter is taken as the unit for the stage micrometer and this unit is divided into spaces of T J T and T ^ ¥ millimeters. If with a given optical combination and tube-length it requires 10 spaces on the ocular mi- crometer to include the real image of T V millimeter on the stage mi- crometer, obviously one space on the ocular micrometer includes only one-tenth as much, or j 1 ^ mm.-MO^^ mm. That is, each space on the ocular micrometer includes T ^ of a millimeter on the stage micrometer, or T ^ T millimeter of the length of any object under the microscope, the conditions remaining the same. Or, in other words, it requires 100 spaces on the ocular micrometer to include 1 milli- meter on the stage micrometer, then as before, 1 space of the ocular micrometer would have a valuation of -j-J-^ millimeter for the pur- poses of micrometry. The size of any minute object may be deter- mined 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 T ^ T mm. x8= T |finm. or 80 /t (§ 182). See Mark, Jour. Applied Micro- scopy, Vol. I, p. 4. § 189. Micrometry with the Ocular Micrometer. — Use the 3 mm. (_^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. In a word, be sure that all the conditions are exactly as when the valuation was determined, then put the preparation under the microscope and find the same three red corpuscles that were measured in the other ways (§ 184-185). Count the divisions on the ocular micrometer required to enclose or measure the long and the short axis of each of the three cor- i 3 2 MAGNIFICATION AND MICROMETRY [CH. IV puscles, 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. 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 cor- rect and the work carefully enough done, the same results should be obtained by each method. (§ 197.)* * 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 i9 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 , 2 ;, millimeter of the stage micrometer, then obviously one space on the ocular micrometer would include \ of ^ mm. or ^ mm. ; the size of any unknown object under, the microscope would be obtained by multiplying the number of divisions on the ocular micrometer required to include its image by the value of one space, or in this case, 5 ' 5 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 15X^=1. or 0.6 mm. (B) By finding the number of divisions on the ocular micrometer re- quired 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 divi- sions of the ocular micrometer are required to include the image of ,% mm., on the stage micrometer, then evidently it would require 5-=- r 2 5 =25 divisions on the ocular micrometer to include a whole millimeter on the stage microme- ter, 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 in a fraction of a millimeter. Thus, suppose it required 15 divisions of the ocular micrometer to include the image of some part of the fly's wing, the actual size of the part included would be 15-^25=! or 0.6 mm. This method is really exactly like the one in (A), for dividing by 25 is the same as multi- plying by sV- (C) By having the ocular micrometer ruled in millimeters and divisions of a millimeter, and then getting the size of the real image in millimeters. In employing this method a stage micrometer is used as object and the size of the image of one or more divisions is measnred by the ocular micrometer, thus : Suppose the stage micrometer is ruled -fa and T J 7 mm. and the ocular micrometer is ruled in millimeters and fa mm. Taking T % mm. on the stage micrometer as object, as in the other cases, suppose it requires 10 of the T \ mm. spaces or 1 mm. to measure the real image, then the real image CH. IV] MAGNIFICATION AND MICROMETRY 133 § 190. Obtaining the Valuation of the Filar Micrometer. — This micrometer (Figs. 1 18-120) usually consists of a Ramsden's ocular and cross lines. As seen in Fig. 119 A 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 ac- Fig. 1 1 S. 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. At the left is shown a clamping screw to fasten the ocular to the upper part of the microscope 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 accurately. With the ordinary ocular micrometer very small objects frequently fill but a part of an inter- val of the micrometer, but with this the movable cross lines traverse the object [or rather its real image) regardless of the mi- nuteness of the object. [Zeiss' Catalog.) must be magnified ^-§-=-^=5 diameters, that is, the real image is five times as great in length as the object, and the size of an object may be deter- mined 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 mag- nification 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 j\ mm. divisions = f$ 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=! mm., the same result as in the other cases. See also \ 195-196 on the Eikonometer. In comparing these methods' it will be seen that in the first two (A andB) 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 micrometer 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 get- ting them an exact division of a millimeter or of an inch apart. 134 MAGNIFICATION AND MICROMETRY [ CH. IV curate stage micrometer must be used. Carefully focus the -j-J-j 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 (§ 188). Make the vertical lines of the filar micrometer parallel with the lines of the stage micrometer. Take the precau- tions regarding the width of the stage micrometer lines given in § 197 (see also Fig. 123). 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 mi- crometer. 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 part of a revolution required to measure the y-J-j- mm. of the stage micrometer Fig. 119. Filar Micrometer Ocular. This filar micrometer ocular is of the Ramsden type and consists of a positive ocular with a moveable hair line and two reference lines at right angles to each other as shown in A. The moveable line must be carried over the entire length of the object to be meas- ured by rotating the drum. A. Field of the filar micrometer showing the moveable and the cross lines, and the comb. The teeth serve to measure the total revolutions of the drum. (Cut loaned by the Bausch & Lomb Optical Co. ) can be easily noted. Measure in like manner 4 or 5 spaces and get the average. Suppose this average is i}( revolutions or 125 grad- uations on the wheel, to measure the j-^j mm. or 10// (see § 182), then one of the graduations on the wheel would measure io/< divided by i25=.o8;U. In using this valuation for actual measurement, the tube of the microscope and the objective must be exactly as when obtaining the valuation (see § 187, 194). CH. IV] MAGNIFICATION AND MICROMETRY 135 § 191. 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 to edge. Suppose it requires 94 of the graduations to measure the diameter, the actual size of the corpuscle would be 94X .08/^=7.52/*. The advantage of the filar micrometer is that the valuation of one graduation being so small, even the smallest object to be meas- ured 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 measurements, 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. § 192. Obtaining the Valuation of the Combined Ocular Micrometer (Fig. 120). — To obtain the valuation of this ocular micrometer one proceeds exactly as for the micrometer with fixed lines (§ 188), except that a partial stage micrometer space can be measured by rotating the drum until the ocular micrometer exactly coincides with the stage micrometer. One can then count up the number of spaces on the ocular micrometer required to measure one or more spaces on the stage micrometer. To this is then added the 100 hundredths of a space indicated on the drum. For example suppose that it required 7 complete spaces of the ocular micrometer and the drum showed 50 hundredths to measure 3 spaces (3 hundredths mm.) on the stage micrometer, then each space on the ocular micrometer would be equal to 0.03 mm. -i-7. 50= 0.004 mm - or 4/*- One or ^e spaces on the drum which represents one hundredth of an interval on the ocular micrometer would have a valuation under these conditions of only 0.04/4. This gives a clear notion of the minuteness of the objects which can be measured and of the smallness of the error in measuring large objects even if one should get the object a few of the drum divisions too small or too large. § 193. Example of Measurement with the Combined Ocular Micrometer. — Select an oval corpuscle of some lower 136 MAGNIFICATION AND MICROMETRY [CI-/. IV animal (frog, hen, turtle, etc.), Arrange the micrometer ocular so that the long axis of the corpuscle will coincide with the cross line in the micrometer scale (Fig. 121). Get one end of the corpuscle exactly level with one end of the micrometer scale. Note the posi- tion of the drum, and then rotate it until the other end of the corpuscle is exactly against the nearest line of the micrometer. Count up the entire intervals required and the partial interval on the drum. Suppose it requires 5 entire and 0.60 intervals (see explanation of Fig. 121) then the whole corpuscle must be 5.60 intervals multiplied by 4/;, the value of one interval: 5.6X4= 2I.4jK. Fig. 120. Screw Ocular Micrometer with moveable scale. This is a Huygenian ocular with a 5 mm. scale divided into 20 % mm. in- tervals. The pitch of the screw moving the scale is ■4 mm. therefore one com- plete revolution of the drum ■moves the scale one interval or % mm. The drum is divided into 100 gradua- tions thus enabling one to measure 100th of an interval on the micrometer scale. This ocular micrometer combines the advantages of the ocular micrometer with fixed scale and the filar micrometer. To complete the measurement of an object not exactly between any two of the micrometer lines the drum need be revolved only partly around. ( Cut loaned by the Spencer Lens Co. ) Fig. 121. Figure of the scale of the screw ocular micrometer, showing the divisions and the cross line. At the left is shown an object on the scale not quite fill- ing 10 of the intervals. To measure this the drum, need be revolved only sufficianlly to measure the part of the interval filled by the object being measured. Originally the scale -was divided in 50 ,'„ mm. spaces, and no cross line was present. In 100*, the present form of scale was specially prepared from the "writer's specifications, and has since that time been regularly supplied. (Cut loaned by the Spencer Lens Co.) § 194. Varying the Ocular Micrometer Valuation. — Any change in the objective, the ocular or the tube-length of the micro- scope, that is to say, any change in the size of the real image, pro- m / 2 3 III III 5 ffls -ijjjil III III CH. IV] MAGNIFICATION AND MICROMETRY 137 duces a corresponding change in the ocular micrometer valuation (§ 177. 187, 197)- § 195. Eikonometer for Magnification and Micrometry. — The eikonometer is something like an . eye. It has a converging lens serving in place of the crystalline lens to focus the rays from the eye-piece of the compound microscope, or from the simple micro- scope upon a micrometer scale, the scale taking the place of the retina in the eye (Fig. 16). This scale is ruled in -^ mms. Above the scale is a Ramsden's ocular of 25 mm. equivalent focus, giving a magnification of 10. The eikonometer scale therefore is a milli- meter scale when seen at the distance of 250 mm. in the visual field of the normal human eye, and it enables one to put a millimeter scale on the image of any object studied. To use it for magnification a stage micrometer is put under the microscope and carefully focused. Then the eikonometer is put in place over the ocular. The microscopic image of the stage microm- eter and the scale of the eikonometer will then appear in the same field as with the ordinary ocular micrometer (§ 188). The two sets of lines should be made parallel. See how many divisions of the eikonometer millimeter scale are required to measure one or more of the divisions of the image of the stage micrometer. Suppose it requires 6 intervals or millimeters of the eikonometer scale to meas- ure the image of yf^ mm. on the stage micrometer. The size of the object is then yf , mm. and of its image 6 mm. The magnification is therefore (§ 170) 6 nim.-=- r f T =200. For determining the magnification of a simple microscope the eikonometer is placed over the simple microscope as it was over the ocular above. With this instrument as with the camera lucida only one eye is used (§ 176). § 196. Micrometry with the Eikonometer. — In the first place the magnification of the microscope must be determined as described in the preceding, section ; and one must keep in mind the factors which will vary the magnification (§ 177). The object to be measured is put under the microscope and focused and the eikonometer put in position. The virtual image is then measured in millimeters by the eikonometer mm. scale. The size of this virtual image is then divided by the magnification and the result will be the actual size of the object as in (§ 184). 138 MAGNIFICATION AND MICROMETRY \_CH. IV For example suppose the long axis of a Necturus' red blood corpuscle measures 9 mm. on the eikonometer scale. If the magni- fication of the microscope is 200 as found above then the actual length of the corpuscle is 9 mm. ■+- 200=0.045 mm., or 45/*. (See A. E. Wright, Jour. Roy. Micr. Soc, 1904, pp. 261, 279; Princi- ples of Microscopy, pp. 145, 163.) A ^m t>i D E MICROSCOPE EIKONOMETER Fig. 122. Wright's Eikonometer for Magnification and Micrometry. — [From Beck's Catalog.) A. Objective; B. Ocular; D. The object ; E. Virtual image of the microscope ; C. The Eikonometer placed over the ocular. The lens G, produces a real image on the eikonometer scale at F. This scale and real image are then viewed through the Ramsden ocular of 25 mm. equivalent focus, H . \ 197. Remarks on Micrometry. — In using adjustable objectives (\ 27, 114), the magnification of the objective varies with the position of the adjust- ing 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 produces a corresponding change in the magnification of the entire microscope, and the ocular micrometer valuation — therefore it is neces- sary to determine the magnification 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 uniformity of micrometers, and the difficulty of determining the exact limits of the object to be measured. Hence, all microscopic measurements are only approximately correct, the error lessening with the increasing perfec- tion of the apparatus and the skill of the observer. A difficulty when one is using high powers is the width of the lines of the 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 CH. IV} MAGNIFICATION AND MICROMETRY 139 from the inside of one bordering 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. 123). 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 es- pecially remarked upon by Richardson, Monthly Micr. Jour., 1874, 1875,; Rogers, Proc. Amer. Soc. Microscopists, 1882, p. 239; Ewell, North American Pract., 1890, pp. 97, 173. B Fig. 123. The appearance of the coarse stage micrometer and of the fine ocular micrometer lines when using a high objective. {A). The method of measuring the spaces by putting the fine ocular micrometer lines opposite the center of the course stage micrometer lines. (B). Method of measuring the spaces of the stage micrometer by one line of the ocular micrometer {0. m. ) at the inside and one at the outside of the course stage microm- eter lines (s. m.). 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 0.2H is the limit of precision in microscopic measures beyond which it is im- possible to go with certainty." W. A. RogersProc. Amer. Soc. Micrs., 1883, p. 198. In comparing the methods of micrometry with the compound microscope given above (§ 183, 184, 185, 189, 191, 193, 196,), the one given in \ 183 is impracticable, that given in (j 184 is open to the objection that two standards are required, — the stage micrometer, and the steel rule; it is open to the fur- ther objection that several different operations are necessary, each operation adding to the probability of error. Theoretically the method given in \ 185 is good, but it is open to the very serious objection in practice that it requires so many operations which are especially liable to introduce 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 mi- crometer lines. If the ocular micrometer has some quite narrow spaces, and i 4 o MAGNIFICATION AND MICROMETRY \_CH. IV others considerably larger, one can nearly always manage to exactly include the object by some two lines. The ocular screw micrometers (Figs.118-120) obviate 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 no estimation or guess work is necessary. The new method by means of Wright's Eikonometer (g I 195-6) is spoken of very favorably by experts who have employed it. For those especially in- terested in micrometry, as in its relation to medical jurisprudence, the follow- ing references are recommended. These articles consider the problem in a scientific as well as a practical spirit: The papers of Prof. Wm. A. Rogers on micrometers and micrometry, in the Amer. Quar. Micr. Jour , Vol. I. pp. 97, 208; Proceedings Amer. Soc. Microscopists, 1882, 1883, 1887. Dr. M. D. Ewell, Proc. Amer. Soc. Micrs., 1890; The Microscope, 1889, pp. 43-45; North Amer. Pract., 1890, pp. 97, 173. Dr. J.J. Woodward, Amer. Jour, of the Med. Sci., 1875. M. C. White, Article " Blood-stains," Ref. Hand-book Med. Sciences, 1885. Medico-Legal Journal, Vol. XII. For the change in magnification due to a change in the adjustment of adjustable* objectives, see Jour. Roy. Micr. Soc. 1880, p. 702; Amer. Monthly Micr. Jour. , 1880, p. 67. Carpenter-Dallinger, p. 270 and end of \ 196. 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. 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 thf meter FOR f Centimeter (c. m.). i-looth Meter; Millimeter (m.m.), i-ioooth Meter: length 1 Micron (/<),i-ioooth Millimeter; the Micron is the unit in Micrometry (§166). ( Kilometer, 1000 Meters; used in measuring roads and other long distances. he gram FOR f Milligram (m. g.), i-ioooth Gram. weight . . \ Kilogram, 1000 Grams, used for ordinary masses, like groceries, etc. the liter for f Cubic Centimeter (c. c. ), i-loooth loiter. This is more common than the capacity . \ correct form, Milliliter. Divisions of the Units are indicated by the Latin prefixes ; dcci, i-ioth ; cetiti, i-looth ; Milh\ i-ioooth ; Micro, i-l.ooo, oooth of any unit. Multiples are designated by Greek prefixes : deka, 10 times hecto, 100 times ; kilo, 1000 times ; myria 10,000 times ; Mega, 1,000,000 times any unit. CHAPTER V 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. 66), microscopic prepara- tions. DRAWING MICROSCOPIC OBJECTS § 198. Microscopic objects may be drawn free-hand directly from the microscope, but in this way a picture giving only the gen- eral appearance 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 ap- pliance used in obtaining outlines, etc. , of the microscope image is known as a camera lucida. § 199. 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 opti- cal device for superimposing or combining two fields of view in one eye. As applied to the microscope, it causes the magnified virtual image of the object under the microscope to appear as if projected upon the table or drawing board, where it is visible with the draw- ing 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 § 202). 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 142 DRA WING WITH THE MICROSCOPE [CH. V of the rays from the drawing paper, pencil, etc. In some of the camera lucidas from this group (Wollaston's, Figs. 108, 112), the rays are reflected twice, and the image appears as when looking E.P.Y--Y ■1 ll rlr^- <**% Fig. 126 Fig. 124. Abbe Camera Lucida with the mirror at 45°, the drawing surface horizontal, and the micro- scope vertical. Axis, Axis. Axial ray from the microscope and from the drawing surface. A, B. ' Marginal rays of Fig. 124 the field on the drawing surface, a b. Sectional view of the silvered surface on the upper of the triangular prisms composing the cubical prism (P). The silvered surface is shown as incomplete in the center, thus giving passage to the rays from the microscope. Fool. Foot or base of the microscope. G. ' Smoked glass seen in section. It is placed between the mirror and the prism to reduce the light from the drawing surface. Mirror. The mirror of the camera lucida. A Quadrant (Q) has been added to indicate the angle of inclination of the mirror, which in this case is 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. Fig. 125. Geometrical figure showing the angles made by the axial ray with the drawing surface aud the mirror. A, B. The drawing surface. Fig. r26. Ocular showing eye-point, E. P. It is at this point both hor- izontally and vertically that the hole in the silvered surface should be placed (.1203). CH. V~\ DRA WING WITH THE MICROSCOPE 143 directly into the microscope. In others the rays are reflected but once, and the image has the inversion produced by a plane mirror. For drawing purposes this inversion is a great objection, as it is necessary to similarly invert all the details added free-hand. (B) By a camera lucida reflecting the rays of light from the drawing paper, etc. , so that their direction when they reach the eye coincides with the direction of the rays from the microscope (Fig. 65, 124). In all of the camera lucidas of this group, the rays from the paper are twice reflected and no inversion appears. The better forms of camera lucidas (Wollaston's, Grunow's, Abbe's, etc.), may be used for drawing both with low and with high powers. Some require the microscope to be inclined (Fig. in) 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 Fig. 1 30. § 200. Avoidance of Distortion. — In order that the picture drawn by the aid of a camera lucida may not be distorted, it is neces- sary that the axial ray from the image on the drawing surface shall be at right angles to the drawing surface (Figs. 127, 129). § 201. Wollaston's Camera Lucida. — This is a quadrangular prism of glass put in the path of the rays from the microscope, and it serves to change the direction of the axial ray 90 degrees. In using it the microscope is made horizontal, and the rays from the microscope enter one-half of the pupil while rays from the drawing surface enter the other half of the pupil. As seen in figure 127, the fields partly overlap, and where they do so overlap, pen- cil or dividers and microscopic image can be seen together. In drawing or using the dividers with the Wollaston camera lucida it is necessary to have the field of the microscope and the drawing surface about equally lighted. If the drawing surface is too brilliantly lighted the pencil or dividers may be seen very clearly, but the microscopic image will be obscure. On the other hand, if the field of the microscope has too much light the microscopic image will be very definite, but the pencil or dividers will not be visible. It is necessary, as with the Abbe camera lucida ( \ 203 ) , to have the Wollaston prism properly arranged with reference to the axis of the micro* 6Cope and the eye-point. If it is not, one will be unable to see the image well, and may be entirely unable to see the pencil and the image at the same time. Again, as rays from the microscope and from the 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 dis- 144 DRA WING WITH THE MICROSCOPE [CH V tance of 250 mm. at which the drawing surface should be placed when deter- mining magnification {\ 178). § 202. *Abbe Camera Lucida. — This consists of a cube of glass cut into two triangular prisms and silvered on the cut surface of the upper one. A small oval hole is then cut out of the center of the silvered surface and the two prisms are cemented together in the form of the original cube with a perforated 45 degree mirror within it (Fig. 124, a b). 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 reflected by this surface to the eye in company with the rays from the microscope, so that the two fields appear as one, and the image is seen as if on the drawing surface (Figs. 124, 129). It is designed for use with a vertical microscope. [Compare § 205.] Fig. 127. Wollaston's Cam- era Lucida, showing the rays from the microscope and from the drawing surface, and the position of the pupil of the eye. See also Fig. 112. For full explanation see Fig. 108 *For some persons the image and drawing surf ace.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 con- centrated upon the drawing surface rather than upon the object under the microscope (Dr. W. B. Pillsbury) . CH. ir\ DRAWING WITH THE MICROSCOPE 145 § 203. Arrangement of the Camera Lucida Prism. — In placing this camera lucida over the ocular for drawing or the deter- mination of magnification, the center of the hole in the silvered surface is placed in the optic axis of the microscope. This is done by properly arranging the centering screws that clamp the camera to the microscope tube or ocular. The perforation in the silvered surface must also be at the level of the eye-point. In other words the prism must be so arranged vertically and horizontally that the hole in the silvered surface is in the axis of the microscope and coin- Fig. 12S. Abbe Camera Lucida in Position— -The prism over the ocular may be turned aside for direct observation. The light modifiers for drawing surface and microscope are in connection with the prism. The prism has centering screws and may be moved up or down with the whole apparatus by the clamping ring around the top of the draw-tube. This serves to place the prism at the proper vertical level for the eye-point of different oculars. (Cut loaned by the Spen- cer Lens Co.) cident with the eye-point of the ocular. If it is above or below, or to one side of the eye.point, part or all of the field of the microscope will be cut off. As stated above, the centering screws are for the proper horizontal arrangement of the prism. The prism is set at the right height by the makers for the eye-point of a medium ocular. If one desires to use an ocular with the eye-point farther away or nearer, as in using high or low oculars the position of the eye-point may be determined as directed in § 67 and the prism loosened and i 4 6 DRAWING WITH THE MICROSCOPE \_CH. V raised or lowered to the proper level ; but in doing this one should avoid setting the prism obliquely to the mirror. In the latest and best forms of this camera lucida special arrangements have been made for raising or lowering the prism so that it may be used with equal satisfaction on oculars with the eye- point at different levels, and the prism is hinged to turn aside with- out disturbing the mirror (Figs. 128, 132). One can determine when the camera is in a proper position by looking into the microscope through it. If the field of the micro- scope appears as a circle and of about the same size as without the -camera lucida, then the prism is in a proper position. If one side •of the field is dark, then the prism is to one side of the center ; if -the field is considerably smaller than when the prism is turned off the ocular, it indicates that it is not at the correct level, i. e., it is above or below the eye-point. § 204. Arrangement of the Mirror and the Drawing Sur- face. — The Abbe camera lucida was designed for use with a vertical microscope (Fig. 124). On a vertical microscope if the mirror is set at an angle of 45 , the axial ray is at right angles with the table top or a drawing board which is horizontal, and a drawing made under these conditions is in true proportion and not distorted. The stage of most microscopes, however, extends out so far at the sides that with a 45° mirror the image appears in part on the stage of the microscope. In order to avoid this the mirror may be depressed to some point below 45°, say at 40 or 35° (Fig. 129). But as the axial ray from the mirror to the prism must still be reflected hori- zontally, it follows that the axial ray no longer forms an angle of go degrees with the drawing surface, but a greater angle. If the mirror is depressed to 35 , then the axial ray takes an angle of 110° with a horizontal drawing surface (see the geometrical figure Fig. 129 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 drawing board twice as many degrees toward the microscope as the mirror is depressed below 45 °. Practically the field for drawing can always be made free of the stage of the microscope, at 45 , at 40 , or at 35 . In the first case (45 mirror) the drawing surface should be horizontal, in the second case (40° mirror) the drawing surface should be elevated io°, and in the third case (35 mirror) the draw- CH P] DRAWING WITH THE MICROSCOPE 147 ing board should be elevated 20 toward the microscope. Further- more it is necessary in using an elevated drawing board to have the mirror bar project directly laterally so that the edges of the mirror are in planes parallel with the edges of the drawing board, other- wise there will be front to back distortion, although the elevation of the drawing board avoids right to left distortion. If one has a micrometer ruled in squares {net micrometer) the distortion pro- duced 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 p and use a horizontal 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 appear as ellipses in the drawings, the major axis being from right to left. The angle of the mirror may be determined with a protractor, but that is troublesome. It is much more satisfactory to have a quadrant attached to the mirror and an indicator on the projecting arm of the mirror. If the quadrant is graduated throughout its entire extent, or preferably at three points, 45°, 40° and 35 , one can set the mirror at a known angle in a moment, then the drawing board can be hinged and the elevation of io° and 20° determined, with a protractor. The drawing 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. § 205. 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 microscope. All that is requisite is to be sure that the fun- damental law is observed regarding the axial ray of the image and the drawing surface, viz., that they should be at right angles. This is very easily accomplished as follows: The drawing board is raised toward the microscope twice as many degrees as the mirror is de- pressed below 45° (§ 204), then it is raised exactly as many degrees as the microscope is inclined, and in the same direction, that is, , so the end of the drawing board shall be in a plane parallel with the i 4 8 DRAWING WITH THE MICROSCOPE [CH. V Fig. 129 Abbe Camera Lucida in position to avoid distortion. Fig. 129. 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 (Q). The mirror is seen to be at an angle o/_j5°> 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°' makes an angle of 110° with a horizontal drawing board. The board must then be elevated toward the microscope 20° in order that the axial ray may be perpendicular to it, and thus fulfil the requirements neces- sary to avoid distortion ( $ 200, 204) . B. Upper view of the prism of the camera lucida. A considerable por- tion of the face of the prism is covered, and the opening in the silvered surface appears oval. C. Quadrant to be attached to the mirror of the Abbe Camera Lucida to indicate the angle of the mirror. As the angle is nearly always 45 , 40° or 35° , only those angles are shown. en. n DRAWING WITH THE MICROSCOPE 149 stage of the microscope. The mirror must have its edges in planes parallel with the edges of the drawing board also (Fig. 130.) § 206. 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 Fig. 130. 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. (Zeil. wiss. Mikroskopie, vol. VII., 1804, p. 298.) (Zeiss Catalog. ) with very low powers (16 mm. and lower objectives) by covering the mirror of the microscope with white paper when transparent ob- jects 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. One can tell which field is ex- cessively illuminated, for it is the one in which objects are most dis- tinctly seen. If it is the microscopic, then the image of the micro- 150 DRA WING WITH THE MICROSCOPE [ CH. V scopic object is very distinct and the pencil is invisible or very in- distinct. If the drawing surface is too brilliantly lighted the pencil can be seen clearly, but the microscopic image is obscure. When opaque objects, that is objects which must be lighted with reflected light ( § 72), 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. 60). 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. 124 G). If the light in the mi- croscope 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 draw- ing pencil with carbon ink. Sometimes it is easier to draw on a black surface, using a white pencil or style. The carbon paper used in manifolding letters, etc., may be used, or ordinary black paper may be lightly rubbed on one side with a moderately soft lead pencil. Place the black paper over white paper and trace the out- lines 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). (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 some- times desirable to draw the outline of an object with a moderate power and add the details with a higher power. If this is done it should always be clearly stated. It is advisable to do this only with objects in which the same structure is many times duplicated, as a nerve or a muscle. In such an object all the different struc- tures can be shown, and by omitting some of the fibers the others may be made plainer without an undesirable enlargement of the entire figure. CH. V] DRA WING WITH THE MICROSCOPE 151 (E) If a drawing of a given size is desired and it cannot be obtained by any combination of oculars, objectives and lengths of the tube of the microscope, the distance between the camera lucida and the table may be increased or diminished until the image is of the desired size. This distance is easily changed "by the use of a book or a block, but more conveniently if one has a drawing board with adjustable drawing surface like that shown in Fig. 130. (F) It is of the greatest advantage, as suggested by Heinsius (Zeit. w. Mikr. , 1889, p. 367), to have the camera lucida hinged so that the prism may be turned off the ocular for a moment's glance at the preparation , and then returned in place without the necessity of loosening screws and readjusting the camera. This form is now made by several opticians, and a quadrant is added by some. (Fig. 128, 132.) Any skilled mechanic can add the quadrant. § 207. Magnification of the Microscope and size of Drawings 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. 124) to the axis of the mirror and then vertically to the drawing board. In getting the scale to which a drawing is enlarged the best way is to remove the preparation and put in its place a stage micrometer, and to trace a few (5 or 10) of its lines upon one corner of the drawing. The value of the spaces of the micrometer being given, thus : ,i T t!l mm. Fig. 131. Showing the method of indicating thescaleat luhich a drawing was made. The enlargement of the figure can then be accurately deter- mined 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 micrometer were j^ millimeter, then the enlargement is 25-=- T f 7 =5oo. That is, the image was drawn at a magnification, of 500 diameters. 152 DRAW INC WITH THE MICROSCOPE [ CI I. V 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 mag- nification in each case may be determined from the scale accompany- ing the picture. It should be remembered, however, that the con- ditions when the scale is drawn must be exactly as when the draw- ing was made. Fig. 132 A. B. Abbe Camera Litcida. (A.) In I his figure the camera lucida is in position for drawing. The ring or collar supporting the mirror is graduated so that the angle of Hie mirror may be exactly determined. Smoked glasses serve to modify the light from the microscope or from the drawing surface as needed. By means of a clamping ring the instrument may be raised or lowered to accommodate the eye-point, in different oculars. (II.) In this figure the camera lucida prism is turned back so that one may took directly into the ocular. (Cuts loaned by the liauseh & Lomb Opti- cal Co.) § 208. Drawing at Slight Magnification. — .Some objects are of considerable size and for drawings should be enlarged but a few diameters, — 5 to 20. By using sufficiently low objectives and differ- ent oculars a great range may be ■ obtained. Frequently, however, CH. V\ DRA WING WITH THE MICROSCOPE iS3 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 condi- tions, its enlargement can be readily determined (§ 207). -7~ --^»>- -y. ?-r*^ Fig. 133. Room and Apparatus for Drawing with the Projection Micro- scope. R. Radiant, an arc lamp with carbons at a right angle ; L. t. Lamp and microscope table ; C. Condenser with W. a large water bath between the lenses to absorb the heat rays; S. w. Stage and stage water bath on which rests the object and keeps the object cool by radiation as well as by absorption ; O. The objective representing the microscope ; M. Mirror at 45° on a draw- ing table, (Dt.). As the microscope is horizontal so that the axial ray is reflected downward at right angles by the 45° mirror there is no distortion. The scale of the drawing is added exactly as described in \ 207. 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 mag- nification. 154 DRAWING WITH THE MICROSCOPE \_CH. V If one has many large objects to draw at a low magnification, then some form of embryograph is very convenient. (Jour. Roy. Micr., Soc, 1899, p. 223.) The writer has made use of a photo- graphic camera and different photographic objectives for the purpose. The object is illuminated as if for a photograph and in place of the ground glass a plain glass is used and on this some tracing paper is stretched. Nothing is then easier than to trace the outlines of the object. See also Ch. VIII. § 209. Drawing with the Projection Microscope. — Except for the highest powers and for details of cell structure the projec- tion microscope furnishes the most satisfactory means of making drawings. With it one can draw large diagrams or small figures directly from the objects; and if the apparatus is properly constructed one may make diagrams from objects 60 to 70 mm. in diameter down to those of half a millimeter or less. This method was much in vogue and highly commended by the older microscopiste who used the solar microscope (Baker, Adams and Goring). Since the general introduction of electric lighting drawing with the projection microscope has become once more common and is the most satisfac- tory method known especially for the numerous drawings necessary for the preparation of models in wax or blotting paper. See Ch. X. REFERENCES FOR CHAPTER V Beale, 31, 355 ; Behrens, Kossel and Schiefferdecker, 77 ; Carpenter- Dallinger, 278 ; VanHeurck, 91 ; American Naturalist, 1886, p. 1071, 1887, pp, 1040-1043 ; Amer. Monthly Micr. Jour., 1888, p. 103; 1890, p. 94; Jour. Roy. Micr. Soc, 1881, p. 819, 1882, p. 402, 1883, pp. 283, 560, 1884, p. 115, 1886, p. 516, 1888, pp. 113, 809, 798; Zeit. wiss. Mikroskopie, 1884, pp. 1-21, 18S9, p. ' 367, 1893, pp. 289-295. Here is described an excellent apparatus made by Winkel. Greenman Anat. Record No. 7, 1907, pp. 170-178. Gage, Origin and Development of the Projection Microscope. Transactions of the Amer. Micr. Soc, Vol. XXVIII, 1906. Consult also the latest catalogs of the opticians. CHAPTER VI MICRO-SPECTROSCOPE AND POLARISCOPE, MICRO- CHEMISTRY, MICRO- METALLOGRAPHY, TEXTILE FIBERS APPARATUS AND MATERIAL REQUIRED FOR THIS CHAPTER Compound microscope; Micro-spectroscope ($ 210); Watch-glasses and shell vials, slides and covers (§229); Various substances for examination (as blood and ammonium sulphide, permanganate of potash, chlorophyll, some colored fruit, etc., (g 230-239), Micro-polarizer (jS 240); Selenite plate (§ 250); Various doubly refracting objects, as crystals, textile fibers, starch, section of bone; Various chemicals, metals, etc. MICRO-SPECTROSCOPE f! 210. A Micro-Spectroscope, Spectroscopic or Spectral Ocular, is a di- rect vision spectroscope in connection with a microscope ocular. The one de- vised 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 trian- gular prism of heavy flint glass in the middle and one of crown glass on each side, the edge of the crown glass prisms pointing toward the base of the flint glass prism, i. e., the edge of the crown and flint glass prisms point in oppo- site directions. The flint glass prism serves to give the dispersion or separa- tion 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 con- nection with the ocular is the slit mechanism and a prism for reflecting hori- zontal rays vertically for the purpose of obtaining a comparison spectrum (§ 223) . Finally near the top is a lateral tube with mirror for the purpose of projecting an Angstrom scale of wave lengths upon the spectrum (§ 224, Fig. I34-I35)- (S an. Apparent Reversal of the Position of the Colors in a Direct Vision Spectroscope. — In accordance with the statements in § 210 the dispersion or separation into colors is given by the flint glass prism or prisms and in ac- 1 5b MICRO-SPECTROSCOPE AND POLAR /SCOPE [C//. VI Fig. 134 Abbe's, Micro-spectroscope. Fig. 135. Longitudinal Section of Slit Mechanism separately . the whole instrument . {Plan view, Full size. ) ( % Full size.) " The eye lens is adjustable so as to accurately focus on the slit situated be- tween the lenses. The mechanism for contracting and expanding the slit is actuated by the screw F and causes the laminae to move symmetrically (Merz's movement) . The slit may be made sufficiently wide so as to include the -whole visual field. The screw H serves to limit the length of the slit so as to com- pletely fill the latter with the image of the object under investigation when the comparison prism is inserted. The comparison prism is provided with a lateral frame and clips to hold the object and the illuminating mirror. All these parts are encased in a drum on the ocular." "Above the eye-piece is placed an Amici prism of great dispersion -which may be turned aside about the pivot A", so as (o allow of the adjustment of the object. The prism is retained in its axial position by the spring catch L. A scale is projected on the spectrum by means of a scale tube and mirror attached to the prism casing. The divisions of the scale indicate in decimals of a micron the wave length oj" the respective section of the spectrum. The screw P serves to adjust the scale relative to the spectrum." "The instrument is inserted in the tube in place of the ordinary eye-piece and is clamped to the former by means of the screw j)L in such a position that the mirrors A and O, ivhich respectively serve to illuminate the comparison prism and the scale of wave lengths are simultaneously illuminated." [From Zeiss' Catalog.) CH. VI] MICRO-SPECTROSCOPE AND POLAR/SCOPE 157 A 3 c £ola.r £oAiu.nv Ptrrnati.. ^zotaf h.. Fig. 136. Various Spectrums.—All except that of sodium were obtained . h diffused day-light with the slit of such a width as gave the most distinct Fraunhofer lines. It frequently occurs that with a substance giving several absorption bands (e. g., chlorophyll) the density or thickness of the solution must be varied to show all the different bands clearly. Solar Spectrum.— With diffused day-light and a narrow slit the spectrum is not visible much beyond the fixed line B. In order to extend the visible spectrum in the red to the line A, one should use direct sunlight and a piece of ruby glass in place of the watch-glass in Fig. 138. Sodium Spectrum. — 77;,? line spectrum ( \ 213 ) of sodium obtained by light- ing the microscope with a Bunsen or alcohol flame in which some salt of sodium is glowing. With the micro-spectroscope the sodium line seen in the solar spectrum and with the incandescent sodium appears single, except under very favorable circumstances ( % in.). \ 298. Work Room. — It is almo9t self-evident that the camera must be in some place free from vibration. A basement room where the camera table may rest directly on the cement floor or on a pier is excellent. Such a place is almost necessary for the best work with high powers. For those living in cities, a time must also' be chosen when there are no heavy vehicles moving in the streets. For less difficult work an ordinary room in a quiet part of the house or laboratory building will suffice. (S 299. Arrangement and Position of the Camera and the Microscope. — For much photo-micrography a vertical camera and microscope are to be pre- ferred (Fig. 170). Excellent arrangements were perfected long ago, especially by the French. (See Moitessier.) Vertical photo-micrographic cameras are now commonly made, and by some firms only vertical cameras are produced. They are exceedingly con- venient, and do not require so great a disarrangement of the microscope to make the picture as do the horizontal ones. The variation in size of the pic- ture in this case is mostly obtained by the objective and the projection ocular rather than by length of bellows (see below Fig. 170). It must not be forgot- ten, however, that penetration varies inversely as the square of the power, and only inversely as the numerical aperture ( § 40) , consequently there is a real advantage in using a low power of great aperture and a long bellows rather than an objective of higher power with a short bellows. A horizontal camera is more convenient for use with the electric light also (Fig. 180) . For convenience and rapidity of work a microscope with mechanical stage is desirable. It is also an advantage to have a tube of large diameter so that the field will not be too greatly restricted (Fig. 176). In some microscopes the tube is removable almost to the nose-piece to avoid interfering with the size of the image. The substage condenser should be movable on a rack and pinion. The microscope should have a flexible pillar for work in a horizontal position. While it is desirable in all cases to have the best and most conven- ient apparatus that is made, it is not by any means necessary for the produc- tion of excellent work. A simple stand with flexible pillar and good fine adjustment will answer. (S 300. Objectives and Oculars for Photo-Micrography. — The belief is almost universal that the apochromatic objectives are most satisfactory for photography. They are employed for this purpose with a special projection ocular. Two low powers are used without any ocular (Fig. 183). Some of the best work that has ever been done, however, was done with achromatic objec- tives (work of Woodward and others). One need not desist from undertaking photo-micrography if he has good achromatic objectives. From a somewhat extended series of experiments with the objectives of many makers the good PHOTO-MICROGRAPHY [C/J. I'll I modern achromatic objectives were found to give excellent results when used without an ocular. Most of them also gave good results with projection oculars. It must be said however, that the best results were obtained with the apochromatic objectives and projection oculars. It does not seem to require Fig. 171. — Vertical photo-micr graphic cam- era, screen and small table The table is about 45 cen- timeters high and in the legs are large screw eyes for leveling screws. The operator can stand on the floor and perform all the necessary operations, and in adjusting the micro- scope can sit on a low stool. The screen is of zinc and has two heavy lead feet to hold it steady. Near the lower left hand corner of the screen is an aperture for the light to shine through upon the mirror. This opening is closed by a black slide "which is just balanced so that it stays in any posi- tion . In making the ex- posure it is raised suffi- ciently to admit the light to the mirror, but the stage is left in shadozo. This screen shades the microscope and the face of the operator. (Trans. Amer. Micr. Soc. 1901.) so much skill to get good results with apochromatics as with achromatic ob- jectives. The majority of photo-micrographers do not use the Huygenian oculars in photography, although excellent results have been obtained with them. An amplifier is sometimes used in place of an ocular. Considerable experience is necessary in getting the proper mutual position of objective and CH. I 'J/J] Pfh ) TO-MICROGRAPHY 223 amplifier. The introduction of oculars especially designed for projection, has led to the discarding of ordinary oculars and of amplifiers. Oculars restrict the field very greatly, hence the necessity of using the objective alone for large specimens.* Fig. 172. Projection Oculars witli section removed to show the construction. Below are shown the upper end with graduated circle to indicate the amount of rotation found necessary to focus the diaphragm on the screen. No. 2, No. 4. The numbers indicate the amount the ocular magnifies the image formed by the ob- jective as with the compensation oculars, (Zeiss' Catalog. ) fj 301. Difference of Visual and Actinic Foci. — Formerly there was much difficulty experienced in photo-micrographing on account of the difference in actinic and visual foci. Modern objectives are less faulty in this respect and the apochromatics are practically free from it. Since the introduction of orthochromatic or isocbromatic plates and, in many cases the use of colored screens, but little trouble has arisen from differences in the foci. This is especially true when mono-chromatic light and even when petroleum light is used. In case an objective has its visual and actinic foci at markedly different levels it would be better to discard it for photography altogether, for the esti- mation of the proper position of the sensitive plate after focusing is only guess work and the result is mere chance. If sharp pictures cannot be obtained with an objective when petroleum light and orthochromatic plates are used the fault may not rest with the objective but with the plate holder and focusing screen. They should be very carefully tested to see if there is coincidence in position of the focusing screen and the sensitive film as described in \ 296. I 302. Apparatus for Lighting. — For low power work (35 mm. and longer focus) and for large objects, some form of bull's eye condenser is desirable although fairly good work may be done with diffused light or lamp-light reflected bv a mirror. If a bull's eye is used it should be as nearl3' achromatic as possible. The engraving glass shown in Fig. 175 answers well for large *A comparative study both with projection oculars, and without an ocular was made with the achromatic objective 25 mm. (1 inch), 18 mm. (% inch), 5 mm. ( 1 to j/g inch) and 2 mm. ( r V inch) homogeneous immersion of the Bausch & Lomb Optical Co.; Gundlach Optical Co. ; Leitz ; Reichert ; Winkel, Zeiss and the Spencer Lens Co. Good results were obtained with all of these objectives both with and without projection oculars. 224 PHOTO-MICROGRAPHY \_CH. VIII objects. For smaller objects a Steinheil lens combination gives a more bril- liant light and one also more nearly achromatic. For high power work all are agreed that nothing will take the place of an achromatic condenser. This may be simply an achromatic condenser, but preferably it should be an apochroma- tic condenser. Whatever the form of the condenser it should possess dia- Otiilir Ho 2 Fig. 173. Compensation Oculars of Zeiss, with section removed to show the construction. The line A- A is at the level of the upper end of the tube[of the microscope while B-B represents the lower focal points. Zeiss recommends the use of the compensation oculars if one desires a greater magnification than the projection oculars give. Fig. 174. Bull's eye lens and holder. (Bausch & LomhOpl. Co.) CH. VIII] PHOTO-MICROGRAPHY 225 phragms so that the aperture of the condenser may be varied depending upon the aperture of the objective. For a long time objectives have been used as achromatic condensers, and they are very satisfactory, although less conven- ient than a special condenser whose aperture is great enongh for the highest powers and capable of being reduced by means of diaphragms to the capacity of the lower objectives. It should also be capable of accurate centering (192)- I 303. Objects Suitable for Photo-micrographs. — While almost any large object may be photographed well with the ordinary camera and photographic objective, only a small part of the objects mounted for microscopic study can be photo-micrographed satisfactorily. Many objects that can be clearly seen by constant focusing with the fine adjustment, appear almost without detail on the screen of the photo-micrographic camera and in the photo-micrograph. Fig. 175. Engraving glass to serve as a con- denser and for a dissecting lens. (Bausch & Lomb Opt. Co.) If one examines a series of photo-micrographs the chances are that the greater number will be of diatoms, plant sections or preparations of insects. That is, they are of objects having sharp details and definite outlines, so that contrast and definiteness may be readily obtained (§ 107, 118, 157). Stained microbes also furnish favorable objects when mounted as cover-glass prepara- tions, but these give color images (§ 107, 119) and require a color screen (?2 9 l). For success with preparations of animal tissue they must approximate as nearly as possible to the conditions more easily obtained with vegetable prep- arations. That is, they must be made so thin and be so prepared that the cell outlines have something of the definiteness of vegetable tissue. It is useless to expect to get a clear photograph of a section in which the details are seen with difficulty when studying it under the microscope in the ordinary way. Many sections which are unsatisfactory as wholes, may nevertheless have parts in which the structural details show with satisfactory clearness. In such a case the part of the section showing details satisfactorily should be sur- rounded by a delicate ring by means of a marker (see Figs. 70, 72). If one's preparations have been carefully studied and the special points in them thus indicated, they will be found far more valuable both for ordinary demonstra- tion and for photography. The amount of time saved by marking one's speci- mens can hardly be overestimated. The most satisfactory material for making the rings is shellac colored with lampblack. Ten years ago many histologic preparations could not be satisfactorily photographed. Now with improved section cutters, better staining and mounting methods, and with the color screens ($ 291) and isochromatic plates 226 PHOTO-MICROGRAPHY {CH. VIII (\ 290) almost any preparation which shows the elements clearly when look- ing into the microscope can be satisfactorily photographed. Good photo- graphs cannot, however, be obtained from poor preparations. \ 304. Light. — The strongest light is sunlight. That has the defect of not always being available, and of differing greatly in intensity from hour to hour, day to day and season to season. The sun does not shine in the evening when many workers find the only opportunity for work. Following the sun- light the electric light is the most intense of the available lights. Then come magnesium, acetylene, the lime light, the gas-glow or Wellsbach light and petroleum light. The last is excellent for the majority of low and moderate power work. And even for 2 mm. homogeneous immersion objectives, the time of exposure is not excessive for many specimens (40 seconds to 3 minutes). This light is cheap and easily obtained. It has the advantage of being somewhat yellow, and therefore in many cases makes the use of a color screen unnecessary if one uses isochromatic plates. A lamp with fiat wick about 40 mm. (ij^ in.) wide has been found most generally serviceable. For large objects and low powers the flame may be made large and the face turned toward the mirror. This will light a large field. For high powers the edge toward the mirror gives an intense light. The ordinary glass chimney answers well, especially where a metal screen is used as shown in Fig. 171. EXPERIMENTS IN PHOTO-MICROGRAPHY § 305. The following experiments are introduced to show practically just how one would proceed to make photo- micrographs with various powers, and be reasonably certain of fair success. If one consults prints or the published figures made directly from photo-micrographs it will be seen that, excepting diatoms and bacteria, the magnification ranges mostly between 10 and 150 diameters. § 306. Focusing in Photo-Micrography. — For rough focus- ing and as a guide for the proper arrangement of the object one uses a ground-glass screen as in gross photography. With the ground-glass screen one can judge of the brilliancy and evenness of the illumination more accurately than in any other way. For final and exact focusing two principal methods are employed : (A). A focusing glass is used either with a clear screen or in a board screen as described above (§ 282). The latter method is like focusing with the compound microscope and a positive ocular. If the focusing glass is set properly the focus should be easily aud accurately determined. cu. vu i -\ PI 10 TO-MICROCRAPHY 121 Huqj er: Je/j b <" a o « o £ B ° D O fl> •=1 u % .2 'a .23 « JB H 01 O « 01 m 2 '" 01 cd a. . 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VIII} PHOTO-MICROGRAPHY 235 source of illumination, and the discussion of the proper cone of light and lighting the whole field, as given in § 94, 106. Then for each picture the photographer must take the necessary pains to light the object properly. An achromatic condenser is almost a necessity (§ 91). Whether a color-screen should be used depends upon judgment and that can be attained only by experience. In the be- ginning one may try without a screen, and with different screens and compare results. A plan used by many skilled workers is to light the object and the field around it well and then to place a metal diaphragm of the proper size in the camera very close to the plate holder. This will insure a clean, sharp margin to the picture. This metal diaphragm must be removed while focusing the diaphragm of the projection ocular, as the diaphragm opening is smaller than the image of the ocular diaphragm. If the young photo- micrographer will be careful to select for his first trials, objects of which really good photo- micrographs have already been made, and then persists with each one until fairly good results are attained, his progress will be far more rapid than as if poor pictures of many different things were made. He should, of course, begin with low magnifications. § 311. Adjusting the Objective for Cover-Glass. — After the object is properly lighted, the objective, if adjustable, must be corrected for the thickness of cover. If one knows the exact thick- ness of the cover and the objective is marked for different thick- nesses, it is easy to get the adjustment approximately correct mechanically, then the final corrections depend on the skill and judgement of the worker. It is to be noted too that if the objective is to be used without a projection ocular the tube-length is practi- cally extended to the focusing screen and as the effect of lengthening the tube is the same as thickening the cover-glass; the adjusting collar must be turned to a higher number than the actual thickness of the cover calls for (see § 113). § 312. Photographing Without an Ocular. — Proceed ex- actly as described for the lower power, but if the objective is ad- justable make the proper adjustment for the increased tube-length (§ii3-) § 313. Photographing with a Projection Ocular. — Proceed 236 PHOTO-MICROGRAPHY [CH. VIII as described in § 307 B, only in this case the objective is not to be adjusted for the extra length of bellows. If it is corrected for the ordinary ocular, the projection ocular then projects this correct image upon the focusing screen. § 314. Photo-Micrographs at a Magnification of 500 to 2000 Diameters. — For this the homogeneous immersion objective is employed, and as it requires a long bellows to get the higher magnification with the objective alone, it is best to use the pro- jection oculars. For this work the directions given in § 307 B must be followed with great exactness. The edge of the petroleum lamp flame is suf- ficient to fill the field in most cases. With many objects the time required with good lamp light is not excessive ; viz. , 40 seconds to 3 minutes. The reason of this is that while the illumination diminishes directly as the square of the magnification, it increases with the increase in the numerical aperture, so that the illuminating power of the homogenous immersion is great in spite of the great magnification (§ 40). For work with high powers a stronger light than the petroleum lamp is employed by those doing considerable photo-micrography. Good work may be done, however, with the petroleum lamp. It may be well to recall the statement made in the beginning, that the specimen to be photographed must be of special excellence for all powers. No one will doubt the truth of the statement who undertakes to make photo-micrographs at a magnification of 500 to 2000 diameters. If one has a complete outfit with electric arc light the time re- quired for photographing objects is much reduced, i. e. ranging from 1 to 20 seconds even with the color screen. As the light is so in- tense with the arc light it is necessary to soften it greatly for focus- ing. Several thicknesses of ground glass placed between the lamp and the microscope will answer. These are removed before taking the negative. It is well also to have a water bath on the optical bench to absorb the heat rays. This should be in position constant- ly (see Fig. 133, 160). §315. Use of Oculars in Photo- Micrography. — There is much diversity of opinion whether or not the ordinary oculars used CH. fill] PHO TO-MICROGRAPH Y 237 for observation should be used in photographing. Excellent results have been obtained with them and also without them. Fig. 1S3. Zeiss' Apochromatic Projection Objective oj yo mm. equivalent focus, for photo-micrography. ( Zeiss' Catalog. ) is used for visual observation. Fig. 184. Gordon's Photo-Micrographic Apparatus. — In this apparatus there is placed over the ocular of the microscope a tube contain- ing a projection lens which focuses the image on the sensitive plate just as the eye focuses the image on the retina. A. The lube bearing the plate at the top. It is about 150 mm. long . B-C. Photographic plate about 40 mm. square, contained in a cap (C) on top of the tube D. Shutter for making the exposure; F. A flange tilting the draw-tube and supporting the camera (A); G. The microscope with a metal block which may be clamped in position to prevent the descent of the body of the microscope during the exposure; E. A focusing ocular of high power placed on the tube of the microscope to ensure a perfect focus. If one has perfectly normal eyes the focus with the ordinary ocular gives a sharp image. With this apparatus the only change needed in the -microscope is the addition of the camera {A ) and the clamping of the metal block (G). Then the exposure may be made. The use of a color screen and properly sensitized plates apply here as with any apparatus. " One of the chief advantages of this extremely simple method of photomicrography is that the performance of the microscope is exactly the same as -when it ' four. Roy. Micr. Soc, 1905, p. 651. 2 3 8 PHOTO-MICROGRAPHS \_CH VIII For great magnification Zeiss recommends the use of the com- pensation oculars with the apochromatics. The Zeiss projection oculars may be used with achromatic ob- jectives of large aperture as well as with the apochromatics. Name NEGATIVE RECORD No. Location Camera Objective Ocular Condenser Diaphragm Object Stained with. Color Screen Plate Light and Hour Date Exposure . Developer . Fixer Mag. X __- Remarks - . PHOTOGRAPHING OPAQUE OBJECTS AND METALLIC SURFACES WITH A MICROSCOPE All of the objects considered in the first part of this chapter are opaque and some of them were to be photographed somewhat larger than natural size. To meet the needs of modern work, especially with metals and alloys one must be able to examine and photograph prepared surfaces at magnifications ranging from five or ten to five hundred or more diameters. \ 317. Microscope for Opaque Objects. — If one does not need to magnify more than about 100 diameters, any good microscope will answer. For the higher powers it is far more convenient to employ a special microscope for metallography (micro-metalloscope.) (German, Metallmikroskop; French, Microscope pour l'dtude des surfaces m€talliques et des objets opaque). CH. Villi Pin > TO-MICROGRAPH i ' 239 Such a microscope has the following general characters: The stage is movable up and down with rack and pinion, it is rotary and more or less mechanical by means of centering screws. With some at least the stage may be removed entirely. No substage condenser is present, and a mirror is onlv necessary for occasional transparent objects. A revolving nose-piece is not so good as an objective changer. See Fig. 176. Fig. 185 Fig. 186 Fig. 185. Leilz' Vertical Illuminator. (From Lei/.:' Catalog.) Fig. 186. Zeiss' Vertical Illuminator. (From Zeiss' Catalog.) I 31S. Illumination of Opaque Objects.— (A) for 25 to 100 diameters. The directions of Mr. \Valmsle3' are excellent (Trans. Amer. Micr. Soc, 1898, p. 191). "Altogether the best light for the purpose is diffused daylight. Proper lighting is more easily obtained with a vertical camera. An even illum- ination avoiding deep shadows is preferable in most cases and is more easily attained with the object in a horizontal position. For many objects it is better not to use a bull's eye or any form of condenser but for others the condenser may be needed, but when the condenser is used one must avoid too much glare. The now little used parabolic reflector and Lieberkubn serve well in man} 7 cases, but he adds " the majority yield better results under the most simple forms of illuminanion," i. e., with the diffused light from the window. This has been the experience of the writer also. In case diffused daylight is employed the camera should be near a good sized window, and the object should be somewhat below the window ledge so that the illumination is partly from above and from the side. (This is easily attained with the small table and vertical camera shown in Figs. 165, 170, 171). The vertical illuminator is advantageous for these powers also. See (B. ). (B) For 100 to 500 diameters, — For the magnification above 50 it is 240 PHOTO-MICROGRAPHY [CH. VIII desirable and for those above ioo it is necessary to use some form of ' ' vertical illuminator," that is some arrangement by which the light is reflected down through the objective upon the object, the objective acting as a condenser, and from the object back through the objective and ocular to the eye of the observer. This is accomplished in two ways: (i) By means of a small speculum-metal mirror in the tube of the micro- scope. This is set at an angle of 45 degrees and the light thrown into the tube upon it is reflected straight down through the objective upon the object. The speculum metal being opaque cuts out a part of the light. Instead of a metal mirror a circular disc of glass is now more frequently used. This allows the major part of the light reflected from the object to pass up through the objective, to reach the eye. (2) By means of a small glass 45 degree prism inserted into the side of the objective or of a special adapter. The light is from the side of the micro- scope, and is reflected by the prism straight down through the objective upon the object as before.* See Figs. 185-186. § 319. Light for the Vertical Illuminator. — For moderate powers one may place the microscope in front of a window, or one may use a petroleum or gas lamp. For the higher powers acetylene or preferably the electric arc light is used. In either case it may be necessary to soften the light somewhat either by a color screen or by some ground glass. The light should be concentrated upon the exposed end of the prism or into the hole leading to the glass disc. Both the prism and the disc should be adjustable for different objec- tives and different specimens. ■ The cone of light, especially with the electric arc lamp, should be enclosed in a hollow metal or asbes- tos cone, to avoid the glare in the eyes of the operator, and it may *The idea of the vertical illuminator apparently originated with Hamilton L. Smith. He used the metal reflector. Beck substituted a cover-glass and Powell and Lealand a disc of worked glass; i. e. glass that had been carefully polished and leveled on the two sides. Carpenter-Dallinger, pp. 336-338. The use of the prism with the objective is due to Tolles (See Jour. Roy. Micr. Soc, vol. iii, 1880, pp. 526, 574). In Zeiss' catalog the prism form is figured. In the catalog of Nachet both the glass disc and the prism forms are figured. For both these devices uncovered objects are most successful or if the object is covered it must be in optical contact with the cover-glass. Naturally good reflecting surfaces like the rulings on polished metal bars give most satisfactory images, hence this method of illumination is especially adapted to micro- metallography. Indeed, without some such adequate method of illumination the study of metals and alloys with high powers would be impossible. So suc- cessful is it that oil immersion objectives may be used. (Carpenter-Dallinger, PP- 335-338). CH. 1 7/7] PHOTO-MICROGRAPHY ■ 241 be uecessary to soften the light with ground glass before attempt- ing to focus and arrange the specimen. This ground glass would in most cases be removed before making the exposure (§ 314.) With the electric light and for long exposure or observation, a water bath to absorb the heat rays is necessary to avoid injuring the lenses. As it is somewhat difficult to adjust the light in a way to give the best effect, one can see the advantage of the adjustment for raising and lowering the stage. This serves for all but the finest focusing, and thus avoids moving the focusing tube enough to throw the lighting out of adjustment. It might be advantageous to have a fine adjustment on the stage also. I 320. Mounting of Objects. — For observation only and with low powers, objects may be mounted either in a liquid or dry as seems best. There should be a black background for most objects, then light will reach the eye only from the object. A light background is sometimes desirable, especially where one cares only for outlines. I 321. Preparation of Metallic Surfaces. — In the first place a flat face is obtained by grinding or filing, and then this is polished. For polishing, finer' and finer emery or other polishing powders are used, (rouge or diamantine, or specially prepared alumina, etc). The aim is to get rid of scratches so that the surface is smooth and free from lines. I 322. Etching. After the surface is polished it should be etched with some substance. This etching material corrodes the less resistant material, the edges of crystals, etc., so that the structure appears clearly. For etching,, tincture of iodine, nitric acid in various degrees of strength, hydrochloric acid, etc., are used or one may use electricity, the metal being immersed in an indif- ferent liquid. See numerous articles in the Metallographist for methods and micrographs. After etching, the surface should be washed well with water to remove the etcher. Le Chatelier recommends that the etched surface when dry be covered with a very thin coating of collodion to avoid tarnishing. The preparation will then last for several months untarnished. I 323. Mounting Preparations of Metal. — In order to get a satisfactory image the flat, polished and etched face should be at right angles to the optic axis. For preliminary observation one can approximate this by mounting the specimen on a piece of bees-wax. (Behrens). Very elaborate arrangements of the stage have also been devised (Reichert). \ 324. Photographing Opaque Objects.— -The general directions given in I 282 should be followed with the necessary modifications. The time of exposure is usually considerably greater with opaque objects than with trans- parent ones. Very few such objects can be photographed in less than 30 242 PHOTO-MICROGRAPHY \_CH. VIII seconds, even with daylight. For metallic surfaces and magnifications of ioo, 150, 250 to 500, with the electric arc light as illuminant the time required for favorable objects is 1, 2, 4 and 7 seconds; with the Wellsbach lamp the time is 5, 10, 30 and 60 minutes (Sauveur). ENLARGEMENTS ; LANTERN SLIDES ; PHOTOGRAPHING BACTERIAL CULTURES \ 325. Enlargements. As a low power objective has greater depth of focus or penetration than a higher power ($ 40), it is desirable in many cases to make a negative of an object with considerable depth at a low magnifica- tion, and then to enlarge this picture to the desired size. As a rule negatives will not bear an enlargement of more than five diameters. For this work the camera shown in Fig. 169 is excellent, and the special microscope stand shown in this figure and in Fig. 165 enables one to get an exact focus. One must select an objective for the enlargement with a field of sufficient size to cover the part of the negative to be enlarged. An objective of 60 to 100 mm. focus will answer in most cases. For the illumination the camera can be elevated against the sky, or artifi- cial light may be used. It is not easy to light so large a surface evenly by artificial light. (A) Enlargement on Bromide Paper. — For this' the negative is put in place and by pulling out the bellows the proper amount, one gets the right magnification. Focus now as for any other object, using the fine adjustment and focusing glass. For great exactness one must put a clear glass in the plate holder and focus on the surface away from the objective. Then place the bromide paper on this clear glass and put another over it to hold it flat against the first plate of glass. The sensitive surface will then be in the exact plane of the focus and the picture will be sharp. For the development and subsequent treatment of the paper, follow the directions of the makers. (B) Enlargement on a Glass Plate.— One may proceed in enlarging as for making lantern slides and make a positive on a glass plate. If it is then desired to get a negative for printing, place this positive on the microscope stand and make a negative from it as if it were an object. Or one may make a contact impression as is frequently done in lantern slide making. By this method one must make three separate pictures, (J) the original photo-micro- graphic negative; (2) the enlarged positive from this; (3) a negative from the enlarged positive. With this negative one may print as from the original negative. (i 326. Lantern Slides from Negatives. — In preparing lantern slides from photo-micrographic or ordinary negatives one may use the contact method, or the camera. With the camera one can enlarge or reduce to suit the particular case. The camera and special microscope stand shown in Fig. 169 are admir- CH. Vllf] PHOTO-MICROGRAPHY 243 able for the purpose. For lantern slide work a photographic objective is used and the cone for enlargement removed. One may put the objective in the front of the camera or in the middle segment, making use of the little side door. I 327. Photographing Bacterial Cultures in Petri Dishes.— For the suc- cessful photographing of these cultures dark ground illumination is employed on the principle stated in \ 103. That is the preparation is illuminated with rays so oblique that none can enter the objective. These striking the culture are reflected into the objective. The clear gelatin around the growth or col- onies does not reflect the light and therefore the space between the colonies is dark. For supporting the Petri dishes a hole is made in a front board for the camera. This hole is slightly larger than the dish. Over it is then screwed or nailed a rubber ring slightly smaller than the Petri dish. This will stretch and receive the dish, and grasp it firmly so that it is in no danger of falling out when put in a vertical position. If the camera has two divisions like the one shown the board with the Petri dish is put in the front of the camera, and the objective in the middle division through the side door. Otherwise the board holding the Petri dish must be on a separate support. The illumination is accomplished by the use of two electric lamps with ■conical shades. (The cheap tin shades with white enamel paint 011 the inside are good). The lamps are placed at the sides so that a bright light is thrown on the culture, but at such an angle that none of it enters the objective directly. A piece of black velveteen is placed 10 to 20 cm. beyond the culture. This prevents any light from being reflected through the clear gelatin to the objec- tive. Unless some such precaution were taken the background would be gray instead of black. One may use daylight by putting the culture in a support just outside a window, leaving the camera in the room. The rays from the sky are so oblique that they do not enter the objective. 0:ie must use a black non-re- flecting background some distance beyond the dish as in using artificial light (Atkinson). I 328. Photographing Bacterial Cultures in Test-Tubes. — Here the lighting is as in the preceding section, but a great difficulty is found in getting good results from the refraction and reflections of the curved surfaces. To overcome this one applies the principles discussed in § 157, and the test-tubes are immersed in a bath of water or water and glycerin. The bath must have plane surfaces. Behind it is the black velvet screen, and the light is in front as for the Petri dishes. As suggested by Spitta it is well to employ a bath sufficiently thick in order that streak cultures may be arranged so that the slop- ing surface will all be in focus at once by inclining the test-tube. REFERENCES FOR CHAPTER VIII See the works and journals dealing with photography. For Photo-Micrography see Pringle, Bousfield, Neuhauss, 3rd ed. Stern- 244 PHO T( ^MICROGRAPH ) ' [CH. VIII berg, Francotte, Spitla and the special catalogs on photo-micrography and projection issued.'by the great opticians. The Journal of the Royal Micro- scopical Society and of the Quekett Micr. Club; Zeit. wiss. Mikroskopie; the Trans. Amer. Micr. Soc,; the Amer. Monthly Micr. Journal; the Journal of Applied Microscopy. For the photography of metallic surfaces, see the various journals of engineering and; metallurgy, but especially Sauveur's journal, the Metallo- graphist, begun in 1898; Jour. Roy. Micr. Soc. See the works on; photo-micrography and photography for the details of lantern slide making. See for the Petri dishes and test-tubes, Atkinson, Botanical Gazette, xviii (1893), p. 333; Spitta, Photo-Micrography (1899), P- 2 6. For photography with ultra-violet light see Zeiss special catalogs. Jour- nal of the Royal Microscopical Society, Zeitschrift fur wiss. Mikroskopie; Dr. August Kohler, Zeit. wiss. Mikr. Bd. xxi, 1904, pp., 129-165, 273-304; six plates; Band 24, 1907, pp. 360-366. Dr. H. C. Ernst of the Harvard Medical .School; Jour. Med. Research N. S. Vol. 9, 1905-6 pp. 463-468, plates. Ptrman.. Various Spectra.— These spectra illustrate some of the points in the dis- cussion of color screens (§ 291). The Solar spectrum shows that all the wave lengths of light are present except for the very narrow dark lines (Fraunhofer lines, \ 214). The Sodium spectrum is an example of the spectrum of an incandescent gas ; it is also an extreme example of monochromatic light. Sodium light is very brilliant, but the appearance of surrounding objects gives one a good idea of the changed appearance which the universe would assume if illuminated by monochromatic light. The spectra of permanganate and methemoglobin illustrate well the ab- sorption spectra of colored substances. If one were to use permanganate for a color screen the object photograph- ing most successfully would be one transmitting light in the E region of the spectrum. Methemoglobin would answer well as a color screen for an ^object trans- mitting light at the violet end of the spectrum and between the lines DE. CHAPTER IX SLIDES AND COVER-GLASSES; MOUNTING; ISOLATION; LABELING AND STORING MICROSCOPIC PREPARATIONS; REAGENTS SLIDES AND COVER-GLASSES ? 329. 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 ■ 76 millimeters (1 3 inches). For rock sections, slides 25 X 45 mm. or 32 X 32 mm. are used; for serial sections, slides 25 76 mm., 50 - 76 mm. or 38 > 76 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. Fig. 187. Glass slide or slip of the ordinary size for microscopic work (3 xi in., 16 x 25 mm. ). (Cut loaned by the Spencer Lens Company). Thick slides are preferred by many to thin ones. For micro-chemical work Dr. Chatnot recommends slides of half the length of those used in ordi- nary 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 excel- lent for ordinary microscopic work should be easily attacked by all liquids; even water extracts a relatively enormous amount of alkalies and alkaline 246 SLIDES AND COVER-CLASSES [C/L IX earths. The slips of greenish glass, while not as neat or desirable for general microscopy, seem to be decidedly more resistant, and are therefore preperable. ' Transparent celluloid slides are recommended by Behrens for work where hy- drofluoric acid and its derivaties are to be examined. (Chamot, Jour, Appl. Micr. vol. iii, p. 793). § 330. Cleaning Slides for Ordinary Use. — Place new slides that are to be wiped at one sitting in a glass vessel of distilled water containing 5 f, „ ammonia (Fig. 188-189). For wiping the slides use a so-called glass towel or other well washed linen towel. One may avoid large wash bills by using absorbent gauze.* In handling the slides grasp them by the edges. Cover the fingers of the right hand with the wiping towel or the gauze and rub both faces with it. When wiped thoroughly dry, place the slide in a dry glass jar like that shown iu Fig. 189, or for larger numbers use a museum jar (Fig. 190). Soap and water are also recommended for new slides. \ 331. Cleaning Used Slides. — If only watery substances or glycerin or glycerin jell} 7 have been used one may soak the slides over night in ammonia water, then changing the water for fresh and wiping as described in \ 330. When balsam or other resinous media (§ 353) have been used it is best to lift, >. Fig. 188. Round glass aquarium jar suited for an aquarium, for cleaning slides or for any other purpose where a wide open glass dish is needed. Fig. 189. Covered glass dish known as an " ointment jar " of the right height to hold slides on end. (Cuts 146, 147 loaned by the Whitall Taium Co.). * The gauze mentioned is No. 10, "Sterilized absorbent gauze", of the Griswoldville Mf'g Co. of N.Y. It is sometimes called bleached cheese cloth. In the author's laboratory it is cut into pieces, ){, \i, ^ of a yard. When a piece is soiled it is thrown away. CH. AY] SLIDES AND COVER-GLASSES 247 heat the slides over a Bunsen flame and remove the cover-glass. Place the cover in cleaning mixture (§339). The slide may also be placed in cleaning mixture or in some hot water containing 10% gold dust or other strong alka- line cleaner. When the metal basin— preferably an agate ware basin— is two thirds full of the slides, heat until the water comes to a boil. Then let it cool. Add fresh water and most of the slides may be wiped clean. Fig. 190. Museum jar with clamp top for storing cleaned slides and for preserving specimens. [Cut loaned by the Whitall Ta- tum Co.) If dichromate cleaning mixture is used the best method is to have a museum jar of it and drop the slides in as they are rejected, or a large number at once as is most convenient. It may require a week or more to clean the slides with cleaning mixture. As this is a very corrosive mixture for metals use only glass dishes in dipping into it. When the slides are freed from balsam etc. pour off the cleaning mixture into another glass vessel and allow a stream of water to flow over the slides until all the cleaning mixture has been washed away. Then add distilled water and wipe the slides from that. An}' slides still not freed from the balsam should be put back into the cleaning mixture. Apparently the slides are not injured by a prolonged stay in the mixture. \ 332. Cleaning Slides for Special Uses. — In making blood films, for micro-chemistry and whenever an even film is desired every particle of oily substance must be removed. The slides should be placed in the dichromate cleaning mixture (J 329) one day or more, thoroughly washed with clean water and then in distilled water, or in 50% to 75% alcohol. The}' are taken from the water or alcohol and wiped dry as needed. In wiping keep two or more layers of the absorbent gauze over the fingers. Only one slide is wiped with each piece of gauze. The surface to touch the slides should never have 248 SLIDES AND COVER-CLASSES [CH. IX been touched by the hands for a minute amount of oily substance leaves a stratum on the slide which causes the liquids used to heap up instead of flow- ing out perfectly flat. That is, the slide is wet with difficulty and the liquid instead of forming a film tends to assume the spheroidal state. Sometimes new gauze or other cloth used may not be wholly free from oily substance, or the soap was not wholly eliminated in washing. Such wiping cloths will not make the slides ready for good films. Some workers soak the gauze in sulfuric ■ether to remove the last traces of oily substance. This is done more especially in cleaning cover-glasses for films, see below. Burnett, p. 22, in speaking of blood smears says : "The slides should be thoroughly clean. Unused slides may be cleaned in strong soap or "gold dust" solution, well rinsed in water, then placed in alcohol from which they are wiped and polished." I 333. Cover-Glasses or Covering Glasses. — These are circular or quad- rangular pieces of thin glass used for covering and protecting microscopic ■objects. They should be very thin, o.ioto 0.25 millimeter (see table, \ 32-34). It is better never to use a cover-glass over 0.20 mm. thick, then the prepara- tion may be studied with a 2 mm. oil immersion as well as with lower objec- tives. Except for' objects wholly unsuited for high powers, it is a great mis- take to use cover-glasses thicker than the working distance of a homogeneous objective (§ 69). Indeed, if one wishes to employ high powers, the thicker the section the thinner should be the cover-glass (see. \ 337). The cover-glass should always be considerably larger than the object over which it is placed. Figs. 191-192. Figures of square and of circular cover-glasses. (Cuts loaned by the Spencer Lens Co. ) \ 334. Cleaning Cover-Glasses for Ordinary Use. — Covers may be cleaned -well by placing them in 82% or 95%' alcohol containing hydrochloric acid one per cent. They may be wiped almost immediately. Remove a cover from the alcohol, grasping by the edge with the left thumb and index. Cover the right thumb and index with some clean gauze ■or other absorbent cloth; grasp the cover between the thumb and index and rub the surfaces keeping 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 dry 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 clean 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. CH. IX] SLIDES AXD COVER-GLASSES 249 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. 335. Cleaning Cover-Glasses for Special Uses. — As with slides, covers intended for films or other purposes where the last particles of oily substance must be removed, are best put one by one into dichromate cleaning mixture (J 339). After a day or more this is poured off and a stream of fresh water allowed to run on the covers until all the cleaning mixture is removed. Then distilled water is added and allowed to stand a few minutes. This is poured off and S 2 ' ', , orgs 11 ,, alcohol added. The covers remain in this until needed. In wiping use the precautions given with slides (jj 332). Figs. 193-194. Glass box and Petri dish for clean cover-glasses. [Cuts loaned by the Whitall Tatum Co.). \ 336. Cleaning Large Cover-Glasses. — For serial sections and especially large sections, large quadrangular covers are used. These are to be put one by one into 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 desirable that these arge covers should be thin — not over 0.15-0.20 mm. otherwise high objectives cannot be used in studying the preparations. Fig. 193. Micrometer Calipers {Brown and Sharpe). Pocket Calipers, graduated in inches or millimeters, and well adapted for measuring cover- glasses. \ 337. Measuring the Thickness of Cover-Glasses. — It is of great advan- tage to know the exact thickness of the cover-glass on an object; for, (a) in studying the preparation one would not try to use objectives of a shorter work- ing distance than the thickness of the cover ( \ 69); (b ) In using adjustable objectives with the collar graduated for different thicknesses of cover, the 250 SLIDES AND COVER-GLASSES [C/f. IX collar can 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, \ 33). Furthermore, if there is a variation from the standard, one may remedy it, in part at least, by lengthen- ing the tube if the cover is thinner, and shortening it if the cover is thicker than the standard (}, 113). Among 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. It is then strongly recommended that every preparation shall be covered with a cover-glass whose thickness is known, and that this thickness be indi- cated in some way on the preparation. \ 33S. Cover-Glass Measures, Testers or Gauges. — For the purpose of measuring cover-glasses there are two very excellent pieces of apparatus. The micrometer calipers (Fig. 195) used chief!}' in the mechanic arts, are con- venient and from their size are easily carried in the pocket. The cover-glass measurer specially designed for the purpose is shown in Fig. 196 by which covers may be more rapidly measured than with the calipers. Fig. 196. Zeiss' Cover-Glass Measurer. Willi this the knife edge jaws are opened by means of a lever and the cover j use rled. The thickness may then he read off on the face as the pointer indicates the thick- ness in hundredths millimeter iu the outer circle and in Ihous- ™|1P andths inch on the inner circle- With 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 glass boxes and properly labeled. Unless one is striving for the most accurate possible results, cover-glasses not varying more than 0.06 mm. maybe 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 contain covers of o. 12, 0.13, 0.14, O.15, 0.16, o 17 and 0.1S mm. I 339- Dichromate Cleaning Mixture for Glass.— The cleaning mixture used for cleaning slides and cover-glasses is that commonly used in chemical laboratories : (Dr. G. C. Caldwell's Laboratory Guide in Chemistry). CH. AY] MOUNTING PREPARATIONS 251 Dichromate of potash (K,Cr,0 7 ) - 200 grams Water, distilled or ordinary - 800 cc. Sulphuric acid (H 2 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 (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 breakage from the great heat developed upon the addition of the sul- phuric 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. It is not necessary to employ chemically pure materials. This is an excellent cleaning mixture and is practically odorless. It is exceedingly corrosive and must be kept in glass vessels. It may be used more than once, but when the color changes markedly from that seen in the fresh mixture it should be thrown away. An indefinite sojourn of the slides and covers in the cleaner does not seem to injure them. . MOUNTING, AND PERMANENT PREPARATION OF MICROSCOPIC OBJECTS \ 340. Mounting a Microscopic Object is so arranging it upon some suitable 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 it is false economy to crowd them too closely together. \ 341. 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 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. Some- times it is not easy or convenient to get the natural medium, then some liquid that has been found to serve in place of the natural medium is used. For many things, water with a little common salt (water 100 cc, common salt T % gram ) is employed. 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 on with fine forceps, as shown in Fig. 197. After the 25 2 MOUNTING PREPARATIONS \CH. IX 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. 210). pi n£ JorctJiK Fig. 197. To show the method of putting a cover- glass upon a microscopic preparation. The cover is grasped by one edge, the opposite e dg e is then brought down to the slide, and the cover gradually lowered upon the object. \ 342. Permanent Mounting. — There are three great methods of making permanent microscopic preparations. 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 experiment. 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 (§ 343); (B) In some medium miscible with water, as glycerin or glycerin jelly ($ 348) ; (C) In some resinous medium like Canada Balsam (g 353). § 343. 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. \ 344. Order of Procedure in Mounting Objects Dry or in Air. 1. A cell of some kind is prepared. It should be slightly deeper than the object is thick (§ 346). 2. The object is thoroughly dried (desiccated) either in dry air or by the aid of gentle heat. 3. If practicable the object is mounted on the cover-glass; if not it is placed in the bottom of the cell. 4. The slide is warmed till the cement forming the cell wall is somewhat sticky, or a very thin coat of fresh cement is added ; the cover is warmed and put on the cell and pressed down all around till a shining ring indicates its adherence (g 347). 5. The cover-glass is sealed. 6. The slide is labeled. 7. The preparation is cataloged and safely stored. \ 345. Example of Mounting Dry, or in Air. — Prepare a shallow cell and dry it (g 346). Select a clean cover-glass slightly larger than the cell. Pour upon the cover a drop of io° * solution of salycilic acid in 95% alcohol. Let it dry spontaneously. Warm the slide till the cement ring or cell is some- CH. IX] MO UN TlNi ; PR EPA RATI I WS 2 53 what sticky, then warm the cover gently and put it on the cell, crystals down. Press on the cover all around the edge (J 347) seal, label and catalog. 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 (\ 232). Fig. 19S. Turn-Table for sealing cover-glasses and making shallow mounting cells. ( Cut loaned by the Bausch & Tomb Opt. Co. ). ''/, 346. Preparation of Mounting Cells. — (A) Thin 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. 198) 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 brnsh lightly on the slide just over the guide ring selected. An even ring of cement should result. If it is uneven, the cement is too thick or too thin, or too much was on the brush. After a ring is thus prepared remove the slide and allow the cement to dry spontane- ously, or heat the slide in some way. Before the slide is used for mounting, the cement should be so dry when it is cold that it does not dent when the finger nail is applied to it. A cell of considerable depth may be made with the shellac by adding successive layers as the previous one dries. (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. 254 MOUNTING PREPARATIONS [CIT. IX \ 347. 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 intimate contact. In doing this the the convex part of the fine forceps or some other blunt, smooth object; it is also necessary to avoid pressing on the cover except immediately over the wall of the cell for fear of breaking the cover. When the cover is in contact with the wall of cement all around, the slide should be placed on the turn-table and carefully 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. 210). If the cover-glass is not in contact with the cell wall at any point and the cell is shallow, there will be great danger of the fresh cement running into the cell and injuring or spoil- ing the preparation. When the cover-glass is properly sealed, the prepara- tion is put in a safe place for the drying of the cement. It is advisable to add a fresh coat of cement occasionally. Fig. 199. Centering Card. A card with stops for the slide and circles in the position occupied by the center of the slide. If the slide is put upon such a card it is easy to arrange the object so that it will be approximately in the center of the slide. The position of the long cover used for serial sections is also shown. (From the Microscope, December, 1886). I 348. 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. CH. 1X~\ MOUNTING IN GL YCERIN 255 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. \ 349. Order of Procedure in Mounting Objects in Glycerin. 1. A cell must be prepared on the slide if the object is of considerable thickness (§ 346). 2. A suitably prepared object 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. 199). 3. A drop of pure glycerin is poured upon the object, or if a cell is used, enough to fill the cell and a little more. 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. 197). The cover is then gently pressed down. If a cell is used, a fresh coat of cement is added before mounting. -•(~X3 Fig. 200. Slide and cover-glass showing method of anchoring a cover-glass with a glycerin prepara- tion when no cell is used. A cover-glass so anchored is not liable to move when the cover is being sealed (« 35')- Fig. 201. Glass slide with cover-glass, a drop of reagent and a bit of absorbent paper to show method of irrigation. 5. The cover-glass is sealed. 6. The slide is labeled. 7. The preparation is cataloged and safely stored. g 350. Order of Procedure in Mounting Objects in Glycerin Jelly. 1. Unless the object is quite thick no cell is necessary with glycerin jelly. 2. A slide is gently warmed and placed on the centering card (Fig. 199) and a drop of warmed glycerin jelly is put on its center. The suitably pre- pared 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. 197) 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 {\ 347). 256 MOUNTING IN GL ) CERIN JELL Y \CH. IX 6. The slide is labeled. 7. The preparation is cataloged and safely stored. (J 351.- Sealing the Cover- Glass when no Cell is used. — (A) For glycerin mounted specimens. The superfluous glycerin is wiped away as carefully as possible with a moist cloth, then four minute drops of cement are placed at the edge of the cover (Fig. 200), and allowed to harden for half an hour or more. These will anchor the cover-glass, then the preparation may be put on the turn-table and ringed with cement while whirling the turn-table. c Fig. 202. A — Simple form of moist chamber made with a plate and bowl. B, bowl serving as a bell jar; P, plate containing the water and over which the bowl is inverted ; S, slides on which are mounted preparations which are to be kept moist. These slides are seen endwise and rest upon a bench made by cementing short pieces of large glass tubing to a strip of glass of the desired length and width. B — Two cover-glasses (C) made eccentric, so that they may be more easily separated by grasping the projecting edge. C — Slide (S) with projecting cover-glass (C). The projection of the cover enables one to grasp and raise it without danger of moving it on the slide and thus folding the substance tinder 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 a resinous medium is used. Then the slide is put on a turn-table and a ring of the shellac cement added. (C) Balsam preparations may be sealed with shellac as soon as they are pre- pared, but it is better to allow them to dry for a few days. One should never use a cement for sealing preparations in balsam or other resinous media if the solvent of the cement is also a solvent of the balsam, etc. Otherwise the cement will soften the balsam and finally run in and mix with it, and partly or wholly ruin the preparation. Shellac is an excellent cement for sealing balsam preparations, as it never runs in. Balsam preparations are rarely sealed. CH. IX ] MOL 'NTING IN BALSAM 257 '{ 352. Example of Mounting in Glycerin Jelly. — For this select some stained and isolated muscular fibres or other suitably prepared objects. (See under isolation \ 357). Arrange them on the middle of a slide, using the cen- tering card, and mount in glycerin jelly as directed in \ 350. Air bubbles are not easily removed from glycerin jelly preparations, so care should be taken to avoid them. \ 353. Mounting Objects in Resinous Media. — While the media tnisci- ble with water offer many advantages for mounting animal and vegetable tis- sues the preparations so made are liable to deteriorate. In many cases, also, thej' do not produce sufficient transparency to enable one to use high enough powers for the demonstration of minute details. By using sufficient care almost any tissue may be mounted in a 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 resi- nous mounting medium. There are two methods of bringing this about : (A) By drying or desiccation (e above a groove in the floor of the com- partment, ib) One end of the slide is seen to be uplifted by depressing the other into the bevel. Fig. 211. Cabinet for Microscopic Sped in e n s , showing the method of ar- rangement and of number- ing the drawers and indi- cating the number of the first ami /ail compartment in each drawer. It is bet- ter to have the slides an which the drawers rest somewhat shorter, then the drawer front may be entire and not notched as here shown. (From I'roc. Amer. Micr. Soc, 1883. ) 266 CABINETS AND TRAYS FOR PREPARATIONS \_CH. IX (5). On the front of each drawer should be the number of the drawer in Roman numerals, and the number of the first and last compartment in the drawer in Arabic numerals (Fig. 211). H A Q Fig. 212. Trays 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 may be easily pulled out of a pile or reinserted. The screw eye shown in A makes it 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) Sectional view of the -whole tray, 1 C) Sectional view of one side (natural size) to show the construction more clearly. These trays are about 30 x 44 centimeters (ri 3-4.1- 17 r-4 in.), and hold 50 1x3 in. slides, i. e., 5 rows 10 in a row. Trays of this kind are so cheap ($17.50 per hundred for those holding 50 to 60 slides), that a laboratory can have all that a re needed. (Trans. Amer. Micr. Soc, 1899, p. 107.) I 371. Trays for Slides and Ribbons of Sections. — Early in 1S97 the writer devised the simple tray shown in Fig. 212. It was designed especially for the ribbons of sections in preparing embryologic series and for material for class work. As will be seen by the figure the two sides are alike and the tray is very shallow. It was soon found that the wood forming the bottom of the tray was too rough for ribbons of sections and smooth white paper was put in the tray before the ribbons were laid upon it. These trays were soon used for the mounted preparations as well as for the ribbons of sections. They were made of a proper size to fit the laboratory lockers (Fig. 214); and naturally came to be used for storage instead of the expensive slide cabinets shown in Figs. 210-21 1. For this purpose five could be put in a single compartment of the locker or 35 in an entire locker. As each tray holds fifty slides 1 x 3 in; 37, 1 } 2 x 3 and 25 slides 2x3 in.,the sav- ing of space was very great. CH. IX} CABINETS AND TRAYS FOR PREPARATIONS 267 \ 372. Slide Trays with Tongue and Groove. — In the first trays the edges were square and sharp. These were rounded in later trays, but there still re- mained a defect, for if one wished to pile up five to twenty trays on the table, they would not stay in an even stack. To remedy this defect the long way of the frame was tongued on one side and grooved on the other as shown in Fig. 213. This is a great improvement as one can make even stacks of 25 or 50 trays, and they will stay in position. Furthermore it renders the groups of 5 trays stored in the locker compartments much easier to manage, as one can re- move any of the five trays without getting the others disarranged as so often occured with the old form, lacking tongue and groove. W =*] Fig. 213. Slide Tray with Cross Pieces on one Face to retain the Slides in Rows. {Dr. Greenman's improvement.) A tongue and groove serve to hold the trays in position when they are piled up. {A. about i-S, and C. about natural size. ) The corners of the tray frame are held in place by the corru- gated pieces of iron used in the construction of picture frames. \ 373. Slide Trays with One Side Divided. — A defect of the trays for storage is the ease with which the slides get disarranged unless the tray is en- tirely full. To avoid this defect Dr. M. J. Greenman of the Wistar Institute divides one face into rows of the righf width for receiving the slides. Then while the slides in any single row might get displaced those of neighboring rows cannot become mixed- (Fig. 213 A. ). One side of this tray is smooth and can be used for ribbons of sections like the original tray. Dr. Greenman stores the trays in metal cabinets, each tray having a separate pair of " runs" as is shown in Fig. 2ii. The author of this book adds the cross pieces to divide the tray into rows and also has the frame grooved and tongued (fi 372). Thus 268 PREPARATION OF REAGENTS [CH. IX constructed the tray is very reasonable in price and most useful for the needs of a modern biologic laboratory.* ooo :x30 ooo ©oo, 3 INCH HOLE5. . OOOO oooo OOOO oooo §000 gooo 1, 2 8r 2i INCH HOLES. SUDES rOR« , ' ' NCH H0LE5 ' REAGENT BOARDS PLANS AMD DRAWERS. or REAGENT BOARD5 REAGENT BOARDS AMD DRAWERS ARE INTERCHANGEABLE THROUGHOUT. ELCVATIOM. V L0CKCR5 IM LABORATORIES. Fig. 214. Student Locker with trays and reagent boards. {Jour. Apt. Mia: 1898, p. 127. ) PREPARATION OF REAGENTS I 374. General on Preparation of Reagents. — In preparing reagents both weights and measures are used. As a rule the amounts given are those which experience has -shown to give good results. Variations in the proportions of the mixtures are sometimes advantageous, and in almost every case a slight change in the proportions makes no difference. Most laboratory reagents are *In Ithaca, these trays are made and furnished by the H. J. Bool Furni- ture Co. The cost per 100 of the original form is $17.50 (§ 371); for the form with tongue and groove, it is $22.50 ; and for the form with tongue and groove and one side divided into rows (# 373), the cost is $30 per hundred. CI I. IX] PREPARATION OF REAGENTS 269 like food, good even under quite diverse proportions and methods of prepara- tion. With a few, however, it is necessary to have definite strengths. a Figs. 215-217. Graduates of various forms for measuring liquids. ( Cuts loaned by the Whitall Tatum Co. ) By a saturated solution is meant one in which the liquid has dissolved all that it can of the substance added. This varies with the temperature. It is well to have an excess of the substance present then the liquid will be satu- rated at all temperatures usually found in the laboratory. I 375. Solutions less than 10 per cent. — In making solutions where dry substance is added to a liquid if the percentage is not over 10%, the custom is to take 100 cc. of the liquid and add to it the number of grams indicated by the per cent. That is for a 5% solution one would take ico cc. of the liquid and 5 grams of the dry substance. This does not make a strictly $'> solution. For that one should take 95 cc. of liquid and 5 grams of the dry substance ; or if the percentage must be exact then one should weigh out 95 grams of the liquid aud add 5 grams of the dry substance. W .I fl JIIIIIIII IHIPIIl mm> Figs. 218-219. Scales for weighing chemicals. (Cuts loaned by the Bausch & Lomb Optical Company.) \ 376. Solutions of 10 per cent and more. — When the percentage is 10% or 270 PREPARATION OF REAGENTS \_CH.IX over it is better to weigh out the number of grams representing the percentage and add to it the right amount of liquid in cubic centimeters. For example if one were to make a 35% aqueous solution of caustic potash in water then one would add 35 grams of caustic potash to 65 cc. of water. If one wished to make a 10% alcoholic solution of caustic potash he would add 10 grams of caustic potash to 90 cc. of alcohol. But here is a case where the alcohol being of less specific gravity than water the mixture would not weigh 100 grams ; and to make the mixture weigh 100 grams giving therefore an exact percentage, one should take 90 grams of alcohol and add to it 10 grams of caustic potash. In practice in making solutions of collodion or celloidin one usually mixes alcohol and 95% or absolute alcohol in equal volumes and then for a 10% solution 10 grams of the dry soluble cotton or celloidin are added to 90 cc. of the ether-alcohol mixture. But ether is much lighter than water and the alcohol somewhat lighter, so that the percentage in this case would be more than 10% because the 90 cc. of alcohol and ether would weigh considerably less than 90 grams. \ 377. Mixtures of Liquids to Obtain a desired Percentage. — It frequently happens that it is desired to obtain a lower percentage or strength of a liquid than the one in stock. This is very readily done according to the general for- mula: Divide the percentage of the strong solution by the percentage of the desired solution and the quotient will give the number of times too strong the solution is. To obtain the right strength take 1 of the strong solution, and of the diluting liquid one less than the quotient obtained by dividing the per- centage of the strong solution by the percentage of the weak solution, thus ; Suppose it is desired to obtain a 5% solution of formaldehyde. As the strong solution obtainable in the market is a 40% aqueous solution of formaldehyde gas it is 8 times too strong for the desired solution. To get the proper strength one takes 1 cc. of the 40% formaldehyde and adds to it 7 cc. of water and the resulting mixture will be only y& the strength of the original solution or 5% instead of 40%. \ 378. Mixtures of Alcohol. — For alcohol if one desires a 50% solution it is usually near enough correct to add equal parts of 95% alcohol and water, but this does not actually give a 50% solution. To find the real proportions according to the general formula : 95%-^5o%=i.9 i, e., for every 1 cc. of 95% alcohol should be added 0.9 cc. of water or for each 100 cc. of 95% alcohol, 90 cc. of water. This even will not give an exact mixture of alcohol for a mix- ture of alcohol and water diminishes somewhat in volume. To get true per- centages an alcoholometer for testing the specific gravity is used. A simple method of getting approximately correct mixtures of alcohol is the following : Pour the strong alcohol into a graduate glass (Fig. 215-217) until the volume is the same as the desired percentage, then add water until the volume is the same as the original percentage of the alcohol. Example : To get 50% from 95% alcohol put 50 cc. of 95% into a graduate and fill the graduate to 95 cc. with water, and the resulting mixture will be 50% alcohol, and so with all other strengths. Here the shrinkage is eliminated from con- sideration because the water and alcohol are not measured separately and then mixed, but one is added to the other until a given volume is attained. CI I. /A'] PREPARATION OF REAGENTS 271 SOME OF THE MORE IMPORTANT REAGENTS USED IN MICROSCOPY 5 379. 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 salicy- late of soda is added to prevent putrefactive changes. This must be carefully filtered. For method of use see Ch. X. § 44S. fj 3S0. Alcohol (Ethyl), C, H-, O H.— Ethyl or grain alcohol is mostly used for histologic purposes. (A) absolute alcohol (/. e., alcohol of 99° ) 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) S2°„ alcohol made by mixing 5 parts of 95 " n alcohol with I part of water. (C) 67% alcohol made by mixing 2 parts of 95",, alcohol with 1 part of water. See also \ 37S-379. Fig. 220. Tat um Co.) Reagent bottle. {Cut loaned by the ll'hitall \ 381. Alcohol (Methyl) C-H.., O H.— Methyl alcohol or wood alcohol is much cheaper than ethyl or grain alcohol on account of the revenue tax on ethvl alcohol. It answers well for main 7 microscopic purposes. It has been relined so carefully in recent years that the disagreeable odor is not very noticeable. 'I 382. Denatured Alcohol. — This is Ethyl or grain alcohol rendered 1111- drinkable by the addition of wood alcohol and benzine (Grain alcohol, S9 Vo i Methyl alcohol io",,, and Benzine \i%). In some cases the denaturing sub- stances are somewhat different, but all render the alcohol unusable for drink- ing. It is then free from internal revenue tax. In Great Britain " Methylated Spirits " consists of grain alcohol with 10% methyl alcohol. This is used very largely in microscopic work. In America the addition of the Benzine renders denatured alcohol also unfit for histologi- cal purposes if it is to be diluted. The addition of water makes it milky. If methyl alcohol alone or combined with pyridin or some other substance wholly 272 PREPARATION OF REAGENTS \_CH. IX soluable in water were used as the denaturing substance, denatured alcohol could be used in microscopic work for all the grades. That denatured as indi- cated above can be used only in full strength or very slightly diluted. For educational and other public institutions the U. S. government grants the privilege of using ethyl alcohol without paying the revenue tax, but for private institutions and for individuals it would be a great relief if the dena- tured alcohol could be mixed in all proportions with water without the forma- tion of precipitates. ? 383. Balsam, Canada Balsam, Balsam of Fir. — This is one of the oldest and most satisfactory of the resinous media used for mounting microscopic preparations. The natural balsam is most often used ; it 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 a time. 1 384. Xylene Balsam. — This is Canada Balsam diluted or thinned with xylene (xylol of the Germans). It is recommended by many to evaporate the natural balsam to dryness and then to dissolve it in xylene. For some pur- poses, e. g.: for mounting glycogen preparations, this is advantageous ; but it is unnecessary for most purposes. Xylene balsam requires a very complete desiccation or dehydration of objects to be mounted in it for the xylene is immiscible with water. 2 385. Filtering Balsam. Balsam is now furnished already filtered through filter paper. 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 satisfactor}-. 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, so common in chemical laboratories. The filtering is most successfully done in a very warm place like an incubator or an incubator room. !■ REAGENTS [ CI I. IX chlorid deteriorate, hence it is better to imbed them soon after they are fixed. Crystals of mercury are removed from the sections by the use of iodized alcohol {\ 413). \ 417. Alkaline Methylene Blue. — Methylene blue 2 grams ; 95% or abso- lute alcohol 50 cc; distilled water 450 cc; 1% aqueous caustic potash 5 cc. This stain works best after a mercuric fixer or a fixer containing mercuric ■chlorid, like Zenker's fluid. 5 418. Muller's Fluid. — Potassium dichromate 2% grams ; sodium sul- phate, 1 gram; water ioocc. This is one of the oldest fixers. It must act a longtime, two weeks to 10 or 12 weeks. This longer time is for nervous tissue to be stained for the myelin. Lately this fixer has been combined with mer- cury (see Zenker's fluid below). Before putting the tissue into 67% alcohol it is washed out in running water for 24 hours. Muller's Fluid 10 cc ; normal salt solution go cc, forms an excellent disso- ciator for epithelia, etc. (§ 399). '{ 419. Neutral Red. — This is used especially for staining living animals. It is used in very weak solutions : ^ gram red ; 1000 cc. of water. Put a few cubic centimeters of this solution into the vessel containing the live animal, or animals. Infusoria stain quickly 10 to 20 minutes or less. Vertebrates may require a few days. Try it on infusoria by adding a drop of the red to several drops of the infusion containing the infusoria. Be sure that there are many animals present. Watch them under the microscope and the color will be seen appearing in the granules of the infusoria. Then one may cover and study with a high power. 5 420. Nitric Acid, H-NO.,. — This is employed for dissociation (Nitric acid Dissociator, Water 80 cc; Nitric acid 20 cc); as a fixer, especially for chick embryos in the early stages (Water 90 cc. ; Nitric acid, 10 cc. ), and as a decalcifier (Nitric acid 3 cc; by% alcohol 100 cc). \ i,i\. Normal Liquids. — A normal liquid or fluid is one which does not injure or change a fresh tissue put into it. The perfect normal fluids for the tissues of any animal are the fluids of the body (lymph and plasma) of the animal from which the tissue is taken. The lymph or serum of one species of animal may be far from normal for the tissues of another animal. The commonly used artificial normal fluid is a solution of common salt (sodium Chlorid) in water, the strength varying from fa to fa per cent. As indicated above, this normal salt or saline solution is employed in diluting •dissociating liquids (\ 399). \ 422. Paraffin Wax. — A histologic Jaboratory requires two grades of paraffin for ordinary work. These are hard paraffin, melting at about 54 centigrade, and a softer paraffin melting at about 43° centigrade. Usually a mixture of equal parts answers very well. It is economical for a laboratory to buy the paraffin wax in cases of about 200 pounds. All paraffin for imbedding and sectioning should be filtered through two thicknesses of filter paper. For this, use a metal funnel, heat the paraffin very CH. IX] PREPARATION OF REAGENTS 281 hot in a water bath and then heat the funnel occasionally with a Bunsen flame. The warmer the room the easier to filter paraffin. Filter the paraffin into small porcelain pitchers. If the paraffin oven has a compartment large enough, it is well to keep one of the pitchers in the oven, then the paraffin remains melted and is ready for use at any time. I 423. Picric-Alcohol. — This is an excellent hardener and fixer for almost all tissues and organs. It is composed of 500 cc. of water and 500 cc. of 95% alcohol, to which 2 grams of picric acid have been added. (It is a \% solution of picric acid in 50% alcohol). It acts quickly, in from one to three days. (Proc. Amer. Micr. Soc, Vol. XII, (1890), pp. 120-122). §424. Picro-Fuchsin. — 10 cc. of a 1% aqueous solution of acid fuchsin ; 75 cc. of a saturated aqueous solution of picric acid. Stain deeply with hema- toxylin first, then use the picro-fuchsin. Wash off the picro-fuchsin with dis- tilled water. Mount in non-neutralized balsam or better in acid balsam (Balsam 50 cc. glacial acetic acid 5 drops). If the white connective tissue is not red enough increase the amount of acid fuchsin. § 425. Shellac Cement. — Shellac cement for sealing preparations and for making shallow cells is prepared by adding scale or bleached shellac to 95% alcohol. The bottle should be filled about half full of dry shellac then enough 959-6 alcohol added to fill the bottle nearly full. The bottle is shaken occa- sionally and then allowed to stand until a clear stratum of liquid appears on the top. This clear, supernatant liquid is then filtered through filter paper or absorbent cotton, using a paper funnel (? 358), into an open dish or a wide- mouth bottle. To every 100 cc. of filtered shellac 2 cc. of Venetian turpen- tine may be added to render it less brittle. The filtered shellac will be too thin, and must be allowed to evaporate till it is of the consistency of thin syrup. It is then put into a capped bottle, and for use, into a small spirit lamp (Fig. 203). In case the cement gets too thick add a small amount of 95% alcohol or some thin shellac. The solution of shellac almost always re- mains muddy, and in most cases it takes a long time for the flocculent sub- stance to settle. One can quickly obtain a clear solution as follows : When the shellac has had time to thoroughly dissolve, i. e. , in a week or two in a warm place, or in less time if the bottle is frequently shaken, a part of the dis- solved 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 solution and rise with the gasolin to the top. The clear solution may then be siphoned off or drawn off from the bottom if one has an aspirating bottle. (R. Hitchcock, Amer. Monthly Micr. Jour., July, 1884, p. 131). If one desires to color the shellac, the addition of a strong alcoholic solu- tion of some of the coal tar colors is good, but is liable to dissolve in the mounting medium when shellac is used for sealing, A small amount of lamp- black well rubbed up in very thin shellac and filtered, is good to darken the shellac. \ 426. Silvering. — Intercellular substance stains brown or black with nitrate of silver. Use % or %% aq. sol. on fresh tissue. Stain in the silver 282 PREPARATION OF REAGENTS \_CH. IX for I to 2 minutes then expose to light in water till brown. One may stain afterward with hematoxylin for the nuclei ; mount in glycerin, glycerin jelly or in balsam. \ 427. Sudan III for Fat. — Sudan III or azo-benzene-azo-/3-napthol, was introduced by Daddi into histology in 1896 (Arch. Ital de Biologie, t. 26. p. 142) , as a specific stain for fat. As it is soluble in all forms of fat and oils and in xylene, alcohol, etc., it is impossible to mount speeimens in balsam after staining. As the fat of tissues is removed by the reagents used in the paraffin and collodion methods (see Ch. X), only teased, free-hand or frozen sectioned material fresh or fixed in some non-fat dissolving fixer can be used (Miiller's fluid and 5% formaldehyde are excellent). The tissues cut free-hand or with the freezing microtome or teased can then be stained with a saturated alco- holic solution of the Sudan. It stains all fat a brilliant red. Preparations can be preserved in glycerin or glycerin jelly. This stain is largely used in Pathology. Daddi used the substance to feed animals and thus to stain the fat which was laid down in the body while the Sudan was fed. The fat in the body already deposited remains unstained. This substance then serves to record the deposit of fat in a given period. In 1907 Dr. Oscar Riddle fed Sudan to laying hens, and the fat in the layers of yolk laid down during the feeding was stained red (Science, XXVII, 1908, p. 945). For staining the yolks of hens eggs the hen may be fed doses of 20 to 25 milli- grams of the Sudan. Eggs so colored hatch as usual, and the chick in utiliz- ing the colored yolk stains its body-fat pink (Susanna P. Gage). \ 428. Table Black. — During the last few years an excellent method of dying wood with anilin black has been devised. This black is lustreless, and it is indestructible. It can be removed only by scraping off the wood to a point deeper than the stain has penetrated. It must be applied to unwaxed or unvarnished wood. If wax, paint or varnish has been used on the tables, that must be first removed by the use of caustic potash or soda or by scraping or planing. Two solutions are needed : SOLUTION A Copper sulphate 125 grams Potassium chlorate or permanganate 125 grams Water 1000 cc. Boil these ingredients in an iron kettle until they are dissolved. Apply two coats of the hot solution. Det the first coat dry before applying the second. SOLUTION B Anilin oil 120 cc. Hydrochloric acid 180 cc. Water 1000 cc. Mix these in a glass vessel putting in the water first. Apply two coats without heating, but allow the first coat to dry before adding the second. CH.IX] PREPARATION OF REAGENTS 283 When the second coat is dry, sand paper the wood and dust off the excess chemicals. Then wash the wood well with water. When dry, sand paper the surface and then rub thoroughly with a mixture of equal parts turpentine and linseed oil. The wood may appear a dirty green at first but it will soon become ebony black. If the excess chemicals are not removed the table will crock. An occasional rubbing with linseed oil and turpentine or with turpen- tine alone will clean the surface. This is sometimes called the Danish method, Denmark black or finish. See Jour. Ap. Micr., Vol. I, p. 145 ; Bot. Zeit, Vol. 54, p. 326, Bot. Gazette, Vol. 24, p. 66, Dr. P. A. Fish, Jour. Ap. Micr. , Vol. VI., pp. 211-212. \ 429. Zenker's Fluid.— Miiller's Fluid, ((S 418), 100 cc. ; mercuric chlorid 5 grams. Just before using add 5 cc. of glacial acetic acid to each 100 cc. of the above. Fix fresh tissue 5 to 24 hours. Wash out with running water 24 hours. Then place in 67% alcohol 1 day or more and finally preserve in 82% alcohol. Tissue fixed in Zenker's has mercuric crystals. They may be removed from the tissue by long treatment with iodin, or by putting the slide bearing the sections in iodized alcohol for half an hour or more (§ 413). This is an excellent fixer, combining the good qualities of mercuric chlorid and of the chromium compounds. Tissues fixed with this show well the red blood corpuscles. REFERENCES FOR CHAPTER IX For information concerning this chapter the reader is first of all advised to consult the microscopical periodicals, especially the Journal of the Royal Microscopical Society and the Zeitschrift fiir wissenschaftliche Mikroskopie und fiir mikroskopische Technik. The smaller journals and the proceedings of microscopical societies frequently have excellent articles bearing upon the subjects of this chapter. This is especially true of the Journal of Applied Microscopy and Laboratory Methods, and the Transactions of the American Microscopical Society. Among modern books, Lee's Microtomists' Vade Mecum, Mann's Physio- logical Histology and Ehrlich's Encyclopaedic der mikroskopischen Technik are indispensable in a laboratory. For the history of staining see Mann. pp. 190-195. • CHAPTER X FIXING; MICROTOMES AND SECTION KNIVES; IMBED- DING ; SECTIONING, STAINING AND MOUNT- ING ; SERIES ; MODELS FIXING TISSUES, ORGANS AND EMBRYOS ; MECHANICAL PREPARA- TION FOR STUDY \ 430. Fixation. — By fixing or fixation in histology is meant the prepara- tion of fresh tissues, organs, embryos or small adult animals usually by means of some chemical mixture, called a "fixer" so that the organ etc as a whole and the elements or cells composing it shall retain as nearly as possible the morphologic characters present during life. The more perfect the fixer the nearer will be the preservation of all structural details. Unfortunately no single "fixer" preserves with equal excellence all the structural details, and therefore it is necessary to prepare the fresh tissue in several different ways and to make a composite of the structural appearances found, thereby approximating the actual structure present in the living body. Changes are so rapid after death that the fixation should begin as soon as pos- sible. For the most perfect fixation the living tissue must be put into the fixer. -^ y ■- ^* ~3-.- Figs. 221-222. Class stoppered jars for fixing and storing tissues for histology. (Cuts loaned by I lie Whit- all Talum Co.) With one of the larger animals where the whole animal is to be used for microscopic study it is a great advantage to bring the fixer in contact with all CH. X] FIXATION OF TISSUES 285 parts of the body quickly, and that is done by washing out the vascular sys- tem with normal salt solution and then filling the vascular system with the fixer. This method of "fixation by injection " is of great importance in the histology of animals which are large enough to inject. If the animal is too small for injection or one wishes only a small part of a larger animal, then the pieces for fixation should be small, say one to three cubic centimeters. Often as for Flemming's fluid (§ 405) and for several others it is better to use pieces 2 to 5 cubic millimeters. Large, solid organs, must be cut into several pieces if the whole is needed. For hollow organs the cavity ma3' be filled with the fixer and the organ placed in a vessel of the same. Figs. 223-224. Shell vial and a Comstock bent-neck vial for fixing and storing material for histology. The Comstock bent-neck vial is especially designed for elongated objects like fish embryos, insects, etc., which are liable to become bent in a vertical bottle. (Cut of the bent-neck, from the Whita.ll Tatum Co.) The amount of fixer should be 10 to 50 times that of the piece of tissue. Of the fixers given under "Preparation of Reagents," Picric alcohol, Formaldehyde and Zenker's fluid are suitable for almost every tissue and organ. F"ormalin has the advantage of having strong penetration, hence it preserves whole animals fairly by immersing after filling the abdominal and thoracic cavities. Formaldehyde is excellent where a study of fat is in ques- tion, and it is much used as a fixer where frozen sections are desired (\ 43S). Remember the necessity of removing mercury from sections of tissues fixed with a mercuric fixer (5 473,477). \ 431. Mechanical Preparation of Tissues etc. for Microscopic Study. -- A limited number of objects in nature are small enough and transparent 2S6 MICROTOMES AND SECTION KNIVES [CM. X enough, and a limited number of the parts of higher animals are suitable for microscopic study without mechanical preparation except merely mounting them on a microscopic slide. Usually the parts of animals are so large and so opaque that the histologic elements or cells and their arrangement in organs can only be satisfactorily studied with a microscope after the tissue, organ, etc., have been teased apart with needles, (jj 357) orsectioned into thin layers. K'jlfflltai Fig. 225 Fig. 226 Figs. 225-226. Washing apparatus for tissues fixed in osmic and chro- mium mixtures. As shown in the figures the apparatus is connected with the water pipe by a small side cock. It is composed of a double vessel, the inner one being made of perforated brass. There are special perforated dishes to insert in the little compartments. For ova and other small objects a piece of gauze is used in the compartment. This apparatus is convenient for washing cover-glasses, for the washing out for iron hematoxylin, etc. The deeper box at the right answers for the slide baskets or holders (Fig. 244). MICROTOMES AND SECTION KNIVES # 432. The older histologists, those who laid the foundations and whose understanding of the finer structure of the body was in many ways superior to the knowledge possessed by workers at the present time, did their mechanical CH. A"] MICROTOMES AND SECTION KNIVES 287 preparation with needles and with sharp knives held in the hand. They dealt also with fresh tissue more largely than we do at the present day, and learned also to distinguish tissues by their structure rather than by their artificial coloration. It was not, however, on account of the lack of elaborate mechanical de- vices for sectioning and complicated staining methods of the present day, but because they put intelligence and zeal into their work that made them so suc- cessful. Only those who were "called" made for themselves a laboratory and saw with their brain. Now many are "sent," but few who use the central organ of sight. If the reader is interested in the mechanical means for sectioning he is referred to Dr. C. S. Minot's papers on the history of the microtome in the Journal of Applied Microscopy, Vol. VI. Inaword.it is now possible with the almost perfect automatic microtomes to make thousands of perfect sections ' where in i860 only occasionally could the most expert get tens with his hand sectioning. $ 433. Types of Microtomes. — There are two great types : (1) The early type in which the preparation to be sectioned is held mechanically and moved up by a screw, the section knife being held in the hand and moved across the object usually with a drawing motion as in whittling (Fig. 228). (2) The mechanical type in which both specimen and knife are mechani- cally held and guided, and the operator simply supplies power to the machine. In the highest types of the second class — automatic microtomes — the operator only needs to put the knife and specimen in position and supply the power and sections of any thickness and any number may be produced in a short time. A skilled and experienced person can get better results here as well as with free-hand sectioning or the hand microtome. Even automatic machines work better for skilled workmen. As is seen by the accompanying cuts, sometimes the knife is fixed in posi- tion and the object to be sectioned moves, while in other forms the object to be sectioned remains fixed and the knife moves. Furthermore for sectioning paraffin, the knife meets the object like a plane (straight cut), while for col- lodion sectioning the knife is set obliquely and there results an oblique or drawing cut as in whittling. I 434. Section Knives. — A section knife should have the following char- acters. (1) The steel should be good. (2) The blade should be slightly hol- low ground on both sides. Why some makers persist in grinding one side flat is a mystery. (3 ) The edge of the knife should be straight, not curved as in a shaving razor. (4) The back should be parallel with the edge. (5) The blade should be long, 12 to 15 centimenters, as it takes no more time or skill to sharpen a large than a small knife. (6) The blade should be heavy. There was formerly a fashion of making very thin bladed section knives, but that is a great mistake, for the thin blade bends and vibrates in cutting firm tissue and large pieces. There is no possible advantage in a thin bladed section knife for microtome work, but much disadvantage from the lack of rigidity. 288 MICROTOMES AND SECTION KNIVES \_CH. X The microtome knives shown on the various instruments figured in this chapter illustrate well the proper form of section knives. (Figs. 227, 238.) Fig. 227. Section knife with the honing back in position (Cut loaned by the Spencer Lens Co. ) § 435. Sharpening Section Knives ; Hones and Strops. — Perhaps it should be taken for granted that any one would appre- ciate the impossibility of making good sections with a dull section knife, but experience teaches the contrary. Students are prone to believe that with one of the elaborate automatic microtomes, good sections may be made with any kind of an edge on the knife. It is forgotton that the knife is the most important part, all the other mechanism is simply its servant. For sharpening, select a fine, yellow Belgian hone, and a very fine Arkansas hone. As a rule hones from the factory are not suffi- ciently plane. They may be flattened by rubbing them on a piece of plate glass covered with moderately fine emory or carborundum wet with water. Round the corners and edges of the hones on the plate glass or on a grindstone. In using the Belgian hone for sharpening knives, wet the surface well with a moderately thick solution of soap. With the Arkansas stone use some thin oil — xylene or kerosene mixed with a little olive oil or machine oil. Honing. Before honing a section knife, make sure that the edge is smooth, that is that it is free from nicks. Test this by shaving off the surface of a block of paraffin. If nicks are present the cut surface will show scratches. It is advisable also to look at the edge of the knife with a magnifier and with a low power (50 mm. ) objective. If nicks are are present remove them by draw- ing the edge along a very fine Arkansas hone. CH. X ] SECTIONING 289 A saw edge may be all right for rough cutting and for shaving razors, but if one wishes to get perfect sections 2 to lOfA. in thick- ness a saw edge will not do. In removing the nicks one should of course bear on very lightly. The weight of the knife is usually enough. In honing use both hands, draw the knife, edge foremost, along the hone with a broad curved motion. In turning the knife for the return stroke, turn the edge up, not down. Continue the honing until the hairs on the arm, wrist or hand can be cut easily or until a hair from the head can be cut within 5 mm. from the point where it is held. The sharper the knife becomes the lighter must one bear on. One should also use the finest stone for finishing. If one bears on too hard toward the end of sharpening, the edge will be filled with nicks. In honing and stropping large section knives, there has come into use during the last few years the so called " honing backs" . These elevate the razor slightly so that the wedge is blunter and one does not have to grind away so much steel, (Fig. 227). Strop. A good strop may be made from a piece of leather (horse hide) about 50 cm. long and 5 to 6 cm. wide, fastened to a board of about the same size. The strop is prepared for use by rubbing into the smooth sur- face some carborundum powder, i. e. 60 minute carborundum, that which is so fine that it remains in suspension in water for 60 minutes, or one may use diamantine or Jewelers' rouge. Stropping. With the back foremost draw the knife length- wise of the strop with a broad sweep. For the return stroke turn the edge up as in honing. Continue the stropping until a hair can be cut 1 to 2 centimeters from where it is held. § 436. Free-Hand Sectioning. — To do this one grasps the section knife in the right hand and the object in the left. I,et the end to be cut project up between the thumb and index finger. One can let the knife rest on the thumb or index finger nail and with a drawing cut make the section across the end of the piece of tissue. By practice one learns to make excellent sections this way. If the whole section is not sufficiently thin, very often a part will be and one can get the information needed. § 437. Sectioning with a Hand or Table Microtome. — 2 go SECTIONING [(//. X The tissue is held by the microtome and moved up by means of a screw. The knife rests on the top of the microtome and is moved across the tissue by the hand. Microtomes of this kind are excel- lent. No one need wait for expensive automatic microtomes to do good sectioning. With a good table microtome the knife being guided by the hand or hands of the operator, he can make straight cuts as for paraffin sectioning, or drawing cuts as for collodion work. (Figs. 228-229). Fig. 228 Fig. 229 Figs. 228-229. Hand and table microtomes. Both have a screw Jot ele- vating the object to be cat and a surface on which to rest the section knife. 228 in held in the hand, 229 is fastened to a table. The knife is held and moved by the hand in both cases. ( Cuts loaned by the Bauch & Lonib Optical Co) . § 438. Sectioning with a Freezing Microtome. — In this method of sectioning the tissue is rendered firm by freezing and the sections are cut rapidly by a planing motion as with paraffin. Now the most usual freezing microtome is one in which the freezing is done with escaping liquid carbon dioxid. The microtome is in general like the one shown in Fig. 229. The knife should be very rigid. A plane blade is often made use of. The tissue may be either fresh or fixed. If alcohol has been used it must be soaked out of the tissue by placing it in water. Sometimes tissues are CH. A] PARAFFIN METHOD 291 infiltrated a day or two in-thick mucilage before freezing. Drop a little thick mucilage on the top of the freezer, put the tissue in the mucilage and turn on a small amount of carbon dioxid. It will soon freeze the mucilage and the tissue as shown by the white appearance. When frozen, cut the tissue rapidly. It is well to have an assistant turn the feed screw up while the sections are cut. When 20 or 30 sections are cut place them in water or normal salt solution. The staining and mounting of the sections will be con- sidered in § 461-471. This is a rapid method of getting sections much used in pathology where quick diagnoses are demanded. In normal histology the freezing microtome is used mostly for organs or parts of greatly varying density. For example if one wishes sec- tions of the finger and finger nail, this apparatus offers about the only means of getting good sections. In that case the bone is decal- cified before trying to make the sections (§ 398). THE PARAFFIN METHOD OF SECTIONING § 439. Object of the Paraffin. — In the early periods in his- tology great difficulty was encountered in making good sections of organs and parts of organs because the different tissues were very, unlike in density. At first tallow and beeswax, elder pith, liver and various other substances were used to enclose or surround the object to be cut. This gave support on all sides, but did not render the object homogeneous. In the early sectioning, a great effort was made to keep all imbedding material from becoming entangled in the meshes of the tissue. This was guarded against by coating the object with mucilage, and hardening it in alcohol. This muci- lage jacket kept the tissue free from infiltration by the imbedding mass and itself was easily gotten rid of by soaking the sections in water. A great advance was made when it was found that the imbed- ding mass could be made to fill all the spaces between the tissue elements and surround every part, the tissue assuming a nearly homogeneous consistency, and cutting almost like the clear imbed- ding mass. Coco butter was one of the first substances to be used for thus "infiltrating" the tissues. The imbedding mass must be removed before the staining and mounting processes. § 440. Infiltration of the Tissue with Imbedding Mass. — 292 PARAFFIN METHOD [C//. X The tissue to be cut in this way is first fixed by one of the fixers used for histology. Several good ones are given in sections 406, 416, 423, 429. (A) The tissue is then thoroughly dehydrated by means of 95% and absolute alcohol. For most objects, especially embryos and other colorless objects it is best, during the dehydration, first to use alcoholic eosin (§ 402), as the most delicate part shows when one cuts the sections. Leave the piece of tissue to be cut over night in alcoholic eosin, and a few hours in uncolored 95% alcohol using 20 times as much alcohol as tissue. For the final dehydra- tion it should be left in absolute alcohol four or five hours or over night, depending on the size of the object. (B) Remove the alcohol by a solvent of the imbedding mass, that is by some substance which is miscible with both alcohol and the imbedding mass (§ 422, 441). Cedar wood oil is most generally used (§ 390). Leave the tissue in cedar oil until the tissue sinks and the thin parts of the specimen become translucent. If the tissue does not sink after a time it means that the tissue was not dehy- drated. Of course this does not apply to lung or other spongy tissue containing rnnch air. It is well to change the cedar oil once. The used cedar oil may be left in an open bottle for the evaporation of alcohol and used over and over again. A Fig. 230. Paraffin dish for infiltrating in the Lillie oven. It is made oj copper and as shown has a handle for easfi in transference. A, the whole dish, B, the dish in section, (four. Appl. Micr. iSgo, p. 266.) (C). Displace the cedar oil by melted paraffin wax. When the tissue is saturated with the oil, transfer it to an infiltrating dish CH. A] PARAFFIN METHOD 293 (Fig. 230), containing melted paraffin. Place in a paraffin oven (Fig. 231) and keep the paraffin melted for from two hours to three days depending on the size and character of the piece to be imbedded. If the tissue was thoroughly dehydrated and well saturated with cedar oil the melted paraffin permeates the whole piece. Fig. 231. The Lillie compartment, paraffin oven for infiltrating tissues with paraffin. Various sizes of this arc made (S, 16 and 24 compartments). Except for the largest laboratories the one with 16 compartments and trays will be found of sufficient capacity. Dr. Lillie has recently omitted a part of the trays and thus gained compartments for receiving dishes in which paraffin is kept melted and ready for use. {Cut loaned by the Spencer Lens Co. ) § 441. Imbedding in Paraffin Wax. — When the object is thoroughly infiltrated imbed as follows : Make of strong writing paper a box considerably larger than the piece to be imbedded. 294 PARAFFIN METHOD ICH. X Nearly fill the box with paraffin wax, ' place on a copper heater (Fig. 241) and allow to remain until bubbles appear in it. Put the box on cold water until a thin stratum of paraffin solidifies on the bottom. Take the piece of tissue from the melted paraffin (Fig. 230) and arrange in the box for making sections in a definite direc- tion. Add hot paraffin if necessary, and then place the box on cold water. The more rapid the cooling the more homogeneous will be the block containing the tissue to be cut. For the best imbedding it is well to drop 95% alcohol on the surface as soon as a film has formed in cooling. In warm climates where cold water is not easy to procure for cooling the blocks, one may float the paper box on 95,% alcohol and w'ith a pipette (Fig. 240) drop strong alcohol on the sides of the box and on the top of the paraffin as soon as a sur- face film has formed. It is very desirable to mark on the box the name of the imbedded object and to indicate which end or face is to be cut. See also under serial sectioning (§ 472-473). Fig. 232. Various forms of scalpels. The one at the left is especially excellent for cutting the ribbons of sec- tions of the proper length for mounting. The large one with straight edge is the best form for trimming the paraffin block square for sectioning. {Cut loaned by the Bausch & Lomb Opti- cal Co.) § 442. Fastening the Block to a Holder. — Use one of the block holders or object discs furnished with the microtome, or a short stove bolt (Figs. 233-236). Heat the larger end and press the paraffin block against the hot metal until it melts the paraffin. Hold the two together while cold water flows over them. When cold the block is firmly cemented to the holder. Pains should be taken to have the axis of the block parallel with the long axis of the holder ; and one should not cut the block so short that th,e holder comes in contact with the tissue when the two are cemented together. * A clamp is sometimes used for holding the paraffin block (Figs. 229, 246-247). CH. X] PARAFFIN J/ET//OD 295 § 443. Trimming the End of the Block for Sectioning. — Sharpen the end to be cut in a pyramidal form, being sure to leave 2 millimeters or more of paraffin over the tissue at the end as well as on the sides. The block is trimmed in a pyramidal form so that it will be rigid. Take particular pains that the opposite faces at the end of the block are parallel, and all the corners right angles. § 444. Making Paraffin Sections. — Put the paraffin block or the metal holder in the clamp of the microtome. Arrange the block so that one side of the pyramidal end is parallel with the edge of the knife, then tighten the clamp, and if an automatic microtome is used make sure that the section knife is also tightly clamped by the proper set screws. It is well to have the knife lean slightly toward the paraffin block (Fig. 239). Fig. 233. The Minot automatic rotary microtome for paraffin sectioning (Sections from 1 /1 to 25 /< may be cut on this instrument. ) (Cut loaned by the Bausch & Lomb Optical Co.). 2g6 PARAFFIN METHOD It'll. X The knife edge meets the paraffin squarely as in planing. The thickness of section is provided for in the automatic microtome by the indicator which may be set for any desired thickness, or one can turn up the screw by hand in the table microtome. (Fig. 229). The paraffin and its contained tissue are cut in a thin shaving. If the tissue was stained in toto with eosin as suggested in § 440 A, it is marked out with great clearness in the containing paraffin. As succeeding sections are cut they push along the previous sections, and if the hardness of the paraffin is adapted to the tem- perature where the sectioning is done the edges of the successive sections will be soldered as they strike. This produces a ribbon as it is called, and if the paraffin block has been properly trimmed at the end the ribbon will be straight and even (Fig. 234). If the rib- bon is curved sideways it indicates that one side of the block is thicker than the other and the sections are slightly wedge shaped. FlG. 234. Automatic rotary microtome for paraffin sectioning. Sections from 111 to 100 11 may be cut on this instrument. (Cut loaned by the Spence) Lens Co. ) It the paraffin is too hard for the room temperature and for a given thickness of section the sections will curl ; if it is too soft the sections will crumple. CH. X ] PARAFFIN METHOD 297 The thinner the sections the harder should be the paraffin or the cooler the sectioning room ; and the thicker the sections and the larger the object to be cut, the softer can be the paraffin and the higher the temperature. If then the sections do not ribbon, make thinner sections or work in a warmer place. If the sections crumple, make thicker sections or work in a cooler room. Of course one can reimbed in a more suitable hardness of paraffin. Fig. 235. The Conklin-Pietzsch automatic lever microtome for paraffin sectioning. This is a modified Ryder microtome and is simpler and therefore cheaper than most paraffin instrutnents. It is designed for sections from 1-300 to 10-300 mm. (3 1-3 to 33 1-311). By means of a special attachment, sections of 211 may be made. ( Cut loaned by Edward Pennock, Philadelphia. ) In the season when steam radiators are used one can get almost any desired temperature by sectioning nearer or farther from the radiator. 298 PARAFFIN METHOD [CH. X In the winter it is a good plan to warm the microtome and sec- tion knife before sectioning. This can be very easily done by put- ting a cloth over the radiator and the microtome something like a tent. \ 445. Electrification of the Paraffin Ribbons. — Some days there is such an accumulation of static electricity in cutting the ribbons that they jump toward anything brought near them. This is very annoying and liable to be so destructive to many of the sections that serial work (g 472) can not be done with safety. Many devices have been tried to overcome this difficulty, like burning a gas jet near the microtome, boiling water near the apparatus etc. , but the safest way is to wait for more favorable conditions. To overcome this electrification, Dixon, (Jour. Roy. Micr. Soc. , 1904, p. 590), recommends fastening a 5 milligram tube of radium bromide on the knife near where the sectioning is done. The radium ionizes the air and ren- ders it a good conductor, and then the static electricity cannot accumulate. I have not been able to try this method. \ 446. Storing Paraffin Ribbons. — The most convenient method for caring for the ribbons as they are cut is to place them on a tray (Fig. 212) lined with a sheet of white paper. It is important to write on the paper full data, giving the name of the tissue, the thickness of the sections, the date etc. It is welt also to number the ribbons and to indicate clearly the position of the first section or the beginning of the ribbon. Ribbons of sections on a tray should be covered by another tray if one wishes to carry them to another room. The slightest gust of air sends them flying. Ribbons on trays may be kept a long time, three or four years at least, if they are stored in a cool place. The sections do not flatten out quite as well after standing a long time as they do soon after they are made. § 447. Spreading the Sections. — Paraffin sections are almost invariably slightly wrinkled or folded in cutting. To remove the wrinkles one takes advantage of the expansion of paraffin when it is warmed. The sections may be floated on warm water when they will straighten out and become smooth, or the usual method is to stretch them on the slide upon which they are to be finally mounted. § 448. Spreading Sections on a Slide. — A double operation is performed in this way, viz; the sections are made smooth and they are also fastened to the slide. Put a minute drop of albumen fixi- tive on the middle of a slide (Fig. 187) and with the ball of one fin- ger spread it over the slide," making a thin even layer. It cannot be too thin. It is liable to stain if it is too thick. CH. X ] PARAFFIN METHOD 299 A Fig. 236 Fig. 237 A Fig. 238 B Fig. 239 3°o PARAFFIN METHOD [ CH. X Fig. 236 A B. A paraffin holder clamp and a razor support for the Minot Microtome for utilizing most of the cutting edge. ( Trans. Amer. Micr. Soc, igoi.) Clamp for the paraffin block holder. In A it is shown in section, in a side view. With this clamp one can use stove bolts as well as the expensive paraffin holders furnished with the instrument. A laboratory can have as many paraffin block holders' as necessary without undue expense. Fig. 237 A B. Razor Support and Razor. (A) Support with heavy base and vertical piece. The base should be capable of moving endwise one or two centimeters to bring the opening in the vertical part opposite the paraffin block. (B) Front piece to the razor. Fig. 238. Razor with straight back and edge. By moving this back and forth on the support nearly the entire cutting edge can be utilized. Fig. 239 A B. The knife support of the microtome with the razor support and razor in position. (A) Front view ; (B) Back view, in which the inclination of the knife toward the paraffin block is shown. With a pipette (Fig. 240) put several drops ot water on the slide and then place a piece of a ribbon on the water; or put the sec- tions on the, albumenized slide and add the water afterward. Heat Fig. 240. Reagent bottle with combined cork and pipette (This is made by taking a cork of the proper size and making in it a hole with a cork borer for the glass tube. It is advan- tageous to have a string tied tightly around the rubber bulb as shown) . the slide carefully over a spirit lamp or gas flame, being sure not to melt the paraffin. As the water warms the paraffin expands and stretches the sections out smooth. A copper heating plate also, Fig. 241, is excellent for spreading sections. CIT. X ] PARAFFIN METHOD 301 After the sections are spread, drain off most of the water, arrange the sections with a needle or scalpel and place the slide in one of the trays (Fig. 212). Allow it to remain over night or preferably longer. The longer the drying in air the more surely do the sections adhere to the glass slide. Fro. 241 Fig. 242 Fig. 241. Copper warming plate on legs. An alcohol or small Bunsen lamp is used zuith this. It is more satisfactory to spread sections by warm- ing the slides on this plate than to heal them directly over the flame. {Cut loaned by the Spencer Lens Co). Fig. 242. Spirit lamp. This is of glass and has the sides flattened so that the lamp rests on one of the sides if it is overturned. (Cut loaned by the Bausch & Lomb Optical Co.). If one is in haste to take the succeeding steps in the preparation , the slide may be dried by putting it into a drying oven at 38° to 40 C. for half an hour or more. The slower drying in air is better if one has plenty of time. Some tissues are very difficult to get perfectly smooth as just described. If fine wrinkles persist one can sometimes overcome the difficulty by letting the slide cool and then covering with a piece of fine tissue paper slightly moistened ; press down firmly with the ball of the finger on the sections. Then take hold of the edge of the paper and roll it off the sections. Unless one is careful the sections are liable to come away with the paper instead of adhering to the slide. As the water dries out the spread sections come in very close contact with the glass and adhere quite firmly to it. The thinner the sections the more tightly do they stick. This makes it possible to perform the rest of the operations on the slide. One has to be careful not to let strong currents strike the sections. 3 02 PARAFFIN METHOD [ CH. X § 449. Deparaffining in Xylene.— This is accomplished by using a solvent of paraffin. The best and safest one to use in a laboratory is xylene. Benzine, gasoline, and even kerosene are used, but xylene is a powerful solvent of paraffin, does not injure the tissue, and is not very inflammable, due to the large amount of carbon in its molecule (see § 392). It requires only a few minutes to dissolve paraffin from the sec- tions, but a day or more in the xylene does no harm. When the paraffin is removed the staining and other operations necessary for a completed preparation may be undertaken (See for these § 461-471). Fig. 243 Fig. 244 Fig. 243. Slide basket or holder and a glass stoppered bottle to contain the same. Xylene is safer than benzin for deparaffining. The slide basket was devised by Dr. A. B. Mix in the author's laboratory in 1898, It is cylindrical and has an unjointed handle or bail. (four. Ap. Micr., vol. i, 1S9S, p. i6y). Fig. 244. Square slide basic/ with hinged bail or handle so that it may be turned down in inserting or removing the slides. In 1000 the hinged bail was added to the round slide baskets, and in 1902 the form was changed from round to square. The square form is more convenient, and suitable for all sizes of slides. ( Cut loaned by the Spencer Lens Co. ) . § 450. Collodionizing the Sections. — Except for carmine stains and perhaps some others, collodion remains practically color- less. While the sections remain quite firmly attached to the slide after they have been spread and dried, thick sections are liable to CH. X] PARAFFIN METHOD 3°3 come off in the many processes of staining, and if one has many sections on a slide some of them may become loosened. To avoid this the sections are covered with a delicate layer of collodion, which holds them down to the slide. The early method was to use a soft brush and paint a thin film over the dried sections before they were deparaffined. Now the sections are deparaffined, and then after draining the xylene from the slide, 10-15 seconds, it is put into a bottle containing ^% collodion (§ 395). In a minute or more the collodion displaces the xylene and penetrates the sections and forms a delicate veil over their free surface. No harm is done by leaving the sections in the collodion a considerable time, but a minute or two is sufficient. The slide is removed, allowed to drain for half a minute, and then put into a jar of 67% alcohol (Fig. 222). The alcohol fixes the collodion and removes the ether. As the 67% alcohol does not hurt the tissue it may stay in the jar a day or more if desired, but half an hour suffices. Steps in Order for the Paraffin Method. — § 439, 450, 461- 471. Name No. Absl. ale. . _ Cedar oil . Date Infilt. _ .. Fixer _ _ _ . . .. _ Temp. bath. .. Imbed, in Time of fix __ .. Sections cut . . -,u's Washed in water. Temp. room. _ 67% ale. -82% ale „ Stains Decalc. g 398.- .67, 82% ale. In toto stain . . Mtd. iu.. ...... Washed in. . ..-_ Remarks _. . 67% ale. .. __82%alc... 95% ale. and eosin. . ... 3 o4 COLLODION METHOD [ CH. X The sections are now ready for the subsequent staining and other operations to make a finished slide. One has to remember that if mucicarmin (§ 389) is to be used in staining, the prepara- tion must not be collodionized as carmin stains collodion. THE COLLODION OR CELLOIDIN METHOD OF SECTIONING § 451. Collodion Method. — In this method the tissue is thoroughly permeated with a solution of collodion which is after- ward hardened. "Unlike the paraffin of the paraffin method, the collodion is not subsequently removed from the tissue, but always stays in the sections. It is transparent and does no harm. The fixing and dehydration with 95% alcohol is the same as for the paraffin method (§§ 430, 440). The paraffin method gives thinner sections than the collodion method and for series and large numbers of sections is superior. The collodion method requires no heat for infiltration, and it does, not render the firmer forms of connective tissue so hard and difficult to cut. It is especially adapted for making sections of large pieces of tissue or organs and when thick sections are desired. It is not easy to cut sections less than 10 ju with collodion, while with paraffin it is possible to make good ribbons of small objects of delicate texture 2 /< to 3 ju in thickness. With a very sharp knife and small delicate object, and one of the better forms of microtomes one can cut short series in i/< sections and get perfect ribbons. Collodion sectioning is sometimes denominated the ' ' wet method ' ' as the tissuejand sections must always be wet with some liquid, while the paraffin method is called the " dry method" as the tissue once infiltrated with paraffin keeps in the air indefinitely and in cutting the sections no liquid is used. § 452. Infiltration with Ether Alcohol.— Transfer the piece of tissue to be cut from 95% alcohol to a mixture of equal parts of sulfuric ether and 95% alcohol and leave in this for a few hours or a day or more as is most convenient. This is to soak the tissue full of a solvent of the collodion. § 453. Infiltration with 1%% Collodion. — Pour off the ether-alcohol from the tissue and add i}4% collodion. Leave in this over night or longer if the piece of tissue is large. CH.X] COLLODION METHOD 305 § 454. Infiltration with 3% Collodion. — Pour off the 1%% collodion and put in its place 3% collodion. Leave the tissue in this half a day or longer. § 455. Infiltration with 6% Collodion. — Pour off the $% and add 6% collodion to the piece of tissue. For complete infiltra- tion with this thick collodion leave the tissue in it for one day at least. If the object is large it is advantageous to leave it in for a week or two. Fig. 245. Slender dish for hardening the collodion in chloroform or in alcohol. {Cut loaned by the Whilall, Tatum Co.). § 456. Infiltration and Imbedding in 8% Collodion. — Pour off the 6% and add 8% collodion. Leave the tissue in this at least one day, and as much longer as possible up to two or three weeks if the piece of tissue is large. (A) For imbedding small pieces use a piece of wood, (deck plug) , vitrified fiber, glass or a good cork for a holder and cover the end with 6% collodion and let it get well set in the air, then put the piece of tissue on the holder and drop 8% collodion upon it at intervals until it is well covered all around. If one takes consider- able time for this the collodion thickens greatly in the air. This is an advantage for it gives a denser block for sectioning. After the collodion is pretty well set, place holder and tissue in a vessel with chloroform to harden. One can put the preparation into the chloro- form or if the vessel is tight it may be above the chloroform, the vapor then acting as the hardener. (B) Imbedding in a box. — If the object is of considerable size it is best to use a paper box for imbedding as with paraffin. If a very small amount of vaseline is rubbed on the inside of the box it prevents the collodion from sticking to the paper. Put first some of the 8% collodion in the box and let it remain in the air until nearly solid, 2 to 3 minutes. Then arrange the specimen to be cut as for imbedding in paraffin, and add gradually 8% collodion until the object is well covered. Let the box stand for a few minutes in air, then place it in a dish like a Stender dish (Fig. 245) and pour some chloroform on the bottom of the dish. Cover and the collodion will harden partly by the chloroform vapor 3°6 COLLODION METHOD [ CI I. X and partly by that which soaks through the paper. It is well to change the chloroform at least once. The used chloroform will con- tain some ether- alcohol, but is good for killing animals. Fig. 246. Microtome for collodion sectioning. A microtome of this form may also be used for paraffin sectioning. In that case the knife is set at right angles in order to cut the block square across instead of with a drawing cut as for collodion. (Cut loaned by the Bausch & Lomb Optical Co.). After 24 or 48 hours the collodion should be firm all through. Then it is placed in 67%" alcohol where it may be left a day or more. If it is to be left an indefinite time the 67 % alcohol should be changed for 82%. § 457. Sectioning by the Collodion Method. — For this one can use a table microtome (Fig. 229) or one of the sliding microtomes (Figs. 246, 247). The sections are made with a knife set obliquely and hence with a drawing cut. The holder with the small piece of tissue is clamped in the microtome and arranged as desired, then the sections are made with an oblique knife which is kept wet with 82% alcohol. The best way to keep the knife wet is to have a dropping bottle over the CH. A'] COLLODION METHOD 3°7 object, the drops falling about every two seconds. As the sections are cut they are drawn up towards the back of the section knife with a soft brush. They can be kept in order in this way and not interfere with succeeding sections. Some operators in drawing the knife across the tissue use a slight sawing motion. However one proceeds, the knife is drawn rather slowly, not rapidly as with paraffin work. Fig. 247. Pietzsch microtome. University of Pennsylvania model. The knife is set very obliquely for collodion sectioning. For paraffin sectioning the knife would be at right angles to the clamp. (Cut loaned by Edward Pen- nock, Philadelphia.) If the imbedding was done in a paper box, remove the box and trim the collodion block suitably. Dry the end away from the tissue, wet it with 3% collodion. Use a piece of wood, a cork or other holder of suitable size. Put some 6% collodion on the holder and let it dry for a minute or so, then press the collodion block down on the holder. Leave in the air for a minute or two and then put into 67% alcohol to harden the cementing collodion. 3 o8 COLLODION METHOD \_CH.X After 15 minutes, or longer if convenient, put the mounted speci- men into the clamp of the microtome and cut as above. Sometimes when the imbedded object is of sufficient size and the collodion block is firm, the block itself is put into the micro- tome clamp, no wood or cork holder being used. §458. Transferring Sections from the Knife' to the Slide. — When one has cut the number of sections for one slide they should be transferred to the slide as follows : Take a piece of white tissue paper about 3x6 centimeters in size and lay it on the knife over the sections. Pr.ess down slightly so the paper is in contact with all the sections. Take hold of the paper beyond the edge of the knife and gradually pull it down off the knife. If there is the right amount of alcohol on the knife the sections adhere to the paper and move with it. This transfers the sections from the knife to a piece of tissue paper. Place the tissue paper with the sections down on the middle of an albumenized slide. Cover with another piece of paper and press down gently. This presses the sections against the slide and absorbs a part of the alcohol. Take hold of one edge of the paper and lift it with a roll- ing motion from the slide. The sections should stay on the slide.* § 459. Fastening the Sections to the Slide. — With a pipette, drop 95% alcohol on the slide of sections, then use a pipette full of absolute alcohol if it is at hand. Drain most of the alcohol away and add a few drops of ether-alcohol. The collodion should melt and settle down closely on the slide. If the collodion does not melt the dehydration was not sufficient and more alcohol must be used. After the collodion has melted down upon the slide let the slide remain a minute or two in the air, and then transfer the slide to ajar of 67% alcohol. (Figs. 243, 251.) After half an hour or longer the preparation is ready to stain, etc. See below for directions (§ § 461-471). * 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 lens paper or thin blotting paper for absorbing the alcohol or oil may be used with it. CH. A'] COLLODION METHOD 3°9 Fig. 24S. Waste bowl with rack for supporting slides and a small funnel in which the slides stand while draining . This outfit is 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 is preferable to tin. A glass dish like that shown in Figs. /SS, 2ji is better than a bowl, as it can be more readily and thoroughly cleaned. {Cut loaned by II 'in Wood & Co.) \ 460. The Castor-Xylene Method of Sectioning. — The preparation of the tissue is the same as described in § 451-456, except that when the collodion is hardened in chloroform it is transferred, not to alcohol, but the block is placed Fig. 249. 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. in Castor-Xvlene (I 392). In a few days the collodion gets as transparent as glass and one can see the tissue within with great clearness. It can remain in the castor-xylene indefinitely. STAINING AND PERMANENT MOUNTING [ CH. X In cutting one proceeds exactly as in \ 457 except that the block is kept wet with castor-xylene and not with alcohol. The sections are arranged on the knife and transferred to the slide in the same way as for alcohol section- ing (J 457-458). For fastening the sections to the slide, as no water is present, one can add the ether-alcohol at once. It is advantageous here to have a mixture of ether 2 parts and absolute alcohol one part for melting the collodion in these oil sections. Allow the slide to remain in the air till the collodion begins to look dull, then the slide may be transferred to a jar of xylene to remove the oil. From the xylene it is transferred to 95% alcohol and then the slide is ready to be stained, etc. as described below {I 461-471). Steps in Order for the Collodion Method. — g 451-460, 461-47 r. Name No. Animal Date Fixer Time of fix Washedin water 67% ale 82 o„ ale. Decalc. (S 398 . 67% ale S2% ale In toto stain Washed in 67% ale 82% ale. 95% ale Ether-ale 1 y 2 % col $% col. ... 6% col 8% col..._ Imbedded Chloroform 67% ale... Or castor-xylene Sections cut /('s Stains Mounted in Remarks . STAINING AND PERMANENT MOUNTING \ 461. Generalities on Stains. — From the standpoint of the object to be stained, dyes may be divided into two great groups : (1 ) (a) Those which select out or differentiate certain parts of the tissue and make them prominent. Such dyes are called then differential or selective. If the nucleus is the part selected, the dye is frequently called a nuclear dye. (b) General or counter stains. These stain all parts of the tissue, and are CH. X] STAINING AND PERMANENT MOUNTING 31 r usually contrasting in color ; blue or purple and bright red are frequent com- binations, e. g. hematoxylin and eosin. (2) From the standpoint of the solvent used in preparing the stains they are called (a) Aqueous, and (b) alcoholic. If one uses an aqueous stain the object must be well wet with water before the stain is applied, and afterward well washed with water before put again into alcohol. If an alcoholic stain is used the object to be stained should be from alcohol of the same strength as that used in making the dye. The dye 19 also washed away from the tissue with the same strength of alcohol ; it may then be put into the stronger alcohols for dehydration. For other classifications of dyes consult the larger works, Lee, Mann, Ehrlich, and the microscopical journals. Fig. 250. Pipette with large rubber bulb for adding liquids to prepara- tions. (Cut loaned by the Bausch & Lomb Optical Co. ) \ 462. Generalities on Mounting. — For permanent preparations one can use a medium like glycerin or glycerin jelly etc. which mixes with water. The method of procedure is given in § 407, 408. For the most permanent mounting resinous media are used, and of these resinous media Canada balsam (§ 383) has been longest, and is now most used. In mounting in balsam one must remember the fundamental principles : (1) the object to be mounted in balsam must not contain water. It must then be dried or desiccated, or it must be rendered anhydrous by some liquid which mixes with water. As all tissues and organs contain much water, to mount them in balsam without drying in the air, which would spoil them in most cases, one must take the following steps ( 1 ) Dehydrate by alcohol which mixes with and displaces the water; (2) Displace the alcohol by some liquid which mixes with it and is also miscible with balsam, e. £■- xylene, etc. (§ 392). (3) As the liquid used just before the balsam usually makes the tissue more or less translucent it is often called a ' ' clearer ' ' . Finally displace the xylene etc. by balsam. If all the water is not removed in some way, the specimen will look turbid. If there is but a trace of water present and one uses natural balsam (? 383 ) for mounting the small amount of water will finally disappear; but it is better to dehydrate the tissue thoroughly before adding the balsam. HEMATOXYLIN WITHOUT AND WITH COUNTERSTAINING § 463. Staining with Hematoxylin. — Take a slide of sections prepared by the paraffin or the collodion method (§ 450, 459 ) from the jar of alcohol and plunge it into a vessel of water to remove the alcohol. For staining put the slide of sections into a jar or shell 312 STAINING AND PERMANENT MOUNTING [ CII. X vial of the hematoxylin solution (Figs. 243, 251 ) or one can lay the slide flat on the staining rack or some other support and add the stain to the sections (Fig. 248 ). It usually takes from 2 to 10 minutes to stain sufficiently with hematoxylin. A good plan when one is learning the process is to wash off the stain after 1 minute either with a pipette (Fig. 250 ) or by putting the slide in a dish of water. Wipe off the bottom of the slide and put it under the micro- scope. Light well, use a low power and one can see the nuclei stained a bluish or purple color as hematoxylin is a nuclear dye. If the color is faint, continue the staining until the nuclei stand out boldly. Sometimes it takes a long time to stain well with hematox- ylin. In such a case the jar of stain may be put into the paraffin oven and the heat will accelerate the staining. One may also heat the individual slides as for spreading sections, but one must be care- ful not to let the stain dry on the sections. As the stain evaporates add fresh stain with a pipette. FlG. 251. Apparatus and regents with which the slide holders are used. With thisjipparatus it is 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 if slides. The bottles for the reagents are glass stoppered specimen or museum bottles. {Mix, Jour. Ap. Jl/icr. 1898, p. ///.) When the sections are well stained with hematoxylin, wash off the hematoxylin with water. If the slide is allowed to stand some- time in ordinary water the color is likely to be brighter. This is due to the action of the alkali ( ammonia, etc. ) usually present in CH. X ] STAINING AND PERMANENT MOUNTING 313 natural waters. One could use distilled water, adding a few drops of a saturated solution of lithium carbonate. Dehydrate in 95% alcohol and absolute if necessary ; clear and mount in balsam as described in the next section (§ 464). Hematoxylin is so nearly a pure nuclear stain for most tissues and organs that the cell bodies are not very evident with this alone, hence some counter stain is generally used also. S 464. Counterstaining with Eosin. — One of the solutions of eosin (§ 401) is dropped upon the sections after the hematoxylin has been washed away with water. This stains almost instantly. One rarely needs to stain with eosin over 10 or 30 seconds. The excess stain is then washed away with a pipette or by dipping the slide into water. §465. Dehydrating, Clearing and Mounting. — Puttheslide directly into 95.% alcohol after it is rinsed with water.* Leave it in the alcohol a short time and transfer to fresh 95 % alcohol or to absolute alcohol a few seconds, 10-20. One must not leave the sections too long in the alcohol or the eosin will all dissolve out. Remove the slide from the alcohol and put it into ajar of clearer (§ 39 2 ) or put it on the rack (Fig. 248, 251) and add enough clearer to cover the sections. Soon the clearer will displace the alcohol and make the sections translucent. It usually requires only half a minute or so. The clearer is drained off and balsam put on the sec- tions, and then a clean cover-glass is added. One soon learns to use the right amount of balsam. It is better to use too much than too little. It is usually better to press the cover down very gently. With some delicate objects like embryos in the early stages this is *In the past the plan for changing sections from 95% alcohol to water, for example, has been to run them down gradually, using 75, 50 and 35% alcohol, successively. Each percentage may vary, but the principle of a grad- ual 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 the alcohol. This plan has been submitted to the severe test of laboratory work, and has proved itself perfectly satisfactory ( 1895-1908). 314 not safe STAINING AND PERMANENT MOUNTING [ CM. X A safe method for all objects is to add a slight weight, and put the slide in a warm place. After the balsam is quite dry the excess may be scraped off the slide with a knife and then the slide and cover cleaned from the remaining balsam by a piece of gauze wet with xylene. Finally the slide should be labeled and stored. Fig. 252. 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. Cut loaned by the Whitall Tatum Co.) CROSS-SECTION SHOWING SLIDES IN POSITION. § 466. Counterstaining with the Eosin in the Clearer. — With this method the eosin is dissolved in the carbol-xylene clearer, and the hematoxylin stained sections are dehydrated with 95% alcohol and absolute alcohol if necessary and then placed in the clearer. The sections are cleared and stained in eosin at the same time. It usually takes half a minute or more for the double process. When the sections are clear and sufficiently red, the slide is removed and the clearer drained off by holding in the forceps or in the drain- ing funnel (Figs. 248, 251). Then the balsam is added, and cov- ered as described above. It is a good plan to rinse off the stained clearer by pure xylene before adding the balsam. This is not absolutely necessary, how- ever. § 467. Hematoxylin and Picro-Fuchsin. — Picro-fuchsin is so selective in its general staining that it is frequently used after hematoxylin. The hematoxylin staining should be intense and after the hematoxylin is washed away add the picro-fuchsin (§ 424). It takes onty a few seconds for it to act, 10 to 30 seconds. Wash with distilled water, or natural water very faintly acidulated. The acid fuchsin is very sensitive to alkalies and fades easily. CH. A'] STAINING AND PERMANENT MOUNTING 315 Dehydrate in 95% and absolute alcohol, clear and mount in acid balsam. Acid balsam injures hematoxylin, but is necessary for the red in the picro-fuchsin. I/Ook out for mercuric chlorid crystals in the sections (§ 413, 477)- § 468. Hematoxylin and Mucicarmin. — Tissues and organs are best fixed in Zenker's or mercuric chlorid. Small intestine is one of the most striking and instructive organs for this double stain . Make the sections by the paraffin method, but do not fasten them to the slide with collodion, for collodion stains with mucicarmin (§ 389). Stain 1 to 24 hours in mucicarmin. Wash off the stain with water and then stain with hematoxylin. Do not stain too deeply. Wash with water, dehydrate, clear and mount in natural balsam. Nuclei will be bluish or purple and the cells containing mucus will be rose red. The goblet cells of the villi stand out like small red goblets, and if any mucus is streaming out of them it will be red. WEIGERT'S ELASTIC STAIN, WITH PICRO-FUCHSIN AND MUCICARMIN § 469. Elastic Stain. — Take a slide of sections made either by the paraffin or the collodion method (§ § 439, 451) from alcohol and put the slide into a jar or a shell vial of the stain. This is an alcoholic stain (§ 461) hence the sections should not be washed in water. Allow the stain to act from y 2 hour to an hour. Wash off the superfluous stain with 95% alcohol from a pipette or by rinsing in a jar of 95% alcohol. • It is better in either case to use the pipette and clean alcohol for the final washing. This stain alone gives a bluish tone to the entire tissue, the elastic tissue being stained a very deep blue. For greater contrast and to bring out the white fibrous tissue, muscle, etc., counter- stain with picro-fuchsin of % the strength given in the regular stain (§ 424, i. e., picro-fuchsin 1 part, distilled water 3 parts). Dip the slide of sections into distilled water, and then into a shell vial of the stain. Stain 15 to 30 seconds on the average- Wash in distilled water and dehydrate in 95 % alcohol and absolute if necessary, then clear in carbol-xylene and mount in acid balsam (§ 3 8 7)- The elastic tissue should be almost black ; white fibrous 316 STAINING AND PERMANENT MOUNTING [ CH. X tissue red, muscle, blood and epithelia yellow or yellowish. Arter- ies are excellent for this combination. § 470. Combined Elastic, Mucicarmin and Picro-Fuch- sin Stain. — For this, one should take some object that is known to contain elastic tissue, mucus, white fibrous tissue and muscle. (The non-cartilaginous part of the trachea is excellent.) The organ should have been fixed in mercuric chlorid or Zenker's fluid ($5 § 416, 429) for this preparation. The sections should be made by the paraffin method (§ 439) and no collodion should be used for fastening the sections to the slide (§ 450) for collodion is stained by mucicarmin. (1) Stain first in the elastic stain 1 hour. Wash well with 95 '/o alcohol and then with water. (2) Stain in a shell vial or jar of mucicarmin (§ 389) from 1 to 24 hours. Wash well with water, but one must be careful in treating these sections as they have no collodion mantle to protect them. (3) Stain 15 to 30 seconds with picro-fuchsin of }(. strength (§ 469). Dehydrate with 95% and if necessary absolute alcohol. Clear in carbol-xylene and mount in acid balsam (§ 387). The elastic tissue will be black or blue black. Mucus will be carmin or rose red, white fibrous tissue will be magenta red, muscle, epithe- lium and blood will be yellow. EOSIN METHYLENE BLUE STAINING §471. Eosin Methylene Blue. — One of the best objects for this stain is a hemolymph gland. Such a gland is easily and surely found by a beginner if he takes the heart and lungs of a veal. In the fat around the heart and behind the pleura will be found red bodies looking almost like blood clots. Remove carefully, fix in Zenker's fluid or mercuric chlorid, (§§ 416, 429). Section by the paraffin method, make the sections $n and io/< thick. Use collo- dion for ensuring the fixation to the slide (§ 450). Stain the sections 5 minutes in alcoholic eosin (§ 402). Wash off the eosin stain with water. (This is an exception to the generalization in § 461, 2.) Stain in methylene blue (§ 417) y 2 to 5 minutes. Rinse well in tap water'. Dehydrate with neutral 95 % alcohol (§ 380) and CH.X~\ SERIAL SECTIONING 317 with absolute alcohol. Work rapidly with only one slide at once. Clear with pure xylene, mount in neutral balsam (§ 386). All nuclei should be blue and all red blood corpuscles, bright eosin red. If one is successful this is a most striking and instructive prepara- tion. Spleen is also very instructive. Eosin-methylene blue staining is also excellent for demonstrat- ing mucus (§ 468). Do not forget that mercury is liable to be present in sections of tissue fixed with any mercuric fixer. Remove them with iodized alcohol (§ 413). This should be done before the staining. One can tell whether the tissues contain mercury by looking at the unstained sections. The mercury looks black by transmitted light, white by reflected light. The substance is often in the form of delicate black pins. MAKING SERIES ; SERIAL SECTIONING § 472. General on Series. — It is coming to be appreciated more and more that in histology as well as in embryology one can only get a complete knowledge of structure by having the entire organ cut in microscopic sections and each section mounted in order. Furthermore it is necessary to have the organ cut in three different planes. In this way one can see every aspect of the structural ele- ments and their arrangement in the organs. In single sections one gets only a partial view. For example, how many students have any other idea of a ciliated cell than that it is a cell with triangular outline with a brush of cilia at the broad end. Probably many would be puzzled if they had a top view of the ciliated end ; and the attached end would be even more puzzling. It may not be possible for every worker to make serial sections of all the organs in all the three planes, but every one who is work- ing seriously in histology can make all his preparations serial, that is the sections which are mounted can be in serial order, then a puz- zling appearance in one section may be perfectly intelligible in one a little farther along. - To get the greatest benefit from serial as indeed also from single sections, the sections should be made in a definite manner, that is, they should be exactly across the long axis of an organ or parallel with the long axis {Transections, and Longisedions) . 3i8 SERIAL SECTIONING [CH.X Or with such an organ as the liver, the skin, etc. , the sections may be parallel with the surface, (Surface Sections) or at right angles to the surface ( Vertical Sections) . ORDER OF THE SECTIONS IN A SERIES § 473. Order of Serial Sections. — Some plan must be adopted in arranging the series or only confusion will result. An excellent plan is to arrange the short pieces of ribbons for a given slide as the words on a page are arranged. That is, section No. 1 is at the upper left hand corner. The next row of sections begins where the first row left off, etc., (Fig. 253). As the paraffin stretches considerably one must cut the ribbons into pieces considerably shorter than the cover-glass to be used. Fig. 253. Slide of an embryologic series showing the method of arranging a sagittal series. This is the 15th slide of the series. The sections are ar- ranged like the words and lines in a book, i. e., from left to right. {From ' ' Guide to Histology and Embryology in Cornell University. ' ' ) Both the paraffin and collodion methods are adapted to the preparation of series. The paraffin ribbons are easier to manage and easier to make than the serial sections in collodion. By arranging the collodion sections as they are cut on the knife in collodion sectioning (§ 457), one can put them on the slide in perfect series by the tissue paper method (§ 458). If the sections are large, as in cutting serial sections of the cen- tral nervous system, the series can be kept in order in a small dish by putting a piece of tissue paper over each section and piling them up. If the vessel is small enough the papers and sections will not shift and get out of order. Or one might put a single section in a Syracuse watch glass and pile them up in series (Fig. 208). Then in mounting the sectious can be taken in order. CH. X] SERIAL SECTIONING 3'9 § 474. Numbering the Serial slides. — For temporary num- bering a fine pen with Higgins' waterproof carbon ink serves well. If the slide is clean one can write on it as well as on paper. When the ink is dry it should be coated with thin shellac or with thin xylene balsam. Sometimes thin collodion is used. It is also im- portant to write the number of the slide with a writing diamond. The double marking is desirable because with wet slides the dia- mond number is hard to see, while the ink marks are clearly visible. One is not so liable to wipe off the sections if the ink mark is present. Fig. 254. Egg pipette. This is made by putting a short piece of soft rubber tubing over the end of a glass pipette with rubber bulb. With this one can handle the eggs both fresh and hardened without any danger of injury, (four. Appl. Micr. 1S9S, p. 120. ) Fig. 255. Lens holder. A lens in such a holder is very convenient for sorting and orienting small eggs or em- bryos in imbedding. One can have the eggs in a watch-glass of melted paraffin on a copper warming plate (Fig. 241) and arrange the eggs or embryos under a lens in such a lens holder. Then if cold ivater is poured on the plate around the watch-glass the paraffin 'will cool and hold them in place. (Cut loaned by the Bausch & Lomb Opt. Co.) FIXING AND STAINING FOR SERIES § 475. Fixing. — The two most used fixers for embryos are Zenker's fluid and Formaldehyde (§406, 4 2 9)- For those unskilled in microscopic technic, or for one who is exceedingly busy the best results are obtained by putting the embryos in formaldehyde, (10 parts of formalin, the formalin of the pharmacy, and 90 parts water answers well). If there is plenty of this the embryos are likely to 320 SERIAL SECTIONS OF EMBRYOS [CH. X be well preserved even though they are left in the membranes, and that is far the best way for small embryos. § 476. Fastening the Sections to the Slide. — For all serial work it is especially desirable to fasten the sections to the slide with collodion (§ 450). This should always be done unless some stain like carmin is to be used on the slide after the sections are fastened. With thin sections, if one is careful enough, an entire series can be carried through without losing a section, but with thick sections (15,/u and thicker) some are almost sure to separate from the slide. § 477. Removal of Mercuric Chlorid from Sections. — It should be remembered that if a fixer containing mercuric chlorid is used the sections are almost sure to contain mercury. By trans- mitted light the mercury appears dark. Often the appearance is as if a multitude of delicate black pins were in the section. Sometimes the mercury is in rounded masses. This should be removed by putting the slides of sections into alcoholic iodin (§ 413). After half an hour or an hour wash off the iodized alcohol with pure 95 % alcohol and the sections are ready for staining. If the embryo was stained in toto and contains mercury, the sections should be passed from the deparaffining xylene to the iodized alcohol (§ 413). After half an hour or more the slides are passed through pure 95% alcohol, and back to the xylene or to carbol-xylene. Then they can be mounted in balsam. § 478. Staining for Series.— There is a great advantage in point of time and safety in staining the entire embryo in some good stain like borax carmin (§ 388). Carmin is a very permanent stain also. For bringing out special structural details the sections are stained on the slide as described in §461-471. The slide baskets are almost a necessity for serial work (Fig. 244, 251), as the slides are handled individually only twice, ( 1 ) when they are spread and dried and put into the baskets, and (2) after all the processes are complete and the sections are to be mounted in balsam. The sections are mounted in balsam directly from the depara- ffining xylene. No alcohol is used unless it is necessary to remove crystals of mercuric chlorid (§ 477). SERIAL SECTIONS OF EMBRYOS § 479. Serial Sectioning Embryos and Minute Animals.— CH.X-] SERIAL SECTIONS OF EMBRYOS 321 Serial sections of these should be made in the three cardinal sectional planes, viz; Transections; Frontal Sections; Sagittal Sections. If models are to be constructed from the sections it may be more conveniently done if the sections are one of the following thicknesses: 5yu, lOyU, 15/*, 20/<, 30/*, 40/*, 50/*, 6o/t, 80/*. § 480. Transections, that is sections across the long axis of the embryo or animal. Imbed the embryo with the right side down, taking the pre- caution to have a layer of paraffin between the embryo and bottom of the box (§ 441). (1) Mount the block of paraffin containing the embryo so that the tail end is next the microtome holder. The head is then cut first. (2) Place in the microtome so that the right side of the embryo meets the edge of the knife. (3) Mount as a printed line and the first or cephalic section is at the upper left hand corner, and the dorsal aspect of the embryo is toward the upper edge of the slide. Under the microscope the rights and lefts appear as in the observ- er' s own body, also the dorsal and ventral aspects so that he can easily locate parts by comparing them with his own body. § 481. Frontal Sections, that is sections lengthwise of the embryo or animal and from right to left (dextral and sinistral), so that the smbryo 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 is then 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 slides as before so that the sections are crosswise on the slides, the tail toward the upper edge. Under the compound microscope the head appears toward the upper edge and the rights and lefts are as in the observer's own body. (4) If the sections are too long to mount crosswise of the slide, cut the sections apart and mount with the head to the right. 322 SERIAL SECTIONS OF EMBRYOS \_CH. X § 482. 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 with the right side down as before. (1) Put the right side of the embryo next the microtome holder, then the left side is cut first. (2) Let the caudal end meet the knife edge if the embryo is small. (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 brings the ventral side up and the head to the right on the slide. Under the microscope the head appears at the left and the dorsal side away from the observer (Fig. 253). (4) For large or long embryos place the right side next the microtome holder as above, but let either dorsal or ventral aspect meet the knife. Cut the sections apart and mount as in (3). § 483. Axes for Sections. — For transections cut across the longest straight line from head to tail. For sagittal sections select the straightest embryo and cut par- allel with the longest axis dorso-ventral. For frontal sections cut parallel with the long axis, dextro- sinistral. § 484. For serial sections with collodion imbedded objects it is a great advantage to have the imbedding mass unsymmetrically trimmed, so that if a section is accidentally turned over it may be easily noticed and rectified. Furthermore it is imperatively necessary that the object be so imbedded that the cardinal aspects, dextral and sinistral, dorsal and ventral, cephalic and caudal, shall be known with certainty. § 485. Thickness of Cover-Glass for Serial Sections. — It is a great advantage 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 (25X76 mm., 1X3 inch) are used, cover-glasses 24 X 50 mm. may be advantageously employed. The combined thickness of the sections on a slide is easily determined by multiplying the number of sections by the thickness of each. CH. A"] SERIAL SECTIONS OF EMBRYOS 323 § 486. 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 information repeated in part on the left end. Fig. 256. Removable mechanical stage. It fits any square stage and has the advantage of large motion in both directions making it especially useful for the study of serial sections. { Cut loaned by the Spencer Lens Co. ) REFERENCES For sectioning staining, etc., in the various ways see : Lee, Mann, Ehrlich, Mallory and Wright, The Microscopic Journals. For the preparation of Embryos see Foster and Balfour's Elements of Embryology. Minot's Laboratory Text-Book of Embryology. Consult also the general Bibliography at the end. 324 DRAWINGS FOR BOOK ILLUSTRATION [ CH. X DRAWINGS FOR PUBLICATION \ 487. Preparation of Drawings. — The inexpensive processes of reproduc- ing drawings bring within the reach of every writer upon scientific subjects the possibility of presenting to the eye by diagrams and drawings the facts dis- cussed in the text. Though artistic ability is necessary for perfect representa- tion of an object, neatness and care will enable anyone to make a simple illus- trative drawing, from which an exact copy can be obtained and a plate pre- pared for printing. A careful study of the cuts or plates used to illustrate the same class of facts as one wishes to show will enable one to produce similar effects. Out- lines which are transferred to the drawing paper may be obtained by the camera lucida, the projection microscope (Figs. 257-258), or from a photo- graph. The drawing should be made so that it can be reduced anywhere from one-eighth to one-half. For ordinary photo-engraving for such line drawings as are used to illustrate this book, use perfectly black carbon ink. A shaded or wash drawing can be reproduced by the half-tone process, also photo- graphs as is illustrated by figures 79, 82, 89-92, 180-182. A crayon drawing on stipple paper with shadows re-enforced by ink lines and high lights scratched out with a sharp knife give admirable results for anatomic figures by the half- tone process. For examples see the various volumes of the American Journal of Anatomy. In vol. iv. pp. 409-443, and in vol. viii, pp. 17-47, one will find in the accompanying plates pure line drawings, half tones from photographs, and half tones from shaded drawings. \ 488. The Lettering on Drawings. — For half-tones this should be done directly on the drawing, as illustrated by the plates just referred to. For photographic reproduction of line work, letters, numerals or words used to designate the different parts can be put on the drawings by pasting the printed letters etc. of the proper size in the right position. In preparing the block the engraver removes all shadows from the edge so that the letters look as if printed on the drawing. If tissue paper were used on which to print the letters the engraver would have less trouble in removing shadows around the edge of the paper. Letters and figures should be distinct, but not so large that they are the most conspicuous feature of the drawing. MODELS FROM SERIAL SECTIONS I 489. General Considerations on Modeling. — Anatomists have for a long time produced models of gross anatomic specimens, and enlarged models for minute details. Naturally after serial sections of embryos and organs came to be made with considerable accuracy and of known thickness, there was a desire to make enlarged models which should be exact representations of the original rather than the generalized approximations built up as an artist produces a statue. CH. X] WAX MODELS 325 Further the difficulty of getting a true conception of the object by study- ing only two dimensions in the sections is very great, hence a model giving all three dimensions becomes almost a necessity for the beginner in embry- ology, and is of enormous advantage to an investigator in working out the true form and relation of complex structures. The principles involved in the construction of a model are exceedingly simple : — 1. It is necessary that the embryo or other object to be modeled should be cut into a series of sections of definite thickness. 2. The sheets of modeling material must be as much thicker than the sections as the model is to be larger than the original. 3. The sections must be drawn as much larger than the actual specimen as the model is to be larger than the object. 4. The drawings with the desired outlines must be made directly upon or transferred to the sheets of modeling material which are then cut out, fol- lowing the lines of the drawing. 5. The different plates of modeling material representing all the sections are then piled up, in order, thus giving an enlarged model of the object with all its parts in proper position and in true proportions. MODELS OF WAX \ 490. Wax Models. — For making wax models, bees-wax 820 grams, paraffin 270 grams, and resin 25 grams, are melted together and thoroughly mixed. To get the sheets of wax of the proper thickness two methods are available : — ( 1 ) The hot wax is poured into a vessel containing hot water. The wax spreads out into an even layer over the hot water and is allowed to cool. While it is solidifying it should be cut free from the edges of the vessel. Of course by calculation and experiment one can put in the right amount of wax to get a plate of a given thickness. (2) One must have a wax-plate machine consisting of a flat surface — planed cast iron is good — with some means of obtaining raised edges. If these are adjustable by a micrometer screw it is simple to set them properly for the desired thickness of plate. Then there must be a hot roller. The hot wax is poured on the plate and with the hot roller resting on the raised edges, the wax is rolled out into a plate. It cools quickly and may be removed for another plate. This is the most rapid and satisfactory method of prepar- ing the plates. By using a brush with turpentine the paper with the drawing can be wet and then with the hot roller cemented to the plate before that has been removed from the machine. The wax plate is cut with a sharp instrument, following the outlines of the object which has been traced upon it by the aid of a camera lucida or the projection microscope. The sections are piled together, some line or lines obtained from a drawing or photograph of the specimen before it was imbedded 326 BLOTTING PAPER MODELS [ CH. X and sectioned being used as a guide by which the correct form of the pile of sections can be tested. Finally the whole is welded into one by the use of hot wax or a hot instrument. Models which illustrate complex internal struc- tures are difficult to prepare, but numerous devices will occur to the worker, as the representation of blood vessels and nerves by strings or wires. A large model will need urach support which can be given by wire gauze, wires, pins or paper according to the special needs. A practical method for wax modeling was first published by G. Born, Arch, f. Mikr. Anat., Bd. xxii, 1883, p. 584. The most detailed statements of im- provements of the method have been published by Born (Bohm u. Oppel) 1904, and by Dr. F. P. Mall and his assistants. See contributions to the Science of Medicine, pp. 926-1045. Proceedings of the Atner. Assoc Anatom- ists, 1901, 14th session (1900) p. 193. A. G. Pohlman, Zeit. wiss Mikroskopie, Bd. xxiii, 1906, p. 41. To overcome the difficulty of cutting outthe wax plates, Dr. E. L. Mark of Harvard University uses an electrically heated wire moved rapidly by a modified sewing machine (Amer. Acad. Arts and Sciences, March, 1907 ; Science, vol. xxv, 1907 ; Anat. Record April, 1907. MODELS OF BLOTTING PAPER § 491. Comparison of Wax and Paper Models. — Wax has certain inherent defects for models : It is expensive, heavy and fragile. It is easily deformed by the temperature of summer, and the amount of time necessary for the preparation of the plates is great. A wax-plate machine is expensive and bulky. It therefore seemed worth while to see if there was not some other material obtainable in the open market which would be more suitable and more generally available. , Blotting paper seemed promising, and an actual trial showed it to be admirably adapted for the purpose. Since making the first model in 1905 it has been constantly used in the laboratory of embryology in Cornell University. Models made from it were demonstrated before the Association of American' Anatomists in 1905 and before the International Congress of Zoology in 1907. " The advantages of blotting paper models are the ease and cleanliness of their production and the lightness and durability of the product. The models are broken with difficulty, are easily packed or transported, and when they cleave apart are easily repaired, thus contrasting with the weight and fragility of wax models and their deformation by heat. " ' ' By this process are secured for the original model recon- structed from microscopic sections, the same qualities which have CH. X\ BLOTTING PAPER MODELS 327 made the Auzoux models molded from papier-mache such useful and lasting additions to laboratory equipment ; and in the hands of Dr. Dwight and Mr. Emerton, of Harvard University, have aided so much in the demonstration of structure and form of special anatomic preparations. " § 492. Thickness of Blotting Paper. — Blotting paper of a uniform thickness of 1 mm. T 9 T mm. and y 2 mm. were found in the market. The 1 mm. is known as 140 lb. A. and costs about two cents for a sheet 61 X 48 centimeters (24X 19 in.).* The thickness is easily tested by cutting out 50 small pieces, piling them, dipping one end in melted paraffin and pressing them together. The whole pile should of course measure 50 mm. if the paper is millimeter paper. § 493. Size of the Model. — In deciding upon the size of the model to be made from a given series of sections one should select the largest section and with the projection microscope throw the image on the table (Fig. 258). By using different objectives and different distances from the microscope one can find a size which seems suitable. The magnification may be found by § 207. Then by multiplying the whole number of sections by the thickness of the sections and this by the magnification one can get the length or height of the model. One must take these preliminary steps and decide upon the magnification to be used or the model is liable to be too large to be manageable or too small to show well the neces- sary detail. (1) Suppose the model is to be 100 times the size of the original object, and the object has been cut into a series of sections io/< thick. Then each section must be represented by a plate or sheet 100 times as long, broad and thick as the object. As the sheets of blotting paper are so large (61X48 cm.) one need be solicitous only about the thickness. As each section is actually io/< thick and the model is to be 100 times enlarged, the thickness representing each section must be ♦Book-stores, paper dealers and job printers are supplied by the paper manufacturers with samples of blotting paper. One can look these samples over, select and order the kinds desired. The millimeter blotting paper mentioned in the text is one of the cheaper grades, costing by the package of 500 sheets about two cents a sheet (sheets 61 X48 centimeters, 24X19 inches). 328 BLOTTING PAPER MODELS \_CH. X io/jX ioo=iooo/< or i millimeter, i millimeter blotting paper is used and every section of the series is drawn. (2) If the blotting paper were only y 9 ^ mm. thick it would be simpler to make the model 90 times the size of the original. If, however, one wished the magnification to be 100, it could be accomplished thus : Each section in the series should be repre- sented by 1 mm. or ioooyu in thickness. But if one uses blotting paper of T 9 T mm. thickness or 900//, there is a loss of 100// for each section and for 9 sections there would be a loss of 900// or the thickness of a sheet of the blotting paper. To remedy this one uses 10 sheets of blotting paper for 9 sections. This keeps the model in true proportion. In practice each of the sections is drawn upon one sheet except one of them and for that two sheets of the blotting paper are united and the sections drawn upon the double sheet. § 494. General Rule for the Use of Blotting Paper.— Divide the thickness by which each section is to be represented in the model by the thickness of one sheet of the blotting paper available. The quotient shows the number of sheets or the fraction of a sheet required for each section. If a quotient is a mixed number reduce it to a fraction. The numerator represents the number of sheets required and the denom- inator the number of sections to go with the sheets. Examples: (a) With a series of 10/i sections to be modeled at 100 enlargement each section of the series must be represented in the model by a thickness of TO/tX 100=1000/* or 1 millimeter. If one uses millimeter or iooo/i paper then iooo/<-i-iooo;U=^-, and one must use 1 sheet for 1 section. (b) With a series of \o)x sections to be made into a model 100 times enlarged, and with blotting paper of -^ mm. or 900/4 thick- ness, each section must be represented by io/«X ioo=iooo/<. If the blotting paper is 900/t thick, then it requires for each section : 1 ooo-i- 900= 1 \ sheets of paper or ] ¥ ° sheets for one section or 10 sheets for 9 sections, that is a double sheet for one of the nine sections. (c) With a series cut 15/1, for a 50 fold model. Each section is represented by a thickness of 1 5/(X 50= 750/*. If one uses 1 mm. or ioooyU blotting paper then each section requires 750-4-1000/^=^ en. -V] DRAWINGS FOR MODELS 329 of a sheet for one or 3 sheets for 4 sections. In this case one omits every fourth section in drawing, thus : 1st, 2d, and 3d sections would be drawn ; then the 5th, 6th and 7th ; 9th, 10th, nth, etc., every fourth being omitted. (d) If for the model just considered one had r 9 ^ mm. or 900/; paper then 750-^-900=4. That is there must be 5 sheets of the paper for each 6 sections. In that case every sixth section would be omitted in the drawing as every fourth section was omitted in (c). Fig. 257. Abbe Camera Lucida in connection with Bernhard' s drawing board. The drazving board is adjustable vertically for a greater or less image distance. It may also be elevated toward the microscope to prevent distortion (Fig. f2g). The base board is hinged so that microscope and board may be i?iclined together (Zeiss' Catalog). It is of course best to use sheets of exactly the right thickness to represent the necessary thickness in the model, (a) but one can produce models with accuracy by duplicating one or more sheets for a group of sections (b) or by omitting certain sections of the series in drawing (c, d). § 495. Drawings for Models. — For drawing one may use the camera lucida (Figs. 128, 132, 257), taking the precautions to 33° DRA WINGS FOR MODELS \_CH. X avoid distortion (§ 204). For getting the exact magnification desired one has recourse to different oculars, objectives and distance of the drawing surfaces (§ 177, 206 E). By far the most satisfactory means for making the numerous drawings, of all sizes of object and all magnifications except the high- est, is the projection microscope ( Fig. 258). One can draw directly upon blotting paper, but it is so import- ant to have a drawing to refer back to that one or more duplicates should be made. This is easily accomplished by putting a sheet of carbon manifolding paper on the blotting paper and a sheet of thin Fig. 258. Room and Apparatus for Drawing with the Projection Micro- scope. R. Radiant, an arc lamp with carbons at right angles; L. t. Lamp and microscope table; C. Condenser with IV. a large water bath between the lenses to absorb the heat rays. S. w. Stage and stage water bath on which rests the object and keeps the object cool by radiation as well as by absorption; O. The objective representing the microscope; M. Mirror at 45° on a draw- ing table, (Dt.) As the microscope is horizontal so that the axial ray is re- flected downward at right angles by the 45° mirror there is no distortion. The scale of the drawing is added exactly as described in \ 207. CH. X] PREPARATION OF MODELS 331 paper over the carbon paper using thumb tacks to hold the blotting paper and the duplicating sheets in position. One should take the precaution to number each drawing as it is made then confusion in the later processes will be avoided. § 496. Cutting out the Sheets for the Model.— " With the blotting paper, if the drawings are small the cutting is easily done with scissors or a knife. When the drawings are large and espec- ially when the model is to be made by representing each section by two or more thicknesses of blotting paper it has been found that an ordinary sewing-machine can be used to do the cutting. By setting the regulator for the shortest stitch an almost continuous cut is made and the parts are easily separated. If a large sewing-machine needle is sharpened in the form of a chisel, the cut becomes consid- erably smoother. It has been found advantageous when long con- tinued or heavy work is to be done to attach to the machine an electric sewing-machine motor. Skill in guiding the work is soon acquired. There are some details of a complicated drawing which are more easily cut by the scissors or a knife after the main lines have been cut by the machine." § 497. Contrasting Colors for Marking Groups of Sec- tions. — "It is a great advantage in any working model to have sec- tions at regular intervals in marked contrast with the body of the material. Blotting paper of a large variety of colors (black, red blue, pink) is easily obtained in the market. In the models made every tenth plate was a bright or light color and every 100th was black, rendering rapid numeration easy." § 498. Putting the Sheets together to Make the Model. — " When the paper sections are thus prepared they are piled and repiled as is usual until the shape conforms to an outline predeter- mined from photographs, drawings, or measurements made before the specimen was cut." ' ' It has been found that an easily prepared support and guide for the model in process of setting up, is made by cutting the out- line to be followed from a block of four or five sheets of blotting paper, marking upon it the lines of direction of every tenth or twentieth section. The colored numerating plates must of course conform to the spacing and direction of these lines." ' ' The preliminary shaping having been accomplished more 332 PREPARATION OF MODELS \_CH. X exact modeling is undertaken. The paper sections slide very easily upon one another. The most satisfactory means of fastening them together is by the use of ribbon pins, ordinary pins, or wire nails of various sizes, depending on the size of the model. No kind of paste or glue was found suitable for this purpose." § 499. Finishing the Model. — "When the model is well formed, inequalities are best removed by rubbing with the edge of a dull knife and smoothing with sand paper. Any dissections of the model for showing internal structures should be planned for at this stage for it is now more easily separated than later. It is also at this time that superfluous "bridges," which have been left in place to support detached parts, would better be removed." "To finish the model it is held together firmly and coated with hot paraffin either by a camels hair brush or by dipping in paraffin and removing the superfluous coating by a hot instrument. One might use a thermo- cautery for this purpose." "The paraffin renders the model almost of the toughness of wood without destroying the lightness of the paper." § 500. Coloring the Surface; Dissectng the Model. — "For coloring the surface of the model, it was found most desirable to use Japanese bibulous paper, lens paper (§ 125) which had been dipped in water color and dried. Any of the laboratory dyes or inks can be used, such as eosin, picric acid, methylene green, black ink, etc. The colored lens paper molds over the surface with ease and is held in place by painting with hot paraffin. All color and enum- eration lines and fine modeling show through the transparent paper." "When the model ceases to be a working model it can be cov- ered with oil paints mixed with hot paraffin and rubbed to any degree of finish desired." "One can dissect a model by a hot knife run along the planes of cleavage or cut across them by a saw." For the literature of blotting paper models see : Susanna Phelps Gage, Atner. Jour. Anat., vol. v, 1906, p. xxm ; Proceedings of the International Zoological Congress for 1907; Anatomical Record, Nov. 1907. (From this paper the above quotations were made). , Zeit. wiss. Mikroskopie. Bd. xxv., 1908, pp, 73-75. ' ' Blotting paper models have also been made and demonstrated by Dr. J. H. Hathaway and by Dr. J. B. Johnston at the Association of American Ana- tomists held in New York, 1906 {Proc. Assoc. Amer. Anatomists, Anat. Record April 1, 1907). BIBLIOGRAPHY The books and periodicals named below in alphabetical order pertain wholly or in part to the microscope or microscopical methods. They are referred to in the text by recogniza- ble abbreviations. For current microscopical and histological literature, the Journal of the Royal Micro- scopical Society, the Zoologischer Anzeiger, and the Zeitscrift ftir wissenschaf tliche Mikros- kopie, Anatomischer Anzeiger, Biologisches Centralblatt and Physiologisches Centralblatt, the Journal of Applied Microscopy and Laboratory methods and the smaller microscopical journals taken together furnish nearly a complete record. See also the list of periodicals. References to books and papers published in the past may be found in the periodicals just named, in the Index Catalog of the Surgeon General's library, in the Royal Society's Catalog of Scientific Papers, and in the bibliographical references given in special papers. A full list of periodicals may also be found in Vol. XVI of the Index Catalog, and in later volumes, the new ones are given. BOOKS Abbe, E. — Gesammelte Abhandlungen. Bd. II. Pp. 346, Illust. Jena, 1906. This volume deals with the microscope, etc. Edited by Dr. E. Wandersleb. Adams, G. — Micrographia illustrata, or the microscope explained, etc. Illust. 4th ed., London, 1771. Adams, George. — Essays on the Microscope. 4 to. Illust. London, 1787. AngstrOm. — Recherches sur le spectre solaire, spectre normal du soleil. Upsala, 1868. Anthony, Wm. A., and Brackett, C. F. — Elementary text-book of physics. 8th ed. Pp. 524, 165 Figs. New York, 1897. Auerbach, F. — Das Zeiss- Werk und die Carl Zeiss-Stif tung in Jena.ihre wissenschaftliche, technische und soziale Entwicklung und Bedeutung. 3d ed. Pp. 166. Illust, Jena, 1907. Babcock, J. F. — The medico-legal examination of Blood and other Stains, Hairs and Fibers. New York, 1894. Bagshaw, W.— Elementary Photomicrography. Pp. 70. Illust. London, 1902. Baily, Frederick R. — A text-book of histology. 2d revised ed. Pp. 497. Illust. New York, 1906. Baker, Henry. — Of Microscopes and the discoveries made thereby. Illustrated by many copper plates. 2 vols. London, 1742-3, 1785. Barker. — Physics. Advanced course. Pp. 902, 380 Figs. New York, 1892. Bausch, E. — Manipulation of the Microscope. A manual for the work table and a text- book for beginners in the use of the microscope. Pp. 200. Illust. New ed. Rochester, N. Y., 190 1. Bausch, Edward.— Use and care of the Microscope (Extracts from Manipulation of the Microscope). Rochester, 1902. This booklet should be in the hands of every beginner in microscopy. It is furnished free to laboratory teachers, and is supplied with each microscope. Bayley, R. Child. — The complete photographer. Pp. 410. Illust. London and New York, 1907. Bayon, P. G. — Diehistologischen Untersuchungs-methodeu des Nervensystems. Pp. 187. Illust. WUrzburg, 1905. Beale, L. S.— How to work with the Microscope. 5th ed. Pp. 518. Illust. London, 1880. Structure and methods. Beauregard, H., et Galippe, V. — Guide de l'felfeve et du praticien pour les travaux pra- tiques de micographie. comprenaut la technique et les applications du microscope k l'histo- logie v£g6tale, a- la physiologie, & la cliuique, a. la hygiene et & la medicine legale. Pp. 904, 570 Figs. Paris, 1880. 334 BIBLIOGRAPHY Beck and Andrews.— Photographic lenses. 2d ed. London, 1903. Many illustrations showing various forms of photographic objectives and the work for which they are adapted. Beck, Conrad.— The Theory of the Microscope. • Cantor Lectures delivered before the , Royal Society of Arts, Nov.-Dec, 1907. Pp. 59. Illust. London, 1908. Behrens, H— Mikrocheraische Technik. 2d ed. Pp. 68. Hamburg, 1900. Behrens, H.— Anleitung zur microchemischen Analyse der wichtigsten organischen Verbidungen. Hamburg, 1895 -1897. Behrens, H. — Transl. by J. W. Judd. A manual of microchemical analysis with an introductory chapter by J. W. Judd, London, 1894. Behrens, W.— The microscope in botany. A guide for the microscopical investigation of vegetable substances. Translated and edited by Hervey and "Ward. Pp. 466. Illu6t. Bos- ton, 1885. Behrens, W.— Tabellen zum Gebrauch, bei mikroskopischen Arbeiten. 3d revised ed. Pp. 237. Braunschweig, 1S98. Behrens, W., Kossel, A., und Schiefferdecker, P.— Das Mikroskop und die Methoden der mikroskopischen Uuntersuchung. Pp. 315, 193 Figs. Braunschweig, 1889+. Bethe, A.— Allgemeine Anatomie und Physiologie des Nerven systems. Pp. 487. Illust. Leipzig, 1903. Boehm, A. A., und von Davidoff, M. — A text-book of Histology, inoluding microscopic technique, from the 2d German edition. Translated by H. H. Cushing and edited by G. C. Huber. Pp. 528. Illust. 2d Araer. ed. Philadelphia and London, 1904. Boehm, A., und Oppel, A. — Taschenbuch der mikroskopischen Technik, 5th edition with directions by Born for making wax models. Pp. 277. Mtinchen, 1904. Bousfield, E. C— Guide to photo-micrography. 2ded. Illust. London, 1892. Bottler.— Die animalischen und vegetabilischen Faserstoffe. Leipzig, 1902. Bowman, F. H.— The structure of the wool fiber, and its relation to technical applications. Illust. New York, 1908. Brewster, Sir David. — A treatise on the mikroscope. From the 7th edition of the Encyc. Brit., with additions. Illust. 1837. Brewster, Sir David. — A treatise on optics. New ed. London, 1853. Brooks, C. P.— Cotton. New York, 1898. Browning, J. — How to work with the micro-spectroscope. London, 1894. Burnett, Samuel Howard. — The clinical pathology of the blood of domesticated animals. Pp. 156. Illust. Ithaca, 1908. Cabot, R. C— A guide to the clinical examination of the blood for diagnostic purposes. 5th ed. Illust. New York, 1904. Cajal, S. Ramon y. — Manuel de Histologia normal y de Tecnica micrografica. 4th ed. Pp. 643. Madrid, 1905. Carnoy, J. B. Le Chanoine. — La Biologie Cellulaire ; Etude compared de la cellule dans les deux regnes. Illust. (incomplete). Paris, 1884. Structure and methods. Carpenter, W. B.— The microscope and its revelations. 6th ed. Pp. 882, Illust. London and Philadelphia, 1881. Methods and structure. Carpenter-Dallinger. — The microscope and its revelations, by the late William B. Car- penter. 8th edition, in which the 1st seven and the 23d chapters have been entirely re-writ- ten, and the text throughout reconstructed, enlarged and revised by the Rev. W. H. Dal- liuger. 22 plates and nearly 900 wood engravings. Pp. 11S1. Loudon and Philadelphia, 1901. Chamberlain, C. J. —Methods in plant histology. 2d ed. Pp. 262, Illust. Chicago, 1905. Chevalier, Charles. — Des Microscopes et de leur usage. Illust. Paris, 1S39. Clark, C. H.— Practical methods in microscopy. 2d ed. Illust. Boston, 1S96. Cohn, A. O. — Tests and Reagents, chemical and microscopical. New York, 1903. Cole, Aaron, H. — Manual of biological projection and anesthesia of animals. Pp. 200, Illust. Chicago, 1907. Comstock, Anna Botsford.— Ways of the Six-Footed. Pp. 152 Boston, 1903. This is one of the most beautiful of the many beautiful books on Insects. Its illustrations are works of BIBLIOGRAPHY 335 art and its descriptions are not only entertaining in the highest degree, but make the reader think concerning the great problems of life. Comstock, John Henry.- Insect Life, an introduction to Nature Study, and a guide for teachers, students, and others interested in out-of-doqr life. Engravings by Anna Botsford Comstock. Pp. 349. New York, 1902. Comstock, John Henry and Anna Botsford.— How to know the Butterflies. A manual of the butterflies of the eastern United States. Pp. 311. Many colored illustrations. New York, 1904. Comstock, John Henry, and Anna Botsford. — A Manual for the Study of Insects. Pp. 701. 7th ed. Ithaca, 1907. Comstock:, John Henry, and Kellogg, V. 1,. — The elements of insect anatomy, an outline for the use of students in entomological laboratories. It gives methods of insect histology. 5th ed. Pp. 145. Ithaca, 1904. Cooke, M. C— One thousand objects for the microscope. Pp. 1S9. 500 figures and a brief description of pretty objects for the microscope. London, 1900. Cotton, A. et Mouton, H.— Les Ultra microscopes et les objects ultramicroscopiques. Pp. 232. Paris, 1906. Crookshank, E. M.— Photography of bacteria. London and New York, 1887. Cross, C. F., and Bevan, E. J.— A text-book of Paper Making. London, 1904. Cross & Cole. — Modern microscopy ; a hand-book for beginners and students. I. The microscope and instructions for its use. II. Microscopic objects : how prepared and mounted. 3d ed. revised and enlarged to which is added III. Microtomes : their choice and use. Illust. Chicago, 1903. Czapski, S. — Grundzfige der Theorie der bptischen Instrumente nach Abbe. 2 Aufl., unter Mitwirkung des Verfassers und mit Beitragen von M. v. Rohr. Pp. 490. Illust. Leipzig, 1904. DaCosta, John C, Jr. — Clinical Hematology. A practical guide to the examination of the blood by clinical methods, with reference to the diagnosis of disease. 2d ed. Pp. 591. Illust. Philadelphia, 1905. Dahlgren, U. and Kepner, W. A. — A text-book of the principles of animal histology. Pp. 508. Illust. New York, 1907. Dana, J. D. — A system of mineralogy. 6th ed. Illust. New York, 1892. Daniell, A. — A text-book of the principles of physics. 3d ed. Illust. London, 1895. Daniell, A.— Physics for students of medicine. Illust. London and New York, 1896. 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Senft, E. — Mikrospopische Untersuchung des Wassers mit bezug auf die in Abw&ssern und Schmutzwiissern vorkommenden Mikroorgauismen uiid Verunreinungen. Pp. 196. Il- lust. Wien, 1905. Silliman, Beuj., Jr. — Principles of physics, or natural philosophy. 2d ed., rewritten. Pp. 710 ; 722 Illust. New York and Chicago, i860. Spencer Lens Company. — How to use and care for the Microscope. A simple treatise on the use of the Microscope, especially adapted to laboratory work. Pp. 29. Buffalo, 1905. This booklet is sent with every microscope, and gives in the briefest and simplest manner the fundamental things to be observed in using a microscope. Spitta, E. J. — Photomicrography. 4 . Pp. 163. Illustrated by half-tone reproductions from original negatives. Text illustrations. London, 1899. Spitta, Edmuud J. — Microscopy, the construction, theory and use of the Microscope. Pp. 468. Illust. London, 1907. Starr, Allen M., with the cooperation of Oliver S. Strong and Edward Learning.— An atlas of nerve-cells. Columbia University Press, New York, 1896. The atlas consists of text, dia- grams, and some of the best photo-micrographs that have ever been published. Sternberg, G. M.— Photo-micragraphs and how to make them. Pp. 204, 20 Plates. Bos- ton, 1SS3. Stevens, Win. C— Plant Anatomy from the standpoint of the development and functions of the tissues, and hand-book of micro-technic. Pp. 349. Illust. Philadelphia, 1907. StOhr, Philipp, and Lewis, Frederic T.— Text-book of Histology arranged upon an Embryological Basis. 6th Amer. ed. Pp. 434. Illust. Philadelphia, 1906. Stokes, A.— Aquatic microscopy for beginners, or common objects from the ponds and ■ditches. Illust. Portland, Conn., 1896. Stokes, Alfred C. — Microscopical Praxis or simple methods of ascertaining the properties of various microscopical accessories. Pp. 244. Illust. Portland, 1894. 2 BIBLIOGRAPHY ' Suffolk, W. T.— On microscopical manipulation. 2d ed. Pp. 227. Illust, London, 1870. Sutherland, W. D.— Blood-stains, Skin Detection and the determination of their source. 1. 178. Illust. New York. Szymonowicz, L. translated by MacCallum, J. B.— A text-book of Histology and Micro- )pic Anatomy of the human body, including microscopic technique. Pp. 435. Illust. Phil- elphia and New York, 1902. Thomas, Mason B., and Wm. R.- Dudley —A laboratory manual of plant histology, ust. Crawfordsville, Ind., 1894. Trelease, Wm. — Poulsen's botanical micro-chemistry ; an introduction to the study of getable histology. Pp. 118. Boston, 1884. Methods. Trutat, M.~ La photographie appliqu^e & histoire naturelle. Pp. 225. Illust. Paris, 1884. Valentin, G.— Die Untersucbung der Pflanzen und der Thiergewebe in polarisirtem. cht. Leipzig, 1861. Van Heurck, H.— The microscope. Illust. London and New York, 1893. Vierordt. Die quantitative Spectralanalyse in ihrer Anwendung auf Physiologic 1876. Vignon, L. — La Soie ; Paris, 1900. Villiers, A., et Collin, E.— Traits des alterations et falsifications des substances alirnen- ire. Paris, 1900. Vogl, A. E.— Die wichtigsten vegetabilischen Nahrungs und Genussmittel. Pp. 575. ust. Wien und Leipzig, 1S99. Vogel, Conrad,— -Practical pocket-book of photography. Pp. 202. Figs. London, 1^93. Vogel, H. W.— Practische Spectralanalyse irdischer stoffe ; Anleitung zur Benutzuug der >ectralapparate in der qualitativeu und quantitativen chemische Analyse organischer and lorganscher KOrper. 2d ed. Figs. Berlin, 1S89. Wall, O. A.— Notes on Pharmacognosy. 2d ed Pp. 703. Illust. St. Louis, 1902. Walmsley, W. H.— The A, B, C of Photo-Micrography. A practical hand-book for :giuners. Plates and text figures. New York, 1902. Weinschenk, E.— Anleitung zum Gebrauch des Polarisationsmikroskopes. Pp. 147- lust. Freiburg, 1906. Wethered, M. — Medical microscopy. Pp. 406, Figs. London and Philadelphia, 1S92. Whipple, G. C. — The Microscopy of Drinking Water. 2d ed. Pp. xii+303. Illust. New ark, 1905. White, T. C. — The Microscope and how to use it. A hand-book for beginners, with chap- rs on marine aquaria and the staining of bacteria. Illust. London, 1907. Whitman, C. O.— Methods of research in microscopical anatomy and embryology. Pp. 5. Illust. Boston, 18S5. Whittaker, E T.— The theory of optical instruments. Pp. 72. Cambridge, 1907. Wilder and Gage —Anatomical technology as applied to the domestic cat. An introduc- m to human, veterinary and comparative anatomy. Pp. 575, 130 Figs. 2d ed. New' York id Chicago, 1886. Wiley, Harvey W. — Foods and their Adulterations. The origin, manufacture and com- isition of food products. Description of common adulterations, food standards and national od laws and regulations. Pp. 625. Illust. Philadelphia, 1907. Wilkinson, F. — The study of the Cotton Plant. New York, 1899. Williams, H. U.— Bacteriology. 4th edition revised and enlarged by R. Meade Bolton. d. 357. Illust. Philadelphia, 1903. Wilson, C. E. A.— Elements of Applied Microscopy. Pp. 168. Illust. New York and jndon, 1905. Wilson. Edmund B., with the cooperation of Edward Learning.— An atlas of fertilization id karyokinesis. Columbia University Press, New York, 1895. This atlas marks an era in tibryological study. It has admirable text and diagrams, but the distinguishing feature is e large number of almost perfect photo-micrographs. Winkelmann, A.— Handbuch der Physik, 2 Aufi. Bd. vi, 1 Optik. Pp. 432. Illust. npzig, 1904. Winslow, Charles-Edward Amory.— Elements of applied Microscopy. A text-book for iginners. Pp. 183. Illust. New York, 1905. BIBLIOGRAPHY 343 Winton, Andrew I,., in Collaboration with Dr. J. Moeller.— The Microscopy of Vegetable Foods. Pp.701. Ilhist. New York, 1906. Wood, J. G. — Common objects for the microscope. Pp.' 132. London, no date. Upwards of 400 figures of pretty objects for the microscope, also brief descriptions and directions for preparation, Wormly, T. G. — The micro-chemistry of poisons. 2d ed. Pp.742. Illust. Phila., 1885. Wright, Sir A. E. — Principles of Microscopy, being a hand-book to the Microscope. Pp. 250. Illust. New York, 1907. Wright, Lewis. — A popular hand-book to the Microscope. Pp. 256. Illust. London, 1885. Wright, Lewis. — Optical Projection, a treatise on the use of the lantern in exhibition and scientific demonstration. 4th ed. Pp.450. Illust. London, 1906. (Beginners will find this book very helpful. ) Wythe, J. H. — The microscopist ; a manual of microscopy and a compendium of micro- scopical science. 4th ed, Pp. 434, 252 Figs. Philadelphia, 1880. Zimmermann, Dr. A. — Das Mikroskop. ein Leitfaden der wissenschaftlichen Mikro- skopie. Illust. Leipzig und Weill, 1895. See also Watt's chemical dictionary, and the various general and technical encyclopedias. PERIODICALS* The American Journal of Anatomy, Baltimore, 1901 +. The American Jourual of Anat- omy including Histology, Embryology and Cytology was established by seven universities, — Harvard, Johns Hopkins, Columbia, Pennsylvania, Michigan, Cornell and Chicago. It is now (1908), published under the auspices of the Wistar Institute of Anatomy and Biology, Philadelphia, and has an editorial board consisting of Charles R. Bardeen, Univer- sity of Wisconsin ; Henry H. Donaldson, the Wistar Institute ; Thomas Dwight, Harvard University; Simon H. Gage, Cornell University; G. Carl Huber, Michigan University; George H. Huntington, Columbia University ; Franklin P. Mall, Johns Hopkins University ; J. Playfair McMur/rich, University of Toronto; Charles S. Minot, Harvard University r George A. Piersol, University of Pennsylvania. Henry McE. Knower, Secretary, Johns Hopkius University. There are also over 80 collaborators connected with different institutions. The American journal of medical research. Boston, 1901+. The American journal of physiology. Boston, 1896+. The American journal of microscopy and popular science. Illust. New York, 1S76-1881. The American journal of science. New Haven, 181S-K The American monthly microscopical journal. Illust. Washington, D. C, i8So-K American Naturalist. A popular illustrated magazine of natural history. Salem and Philadelphia, Boston and New York, 1867+. American quarterly microscopical journal, containing the transactions of the New York microscopical society. Illust. New York, 1878+. American microscopical society, Proceedings. 1878-1894 ; Transactions, 1895+. The Anatomical Record. Baltimore, 1906+. The scope includes the results of original investigations, preliminary reports, reviews, critical notes, courses of study, laboratory plans and events, including appointments, technique. It will also contain the Proceedings of the Association of American Anatomists. The first volume of the Anatomical Record fc NoTE — When a periodical is no longer published, the dates of the first and last volumes are given ; but if still being published, the date of the first volume is followed by a plus sign. See Vol. XVI of the index Catalog of the Library of the Surgeon General's office for a full list of periodicals. See also the later volumes for additions. . Besides the above-named periodicals, articles on the microscope or the application of the microscope appear occasionally in nearly all of the scientific journals. One is likely to get references to these articles through the Jour. Roy. Micr. Soc. or the Zeit. wiss. Mikroskopie. Excellent articles on Photo-micrography occur in the special Journals and Annuals of Photography. 344 BIBLIOGRAPHY was published with the American Journal of Anatomy (Nov., 1906 to Feb., 1908) and under the supervision of the same editorial board. It is now independent, with the following editors : Irving Hardesty, University of California ; G. Carl Huber, University of Michigan; Clarence M. Jackson, University of Missouri ; Horace Jayne, the Wistar Institute ; Thomas G. Lee, University of Minnesota ; Frederick T. Lewis, Harvard University ; Warren H. Lewis, Johns Hopkins University ; Florence R. Sabin, Johns Hopkins University ; George I,. Streeter, University of Michigan. Published by the Wistar Institute of Anatomy and Biology. Philadelphia, 1908-I-. Anatomischer Anzeiger. Centrablatt ftlr^die gesammte wissenchaftliche Anatomic Amtliches Organ der anatomischen Gesellschaft. Herausgegeben von Dr. Karl Bardeleben. Jena, iS86-h Besides articles relating to the microscope or histology, a full record of current anatomical literature is given. Auuales de la soci6t6 beige de microscopic Bruxelles, 1874 -f. Archives d'Anatomie microscopique. Illust. Paris, 1S97. (Balbiani et Ranvier.) Archiv ftlr miroscopische Anatomic Illust. Bonn, 1865+. Bibliographic Auatomique, Paris, 1893+ Centrablatt ftlr Physiologic Unter Mitwirkung der physiologischen Gesellschaft zu Berlin, Heraugsgegebeu von S. Kxner und J. Gad. Leipzig and Wien. 1S87+. Brief extracts of papers having a physiological bearing. Full bibliography of current literature. English mechanic. London, 1866+. Contains many of the papers of Mr. Nelson on Hghting, photo-micrography, etc. Index medicus. New York, 1879-f . Bibliography, including histology and microscopy. International journal of microscopy and popular science. London, 1890-h Journal of anatomy and physiology. Illust. London and Cambridge, 1867+ - Journal of Applied Microscopy and Laboratory methods. Illust. Rochester, N. Y.,i89SH-. Journal de micrographie. Illust. Paris, 1877-1S92. Journal of microscopy and natural science. London, 1885-!-. Journal of the New York microscopical society. Illust. New York, 1885+. Journal of physiology. Illust. London and Cambridge, 1878+. Journal of the American chemical society. New York, 1879+. Journal of the Royal Microscopical Society. Illust. London, 187s- Bibliography of works and papers relating to the microscope, microscopical methods and histology. It also includes a summary of many of the papers. Journal of the Quekett microscopical club. London, 1S68+. The Journal of Comparative Neurology and Psychology. This Journal was founded in 1891 by Clarence L- Herrick, and was then called the Journal of Comparative Neurology. Since 1900 it has had the present name. The editorial board consists of : Henry H. Donald- son, C. Judson Herrick, Herbert S. Jennings, J. B. Johnston, Adolph Meyer, Oliver S. Strong, John B. Watson, Robert M. Yerkes. It is now published by the Wistar Institute of Anatomy and Biology. Philadelphia, 1908. The Journal of Experimental Zoology. 1904-1-. Editors: William K. Brooks, William E- Castle, Edwin G. Conklin, Charles B. Davenport, Herbert S. Jennings, Frank R. Lillie, Jacques Loeb, Thomas H. Morgan, George H. Parker, Charles O. Whitman, Edmund B. Wilson. It is published by the Wistar Institute of Anatomy and Biology, 1908. Journal of Morphology, 1887-1901, 1903, 1908 -|-. The present editors are Edward Phelps Allis, Jr., Edwin G. Conklin, Henry H. Donaldson, Milton J. Greenmau, Ross G. Harrison, G. Carl Huber, Rorace Jayne, Frank R. Lillie, Franklin P. Mall, Charles S. Miuot, Thomas H. Morgan, George H. Parker, Charles O. Whitman, Edmund B. Wilson. The Journal of Morphology is now published by the Wistar Institute of Anatomy and Biology. Philadel- phia, 1908. The Lens, a quarterly journal of microscopy, and the allied natural sciences, with the transactions of the state microscopical society of Illinois. Chicago, 1872-1873. The Metallograpist, a quarterly publication devoted to the study of metals with special reference to their physics, microstructure, their industrial treatment and application. Illus- trated especially by photo-micrographs of metals and alloys. Boston, 1S9S+. The Microscope. Illust. Washington, D. C, 1881-1897. BIBLIOGRA PH Y 345 Microscopical bulletin and science news. Illust. Philadelphia, 1883+. The editor, Edward Pennock introduced the terni "par-focal" for oculars (see vol. iii, p. 31). Monthly microscopical journal. Illust. London, 1869-1877. Nature. Illust. London, 1869-h The Observer. Portland, Conn., 1890-1897. Philosophical Transactions of the Royal Society of London. Illust. London, 1665-)-. Proceedings of the American microscopical society, 1S78 — . Proceedings of the Royal Society. London, 1854-J-. Quarterly journal of microscopical science. Illust. I/>ndon, 1853-!-. Rev. de Metallurgies Paris, 1904-h Science, a weekly journal devoted to the advancement of science. New York. N. S. XS95-. Science Record. Boston, 18S3-4. The Scientific American. New York, 1845+. Zeitschrift f. Angewandte Mikroskopie. 1898+. Zeitschrift fftr Instrumentenkunde. Berlin, 18S1-K Zeitschrift flir physio logische Chemie. Strassburg, 1877-K Zeitschrift ftir wissenschaftliche Mikroskopie und fllr mikroskopische Technik. Illust. Braunschweig, 1884 -f-. Methods, bibliography and original papers. INDEX A ibe apertometer 187 ibe camera lucida 142-152, 329 ibe condenser or illuminator.. 54-58 ibe test-plate 185-187 lerration; chromatic 4, 5, 185 Cover-glass 1- . 64 Spherical 4, 5, 185 isorption spectra 158-160, 167-172 etylene light 42, 6o, 226 hromatic, condenser 49, 50, 229 Objectives 12, 15, 75 Oculars 26 hromatism 15 :tinic focus 223 ljustable objectives 14, 16, 64-68 Experiments with 64 Micrometry 138 Photo-micrography 235 [justing collar 65 fjustment, of analyzer 173 Coarse or rapid, and fine 74 Frontispiece ; of objective_i4, 16,65 Objective for cover-glass 65 rial image 35,37 r bubbles , 103-106 bumen fixative, Mayer's 271 cohol, absolute 271 Ethyl 271 Denatured : 271 Methyl , ! 271 Picric 281, 285 coholic dye 311 nici prism 155 nplifier 123 nplification of microscope 116 lalyzer 163, 173 lgle, of aperture 19, 20 Critical 64 lgstrom and Stokes' law 160 lgular aperture 19, 20 lisotropic 175 )ertometer, Abbe's 187 jerture of objective 19, 23, 187 Illuminating cone ._ 52 Numerical of condenser 52 jlanatic cone 54 Objectives 15 Ocular 15 achromatic condenser 50 Objectives 15, 76, 221 Apparatus and material, 1. 39, 99- »6, 141, 155. 185, 203, 220 Appearances, interpretation. _ .99-1 15 Arrangement, minute objects 261 Serial sections 318 Tissue for sections 317 Artifacts _. 100 Artificial illumination, 42, 56, 60, 223, 228, 233 Axial light : 41 Experiments with 48 Abbe illuminator 56 Axial, point 19 Ray 41 Axis, optic 2, 3, 12 Crystals 175 Illuminator 57 Secondary 3, 6, 57 Back-ground for photographing " . 207, 213. Back combination or system of objective 12-14 Bacterial cultures, photographing, 243 Balsam . : 272 Acid 272, 281, 315 Bottle 257 Mounting in__ 257, 311 Removal from lenses 71 Natural , 272 - Neutral : .272 Removal from slides 246 Xylene 272 Bands, absorption 158 Base of microscope, Frontispiece, Bath, water 153, Bibliography, 1, 38, 114, 139-140, 154, 172, 184, 192, 202, 243, 261, 264, '283, 323,326, 332, 333-345- Binocular 112-114 Black, anilin for tables 1282 Blocks, for shell vials 259 Blood, absorption spectrum of 168 Blotting paper for models 326 Board, reagent -.259, 268 Body of microscope, Frontispiece. Borax, carmin . 273 Bottle for balsam, glycerin or shellac 257 Reagent 271 INDEX 347 Box, glass 249 Brownian movement 109, 115 Bubble, air 103-106 Bull's-eye 60, 224 Engraving glass 225 Burning point 7, 35 Cabinet for microscopic prepara- tions 264-265 Calipers, micrometer or pocket 249 Camera, bed 205 Drawing - 154 Embryos 209 Large, transparent sections 212 Photo-micrographic 219, 222 Testing 220 Vertical 205, 208, 219, 222 Camera lucida 141 Abbe 142-152 Wollaston's 121, 124, 143, 144 Canada balsam : 272 Mounting in 257, 311 Removal from lenses 71 Removal from slides 246 Carbol-turpentine 274 Carbol-xylene 274 Carbon-monoxide lie magi obi n, spectrum of 169 Card, catalog 264 Centering 254 Care of, eyes ._■ 7 2 Microscope, mechanical parts__ 70 Optical parts 69-72 Negatives 207 Water immersion objectives 68 Carmin, borax 273 Mucus 273 To show currents and pedesis, 108, 109 Spectrum 170 Castor-xylene clarifier 273 Cataloging, formula 262 Preparations 261 -264 Cedar-wood oil, bottle for 257 Clearing 273 Oil immersion objectives 273 Cells, deep, thin 253 Isolated preparation 260 Mounting . 253 Staining 260 Cement, shellac 253, 281 Cementing collodion 275, 302 Center, optical _ 2, 3 Centering, arrangement of illum- inator 5°, 54 Card 254 Image of source of illuminarion 51 Centimeter rule 118 Central light 41, 48, 104 Chamber, moist 256 Chemical focus 15 Microscope 178 Rays 15 Scales 269 Chemistry, Micro 176 Chloral hematoxylin 278 Chromatic, aberration 4-5 Correction 15, 185 Objective 14 Circle, Ramsden _.. 37 Clarifier, castor-xylene 273-274 Class demonstrations in histology and embryology 193-202 Cleaning, back lens of objective, "72 Homogeneous objectives 69 Mixture for glass 250 Optical parts 70-72 Slides and cover-glasses 246-249 Water immersion objectives 68 Clearer 273, 274, 311 Clearing, mixture 274 Tissues 311, 313 Cedar- wood oil 273 Clinical microscope 193 Cloudiness, of objective and ocu- lar, how to determine 101 Removal 71 Coarse adjustment of microscope, Frontispiece ; testing 74 Cob-web micrometer 133 Collective lens 28 Collodion 274 Coating glass rod : 107 Cementing,! 275,303 Clarifying 309 Fastening sections to slide 275 Hardening 305 Method 304-310 Collodionizing sections 302 Color, correct photography 2r4 Correction. 15 Images 64, 68 Law of 160 Production of ___I76 Screens i_. 214-217, 244 Colored, minerals spectra of 171 Comparison prism 1163, 164 Spectrum 164, 169 Compensation ocular 28, 29 Complementary spectra 160 Compound microscope, see under microscope. Concave lenses 3 Mirror, use of 42-43 Condenser 48-58 348 INDEX Abbe 54-58 Achromatic 49, 50, 229 Apochromatic 50 Bull's eye 60, 224 Centering 50, 54 Illuminating cone 52 Mirrorwith 55 Non-achromatic 55 Numerical aperture 52 Optic axis 50, 54 Photo-micrography 224, 229 Standard size 30, 55 Substage „ 49 See also illuminator. Condensing lens 40 Cone, aplanatic 54 illuminating 52 Congo-glycerin 275 Red 275 Conjugate foci 4 Construction of images, geometri- cal 5 Continuous, spectrum 158 Contoured, doubly 107 Converging lens 3 Lens system 11 Convex lenses . 3-6 Correction, chromatic, or color 4, 15. 185 Cover-glass 65, 66 Over and under correction 15 Cotton, collodion, gun or soluble. 274 Counterstaining 3 IO_ 3i7 Cover-glass, or covering glass 248 Aberration by 64 Adjustment, specific directions 65 Adjustment for, in photo-micro- graphy 235 Adjustment and tube-length 16, 17, 65 Anchoring 255 Cleaning 248-249 Correction. 64,65 Effect on rays from object__2i, 65 Gauges : 249-250 Measurer 249, 250 Measuring thickness of 249 Non-adjustable objectives, table of thickness 18 No. 1, variation of thickness 250 Putting on 103, 252 Sealing 254 Serial sections 322 Thickness 17, 18, 249,322 Tube-length 174 Wiping .248 Critical angle 64 Crystals from frog for pedesis ..no Systems 180 Crystallization under microscope 58, 177-180 Crystallography 176 List of substances 179-180 Currents, diffusion, avoidance 313 Liquids 108 Cutting sections 289 Dark-ground illumination. 42, 56-60, 198 Abbe illuminator 58 Mirror 57 Dark room for drawing 153 Daylight, lighting 40 Decalcifier 275 Deck-plugs for collodion blocks—305 Dehydration 258, 271, 311 Demonstration, microscopes. .192-194 Micro-projection apparatus 200-201 Denatured alcohol." 271 Deparaffining 302 Designation of oculars 29 Wavelength 165 Determination of field of micro- scope 33 Equivalent focus 190-192 Magnification 116, 191 Working distance 47 Diamond, writing 319 Diaphragms and their employment 42, 43. 50-58 Diffusion currents, avoidance 313 Direct, light 40 Vision spectroscope 15s Disc, Ramsden 37 Dissecting microscope 10, 194 Dissociating liquids 275 Dissociator, formaldehyde 275 Miiller's fluid 275 Nitric acid 261, 275, 280 Distance, principal focal 4, 7, 35 Standard at which the virtual image is measured 123 Working d. of simple micro- scope or objective 47 Working d.of compound micro- scope 13, 39, 47 Distinctness of outline 105 Distortion in drawing, avoidance. 143 Diverging lens 3 Double spectrum 164 Vision 116, 118 Doubly contoured 107 Refracting 175 Draw-tube, Frontispiece. Pushing in 44 INDEX 349 Drawing, with Abbe camera lu- cida 146-153 Board for Abbe camera lucida, 146-149. 3 2 9 Distortion, avoidance 143 Embryograph for * 154 Microscope 140 Models 329 Photographic camera 154 Photo-engraving 324 Room for projection micro- scope 153, 154 Scale and enlargement 151 With simple microscope 153 Drugs, adulteration 182 Dry objectives 14, 20-23 Light utilized 21 Dry mounting 252 Numerical aperture 20 Dry plates, discovery by Mad- dox 218 Dust, of living rooms, examina- tion in On objectives and oculars, how to determine 101 Removal 71 Dye, general staining with 310 Aqueous 311 Alcoholic 311 E . Eccentric diaphragm 5r, 57, 58 Egg pipette 319 Eikonometer 137, 138 Elastic stain 275, 315 Embryograph 154 Embryos, camera for 209 Photographing 209-212 Records : 211 Serial Sections 320 Engraving glass for condenser 225 Enlargements 242 Eosin 276 Equivalent focal length or focus of objectives and oculars__i3, 29, ——33. 190-19 2 Erect image 1 Erecting, binocular microscope 112-113 Etching for metallography 241 Ether, alcohol 276 Sulfuric 276 Ethyl alcohol 271 Examination of dust of living rooms, bread crumbs, corn starch, fibres of cotton, lin- en, silk, human and animal hairs, potatoes, rice, scales of butterflies and moths, wheat in Experiments, Abbe condenser 56 Adjustable and immersion ob- jectives 64 Compound microscope 30 Homogeneous immersion obj ec- tive 68 Lighting and focusing 42, 43 Micro-chemistry 176, 181 Micro-spectroscope 167 Micro-polariscope 174 Mounting 251 Photo-micrography 226 Simple microscope 6 Exposure, of photographic plates. 226, 233, 236, 242 Color-screen 214-217, 244 Extraordinary ray of polarized light 173 Eye and microscope 1, 7, 9, 11,36 Eyes, care of 72 Emmetropic, hyperopic, my- opic, normal 8, 9 Muscae volitantes no Eye-lens of the ocular 25 Bye- piece 25 Micrometer 129 Parfocal 45 Eye -point 7, 37, 130, 142 Ocular, demonstration 37 Eye-shade, adjusting 73 Farrants' solution 276 Fibers, examination in Textile 181 Field 32 Camera lucida 121 Illumination 52, 61 Orthoscopic ocular j 27 Periscopic ocular 27 View with Microscope_32-34, 118, 142-145 Size of, with different objec- tives and oculars 33, 34 Field-lens, of ocular 25-27 Action 37 Dust on 101 Filar, micrometer ocular 26, 30 Ocular micrometer ^-'SS Filtering balsam 272 Fine adjustment, Frontispiece ; Testing .. 74 Fir, balsam of 272 Fixation 284 Fixative, albumen, Mayer's 271 Fixer , 284 INDEX luid, Miiller's 275, a8o', 283 luid, Zenkers' 283 uoritelens . 15 Deal distance, or point, principal 4, 7, 35 Length equivalent .. 13 3CUS 6 Actinic 223 Chemical ; 15 Conjugate 4, 6 Equivalent, of objectives and oculars 13, 29, 190 Principal 4, 5, 7 Virtual 3 Visual 15, 223 Dcusing, 7, 39 Adjustments, testing 74 Compound microscope 39 Experiments 43 Glass 207, 210 High objectives 45 Low objectives 43 Objective for micro-spectro- scope 166 Photo-micrography 226 Screen for photo-micrography 210, 226 Simple microscope 7, 39 Slit of micro-spectroscope 167 ood, detection of adulteration 182 orm of objects, determination 103 ormal 260 ormalin__ _... 276 ormaldehyde . 276 Dissociator 275 Isolation 260 Percentages 270 ormula, for aperture 20 Cataloging 262 Desired percentages 270 Equivalent focus 191 Refraction 62 raunhofer lines 158-159 ree, hand sections 289 Working distance 39 ront combination or lens of ob- jective 12-14 rontal sections 321 uchsin, acid 281 Basic 275 Picro 281 unction of objective 34~35 Ocular 36 G auge, cover-glass 250 auze, absorbent 246 elatin, liquid . 279 Geometrical construction of images 5 Glass, cleaning mixture 251 Ground 34, 210, 225 Rod appearance under micro- scope 106, 107 Slides or slips 245 Glasses, graduate 269 Watch 260 Glue, liquid, preparation of 279 Glycerin 277 Congo _ 275 Mounting objects 255 Glycerin jelly for 277 Anatomic preparations 277 Microscopic preparations 277 Mounting objects 255 Glycogen, iodin stain . 278 Graduates 269 Greeuough's binocular microscope : 112, 113 Ground glass, focusing screen.210, 226 Preparation 34 Gun cotton 274 H Half-tones 324 Hardening collodion 305 Tissue 284 Hematein 278 Hematoxylin, chloral 278 Hemoglobin spectrum 159 High school microscope 75 Histology, physiologic 264 History of photo-micrography 217 Holder, lens 10, 124,319 Slide 302,312 Homogenous immersion, conden- ser 54 Objective 14, 19-23 Cleaning 69 Experiments 68 Numerical aperture I9 _2 3 Homogenous liquid 14 Tester 68, 188 Hones and honing 288 Horizontal camera 233 Huygenian ocular 26, 27, 37, 130 I Illuminating, cone, aperturel 52 Objective 16, 184 Power 24 Illumination 48 Abbe camera lucida 150 Artificial 42, 56, 60 Air and oil 103-106 Centering image 51 INDEX 35 1 Dark ground 42, 56-60,198 Daylight 40 Entire field 61 Lamrj 61 Methods 39-64 Micro-polariscope 174 Micro-spectroscope „^_. 165 Oblique with air and oil 104 Opaque objects 166, 239 Photography 1 226 Photo-micrography 232 Wallaston's camera lucida .^..144 Illuminator L48-58 Vertical 16, 184, 240 See also condenser. Image, aerial , 35, 37 Color 64, 68 Flame 52 Geometrical construction 5 Inverted, real of objective 35 Object,size and position^, 9, 11, 122 Real s, 9, 11, 12, 35-37, 116 Refraction 61, 68 Retinal 7, 11, 36 Swaying of 56 Virtual 5,7, 9, II, 123 Image-power of objectives 22 Imbedding 293, 305 Immersion, fluid or liquid. 14, 68, 273 Illuminator 54 Objective 14, 20, 68-69 Incandescence or line spectra 158 Incident light 4P Index, medium in front of ob- jective 20-23 Refraction 62 Indicator ocular 197 Infiltration, collodion 304 Paraffin 291 Paraffin dish 292 Ink for labels, catalogs, drawing. 324 Interpolation 20 Interpretation of appearances un- derthe microscope 99-115 Iodin stain for glycogen 278 Iris diaphragm 181 Irrigating with reagents 255 Isochromatic plates , 214 Isolation 259 Formaldehyde . 260 Nitric acid 261 Isostigmar objective 206 Isotropic 174 J-K Japanese filter or tissue paper 70 Jar for slides, etc . . 246-249 Jejly, glycerin 277 Jena glass 12 Jurisprudence, micrometry 140 Knife, sharpening 286-289 Support 299 Labels and catalogs 262 Labeling microscopical prepara- tions 262 Photographic negative 207 Serial sections 323 Laboratory compound microscope 74 Table _: 73 Lamp, acetylene 42, 6o, 226 Alcohol or spirit 301 Black 279 Condenser 153, 330 Electric 42, 226 Petroleum 42, 60, 226, 233 Lantern 200 Slides , ^242 Law of color 160 Lens, concave 3 Converging 3 Convex 3-6 Eye 25 Field 25-27 Fluorite 15 Holder 10, 124, 319 Paper 70 System 11 Thick .__, 3 Letters, in stairs 102 Photo-engraving , 324 Lettering oculars 29 Light, with Abbe illuminator 56 Acetylene 42, 60, 226 Artificial 42, 56, 60, 226 Axial 41, 48, 56 Direct 40 Central 41, 48, 104 Electric 42, 226 Incident 40 Mirror 42, 43 Oblique 41, 48, 56 Petroleum 42, 60, 226, 233 Photo-micrography ._ 226 Polarized 173 Reflected 40 Sun 226 Transmitted 41 Utilized with different objec- tives , 21 Vertical illuminator 240 Wave length of 164 Welsbach ' 42, 226 Lighting L 39-80 INDEX Abbe camera lucida 150 Artificial 42, 56, 60, 232 Experiments 42 Horizontal camera 233 Micro-polariscope 174 Micro-spectroscope 165 Mirror 43, 48 Daylight 40, 226 Photography 210, 213 Photo micrography 228 Vertical illvrminator 240 See illumination me spectrum T 58-i59 quid, currents in 108 Homogeneous 14, 68, 273 3cker, Laboratory or Student 268 Dngisection 317 M agnification, of compensation oc- ulars ;_ 28 Effect of adjusting objective__i38 Determination 16-140, 191 Eikonometer 137 Expressed in diameters 116 Initial or independent 191 Microscope 116 Microscope with Abbe camera lucida 151 Microscope, compound 119 Microscope, simple 117 Photo-micrographs 232 Real images 116 Table 126 Projection microscope 117, 330 Varying with compound micro- scope 123 Velocity 108 agnifier, tripod . 9, 117, 207 arker for preparations 80 arking objects 80, 199 Negatives 213, 238 Objectives 32 asks for preparations 201 easure, metric, cover 2nd p 118, 140 Unit of, in Micrometry 127 Wave length 165 easurer, cover-glass 250 easuring thickness of cover-glass . . 249 easurement with microscope and micrometer, Ch. IV. echanical parts of compound mi- croscope, Frontispiece.n, 74-75 Care ._ 70 Testing 73 echanical stage Fig. 76-78 Mercuric chlorid 279 Crystals 279, 283, 315, 317, 320 Metallic surfaces, photography 239 Preparation 1 241 Metallography, microscope 183 Metals, examination 183, 239 Met-hemaglobin, spectrum 157,169,244 Method, collodion 304-310 Paraffin 291-304 Methyl alcohol 271 Methylated spirits 271 Methylene blue ._. 280 Metric measures and equivalents, cover 2nd p ! 118 Micro-chemistry 176-181 Slides . 1 79 Micro-metallography, objects 183 Micrometer 116 Calipers 249 Cob-web 133 Combined ocular __: 135 Filar m. ocular 133, 134 Filling lines 119 Net 147 Ocular and stage 139 Objector objective 119 Ocular or eye-piece 129-138 Ocular, micrometry 131 Ocular, ratio 132 Ocular, valuation 130, 135, 136 Ocular, varying valuation 136 Photo-micrography 232 Screw ocular 133 Stage 119 Table of magnification 126 Micrometry 126-140 Adjustable objectives 138 Comparison of methods 139 Compound microscope 127 Eikonometer 137 Jurisprudence 140 Limit of accuracy in 139 Ocular micrometer 131 Simple microscope 126 Remarks on 138 Unit of measure in 127 Micro-millimeter 127 Micron 127 Measuring wave length of light_i6s Micro-photograph 217 Micro-polariscope 163, 173-176 Micro-polarizer 173 Micro-projection 153, 200 Drawing 153, 330 Magnification 117 Masks for specimens 201 Microscope, care. ., 70 Amplification n6 INDEX 353 Binocular 112-114 Chemical 178 Clinical ^ 193 Demonstration 192-193 Dissecting 10, 194 Erecting . 1 12-1 13 Field 3 2 -34, M2-I45 Focusing 39 Illumination for, Ch. II. Magnification 116 Metallography 183 Micro-chemical analysis 176 Opaque objects 238 Photo -micrography 227 Polarizing 173 Preparation, with erecting prism 113 Projection 153, 154, 200-201 Price 75 Putting an object under 3: Screen , 69 Stand for large, transparent ob- jects 212 Stand, for embryos 209 Solar 200 Traveling 195, 196 Traversing 182 Microscope compound 11 Drawing 140 Figures,Frontispiecei2, 82-98, 1 13, 178, 182, 193,195, 198,202,227, 230 Focusing 39 High schools 74 Laboratory , 74, S2-98 Lamp 60, 61 Magnification 116-126 Magnification of drawing with Abbe camera lucida 151 Mechanical parts 11, 74 Micrometry 127 Optic axis 12-14 Optical parts 11, 74 Quality and cost 75 Testing 73 Varying magnification 123 Working distance 13, 40, 47 Micro-summar 210 Microscope, simple •„ 1 Drawing 153 Experiments 6 Figures 9, 10, 124, 192, 194, 207 Focusing ,7, 39 Images 7 Magnification 137, 117, 124 Micrometry 126 Obtaining focus, 7 Working distance. . 39 Microscopic, objective 11 Objects, drawing 141 Ocular 25 Slides or slips 245 Tube-length 17-19, 66 Vision 25 Microscopic preparations, cabinet . ._ 264-265 Cataloging 262-264 Labeling 261-264 Mounting 251-261 Trays 266 Micro-spectroscope 155-172 Adjusting 160 Direct vision . 155 Experiments : 167 Focusing the slit 161 Lighting 165 Objectives to use 166 Reversal of colors 155 Slit, mechanism 156, 160 Micro-Tessar objective 209 Microtomes 286 Figures„_290, 295, 296, 297, 306, 307 Micrum 127 Mikron 127 Milk globules, to overcome pedsis. 1 15 Minerals, absorption spectra 171 Minot's microtome 295 Minute objects, arrangement 261 Mirror . 12-14 Abbe illuminator __!__ 55 Arrangement for drawing 146 Concave, use of 43 Dark ground illumination 56-60 Light with, central and oblique 48 Lighting 40 Plaue, use of 43 Mixture, clearing 274 Cleaning 250 Models — 324 Blotting paper 326 Drawing 329 Wax 325 Moist, chamber . 256 Molecular movement 109 Monazite sand, spectrum 171 Mounting 251, 311 Cells, preparation 253-258 Low powers 241 Media and preparation 252 Objects for polariscope 174-176 Permanent 252 Temporary 251 Mounting objects 251 Balsam 257, 272, 311 Dry in air 252 Glycerin 255 Glycerin jelly 255, 257 >4 INDEX Media miscible with water 254 Minute objects __2oi Opaque objects 241 Permanent 252 Resinous media, by drying or desiccation 257-258, 311 Resinous media, by successive displacements 258, 311 Temporary 251 iovement, Brownian, or molecu- lar 109 ucicarmin or mucus stain 273, 315-317 tiller's fluid 280, 283 Dissociator 275 uscae volitantes 1 10 uscular fibers, isolation 261 Polarizing object 175 useum jar 246-247 N atural balsam 272 egative, labeling 207,238 Oculars 25 Record 238 Storing 207 et micrometer 147 eutral balsam 272 Red 280 icol. prism 173 itric acid 280 Dissociator 275 omencjature of objectives 13 on-achromatic condenser 54 Objectives 14 on-adjustable objectives 16, 18 ormal liquid 280 Salt or saline solution 275, 280 ose-piece 31 + 46 Marking objectives 32 Thread or screw-thread 77 umerical apeiture, of condenser. 52 Objectives 19, 23, 187 Table 23 O bject, determination of form 103 Image, size of 12, 122 Marking parts 80+ Marking position 197, 199 Micrometer 119 Mounting 251, 311 Putting under microscope 31 Shading 69 Suitable for photo-micrography 225 Transparent with curved out- lines, relative position in microscopic preparations 103 Objective 11-25 Achromatic 12, 15 Adjustable 14, 16, 64-66 Adjustable, micrometry 138 Adjustable, photo-micr 235 Adjustment 64 Aerial image 35 Aperture 19, 20, 187 Aplanatic 15 Apochromatic 15, 221 Back combination 13, 14 Cleaning 70-72 Collar, graduated for adjust- ment : 65 Cloudiness or dust, how to de- termine 101 Designation 13 Dry 34, 20-24 Equivalent focus 13, 33, 190 Field .__ 33, 34 Focusing for micro-spectro- scope 166 Front combination 13, 14 Function 34~35 Glass for 12, 15 High, focusing 45 Homogeneous immersion. ... 14, 19-23,68 Homogeneous immersion, cleaning 69 Homogeneous immersion, ex- periments 68 Illuminating 16, 184, 239 Image, power 22 Immersion 14, 20, 68 Index of refraction of medium in front 2i_ 23 Initial magnification 191 Inverted, real image 35 Isostigtnar 206 Laboratory microscope 75 Lettering 13 Light utilized 21 Low, focusing 43 Magnification 191 Marking, bv Krauss' method__ 32 Micrometallography 16, 183 Micro-polariscope _. 174 Microscopic 11 Microtessar 209 Micro-spectroscope 166 Nomenclature 13 Non-achromatic 14 Non-adjustable 16 Non-adjustable, table 18 Nose-piece 31, 32, 46,83+ Numbering 13 Numerical aperture 19, 23, 187 INDEX 355 Oil immersion 14 Pantachromatic 16 Para-chromatic _. 16 Par-focal .. . 3 1, 46 Photography __ 206-210 Photo-micrography 221 Projection 16 Putting in position and remov- ing 30 Screw-thread 77 Section 16 Semi-apochromatic 16 Table of field 33 Terminology 13 Unadjustable 16 Variable 16 Visual and actinic foci__ 223 Water immersion 19-23, 66 Working distance 13, 39, 40, 47 Oblique light, with Abbe illumi- nator 56 Mirror 48, 57 Ocular, various forms 25-28 Aplanatic 15 Cleaning 70-72 Cloudiness, how to determine and remove 71,101 Compensation 28, 29 Equivalent focus 29, 33, 191 Eye-point 26, 37 Field-lens ._' 37 Filar or screw micrometer 30, 133-136 Function 36 Huygenian 26, 27, 37, 130 Indicator 80, 197 Iris diaphragm 181 Lettering and numbering 29 Micrometer, micrometry 129-136 Negative 25 Parfocal 27, 43 Photo-micrography 221 Pointer 80, 197 Positive 26 Power 29 Projection 29, 223 Searching 28 Spectroscopic 155 Standard size 30 Table 26 Working 28 Oil, and air appearances and dis- tinguishing optically 103-106 Cedar-wood 273 Oil-globules, with central and oblique illuminations 104 Oil immersion objectives 14 Opaque objects, lighting 183, 238 Photography 238 Optic axis 2, 3, 12 Condenser or illuminator 54 Crystals 175 Microscope 12-14 Optical 1 Center 2 Focus 15 Parts of compound microscope Frontispiece and , 11, 74 Section 108 Order of procedure in mounting objects dry or in air 252 Glycerin and glycerin jelly 255 Resinous media by desiccation.257 Resinous media by successive displacement 258, 311 Ordinary ray, with polarizer 173 Orthochromatic plates 214 Orthoscopic ocular, field 32 Outline distinctness 105 Oven paraffin 293 Over-correction 5 Oxy-hemoglobin, spectrum.. 159, 169 Paper, bibulous, filter, lens, or Jap- anese for cleaning 'oculars and objectives 70 Blotting for models .^1 326 Paraffin 280 Filtering 281 Infiltrating 292 Imbedding 293 Method 291-304 Oven 293 Removing from sections 302 Wax 280 Parfocal objectives A 47 Oculars .._ 43-45 Pedesis 109, no Overcoming 115 Polarizing microscope no Penetrating power . 24 Pentration of objective 24 Percentages, of liquids 270 Permanent, mounting 252 Preparation of isolated cells 260 Permanganate of Potash,, spectrum . 157, 168 Petri dish 249 Photographing bacterial cul- tures ... ....243 Petroleum light 42,60, 226, 233 Pharmacological products, exami- nation 182 Photo-engraving, drawing and let- tering 324 356 INDEX Photographic, camera 203-205 Negatives 207-213, 238 Objectives 206, 209, 210 Prints 207 Photography of bacterial cultures_243 Color-correct 214 Colored objects 2r4 Compared with photo-micro- graphy 217-220 Embryos 209 Focusing and exposure 204-206,210 Indebtedness to photo-micro- graphy 217 Large transparent objects. 2 12-2 14 Lighting 206, 213, 239 Metallic objects 239 Objectives 206, 209, 210 Objects in alcohol or water 204 Opaque objects 238, 241 Plates 214 Stage 208 Vertical camera 204-2 10 Photo-micrograph 217 Determination of magnification 232 5-20 diameters 209 20-50 diameters 228 100-2000 diameters 232-236 Metallic surfaces. 238-242 Objects suitable 225 Opaque objects 238-242 Prints 207 Plates 214 Reproductions 234 With and without an ocular ___ 229-237 Photo-micrographic, camera , r 219-222, 233 Outfit : 220 Stand 227, 230 Photo-micrography 203-244 Apparatus 220, 233 Compared with ordinary pho- tography 215, 220 Condenser 49, 224, 229 Distinguished from micro-pho- tography 217-220 Cover-glass correction 235 Experiments 226 Exposure_2ii, 214, 217,231,236,242 Focusing 206, 210, 226 Focusing screen 206 Lighting 210, 223-226, 229, 232, 239, 240, 243 Micrometer formagnification_.232 Objectives and oculars 16, 221, 237-240 Staining preparations 215 Vertical camera 208, 219, 222 With and without ocular. .229-237 Record table 238 Physiologic histology 264 Picric-alcohol 281 Picro-fuchsin 281, 314 Pillar of microscope, Frontispiece. Pin-hole diaphragm 54 Pipette 300, 311 Egg 3'9 Plane mirror, use 43 Plates, color-correct 214 Isochromatic or orthochro- matic 214 Pleochroism 175 Pleurosigma angulatum 48 Point, axial 19 Burning 7, 35 Eye 37, 130, 142 Pointer ocular 80, 197, 176 Polarized light, extraordinary and ordinary ray 173 Polarizer and analyzer 162, 173 Polarizing microscope, pedesis no, 175 Position of condenser 54 Objects or partsof same object. 102 Positive oculars 12, 26 Power, of microscope * 116 Illuminating, penetrating, re- solving, of objective 24-25 Ocular 29 Preparation of reagents 268-283 Preparations, cataloging 261 Cabinet 264 Labeling 262 Permanent 252 Temporary 251 Principal, focus 3, 4, 7 Focal distances 4, 35 Optic axis 2, 7, 12 Prism of Abbe camera lucida. 144-148 Amici 155 Comparison 164 Dispersing 158 Erecting 113 Nicol 173 Slit of micro-spectroscope, mut- ual arrangement 161 Wollaston's camera lucida. 143-144 Prints, photographic 207 Projection, microscope 153, 154, 200- 2or, 330 Objective 16 Ocular 29, 223 Pyroxylin 274 INDEX 357 Q-R Quandrant for camera lucida_i46, 148 Ramsden circle or disc 37 Ratio, ocular micrometer 132 Razor and support 299 Reagent 268-283 Board ., 268 Bottle 271 Real image 6, 9, n, 12, 35-37, 116 Record, of embryos 211 Negatives 213, 238 Record table, collodion method --310 Negatives 238 Paraffin method 303 Red, congo 275 Neutral 280 Reflected light 40 Reflection, total 64 Refraction, images 61, 68 Index 62 Medium in front of objective.. 20-23 Refractive, doubly 175 Highly 107 Singly . -.175 Relative position of microscopic objects 103 Resinous media, mounting objects,257 Resolution and numerical aper- ture 24 Resolving power 24 Retinal image 7, 9, n, 12 Revolving nose-piece 3t, 32 Ribbon sections 296 Deparaffining 302 Electrification . 298 Spreading.: 298 Storing 298 Tray 266, 268 S Sagittal sections 322 Salicylic acid, crystallization 58 Salt solution, normal ....280 Scale, of drawing 151 Size of photographs 204 Wave lengths 164 Scales, chemical 269 Screen, color 215-217 Focusing for photography. 206, 210 Ground glass 34 Microscope 1 69 Screw, society 7° Micrometer 30, 133-135 Sealing cover-glass 254, 256 Searching ocular 28 Secondary axis 3^7 Section knife and sharpening i.. 287-289 Lifter 309 Optical 108 Sections, arrangement of tissue 294 Clearing 274, 311 Cutting 280-323 Dehydration 311 Deparaffining 302 Extending with water 298 Fastening to slide 298-303, 308 Free hand 289 Freezing 290 Frontal 321 Ivongi- 317 Mounting 311 Ribbon 295 Sagittal 322 Serial 317 Spreading or stretching by heat 298 Staining 310 Surface 318 .Trans-. 317, 321 Transferring 308 Vertical 318 Selenite plate for polariscope 176 Semi-apochromatic objective 16 Serial sections 3 T 7~3 2 3 Embryos 320-323 Sharpening section knives 288 Shell vials 259, 285 Shellac cement 253, 281 Significance of aperture 23 Silvering 281 Simple microscope, see under mi- croscope. Sines, table of, 3d page of cover. Slides... 245 Cleaning 245-247 Holder 302 Micro-chemistry 179,245 Tray 266-268 Sliding microtome 306-307 Slips, glass.... 245 Slit mechanism of micro-spectro- scope J 156, 160 Society screw 76 Sodium, lines and spectrum -__ 157, 158, 244 Solar microscope.' 200 Spectrum or s. of sunlight 157. 158, 244 Soluble cotton 274 Solution, Farrants' 276 Percentage 270 Saturated 269 Spectral, colors 158 358 INDEX Ocular 155, 160 vSpectroscope . 155 Direct vision 155, 167 Spectroscopic, examination of color-screens _'. 2 16 Ocular 155 Spectrum 157-172, 244 Absorption 158, 159, 160, 167 Analysis 172 Angstrom and Stokes' law 160 Banded 170 Blood ,168 Carbon monoxide hemogloblin.169 Carmin solution _ 170 Colorless bodies 171 Color screens -244 Comparison 164 Complementary 160 Continuous 158 Double 164 Incandescence 158 Line 158 Met-hemoglobin 157, 244 Minerals, monazite sand __^ 171 Oxy-hemoglobin 159, 169 Permanganate of potash .... ... 157, 168, 244 Single-banded of hemoglobin . 159, 169 Sodium 157, 158, 244 Solar 157, 158, 244 Two-banded of oxy-hemoglo- bin .169 Spherical aberration 4, 5 Test 185 Stage, Frontispiece, mechanical.. 82 Stain, alcoholic and aqueous 311 Counter 310-317 Elastic 315, 275 Staining 260, 310-317 Stand, microscope 75 Photo-micrographic 98, 227 Special for embryos 208 Special for large transparent objects 212 Standard, distance (250 mm. ) at which the virtual image is measured 123 Screw • 77-80 Size for condenser 30, 55 Size for oculars _, 30 Starch, determination by polari- scope 175 Stender dish 260, 305 Stokes and Angstrom's law of ab- sorption spectra 160 Strops and stropping 289 Storing negatives 207 Preparations 264 Ribbons of sections 298 Student locker 268 Substage, Frontispiece. Substances for crystallography 179-180 Sudan III 282 Sulphonal with polarizer 176 Sulphuric or sulfuric ether 276 Support for knife of microtome 299 Surface sections 318 Swaying of image 56 System, back, front, intermediate of lenses ; 12-14 Crystal 180 Metric, cover 2nd p 140 T Table, black 282 Collodion method 3(0 Immersion fluid 189 Laboratory 73 Magnification and valuation of ocular micrometer 126 Oculars 26 Tube-length and thickness of cover-glasses 18 Natural sines, third page of cover. Numerical aperture - 23 Paraffin method 303 Record, photography 238 Size of fields 33 Testing homogeneous liquids.. 189 Valuations of ocular microme- ter 126 Weights and measures, 2d page of cover. Temporary mounting 251 Terminology of objectives 13 Test of chromatic and spherical aberration 185-188 Tester, cover-glass 250 Homogeneous liquids 68, 188 Testing a camera '_ 220 A microscope and its parts 73 Test-plate, Abbe's, method of using 185-187 Textile fibers, examination m, i8r Thickness, of cover-glass for non- adjustable objectives 18 Serial sections 321 Thread, standard for nose-piece and objective 77 Tissues, arranging for sections 294 Fixing or hardening 284-286 Washing apparatus 286 Transections 317, 321 Transferring sections 308 INDEX 359 Transmitted light 41 Traversing microscope 182 Tray for ribbons or slides 266-268 Triplet, Hastings 10 Tripod 9, 117 Focusing glass 207 Tube of microscope, Frontispiece. Tube-length 17-19 Cover-glass adjustment 66-67 Importance 66 Various opticians, table 18 Turn-table 253 u Utramicroscopy 59 Unadjustable objectives 16 Under-correction 5 Unit of measures, in micrometry. 127 Wave length 165 .Valuation of ocular micrometer 126, 130, 135, 136 Variable objective 16 Varying magnification of com- pound microscope 123 Varying ocular micrometer valua- tion 136 Velocity under microscope 108 Vertical, camera 203, 222 Illuminator 16, 184, 239 Sections : 318 Vials, preparation and shell 259, 284-285 Blocks 259, 26S Virtual image 7, 9, 11, 12, 36 Standard distance at which measured 123 Visibility with objectives 24 I Vision, double 1 16, 118 I Microscopic 25 w Washing apparatus for tissues 286 Waste bowl 309 Watch glass- 260 Water immersion objective 19-23, 66 Light utilized 21 Numerical aperture 22, 23 Water, bath 153 1 Wave length, designation 165 Scale 164 Wax, bees 325 Models 325 Paraffin 280 Weigert's elastic stain 275, 315 Weights and measures, see 2d page of cover. Welsbach light ., 226 Wenham's binocular microscope if 2, 114 Wollaston's camera lucida I2i, 124, 143, 144 Work-room for photo-micrography 221 Work-table, position, etc. ._ 73 Working, distance of microscope or objective 13, 39, 40, 47 Ocular 28 Writing diamond 319 X Xylene 273 Balsam 272 Xylol, German form of xylene 273 Zenker's fluid 283 TABLE OF NATURAL SINES Compiled from Prof. G. W. f ones' Logarithmic Tables Minutes i'o. 00029 2 0.00058 3 0.00087 4 0.00116 5 0.00145 6 0.00175 7 0.00204 8 0.00233 9 0.00262 10 0.00291 n 0.00320 12 0.00349 13 0.00378 14 0.00407 15 0.00436 16 0.00465 17 0.00495 18 0.00524 19 0.00553 20 0.00582 21 0.00611 22 0.00640 23 0.00669 24 0.00698 25 0.00727 26 0.00756 27 0.00785 28 0.00814 29 0.00844 30 0.00873 31 0.00902 32 0.00931 33 0.00960 34 0.00989 35 0.01018 36 0.01047 37 0.01076 38 0.01105 39 0.01 134 40 0.01164 41 0.01193 42 0.01222 43 0.01251 44 0.01280 45 0.01309 46 0.01338 47 0.01367 48 0.01396 49 0.01425 50 0.01454 51 0.01483 52 0.01513 53 0.01542 54 0.01571 55 0.01600 56 0.01629 57 0.01658 58 0.01687 59 0.01716 60 0.01745 Degrees and Quarter Degrees up to 90°. 1 i°,i5 i>3° 1. 45 2 2,tS 2,3° 2,45 3 3.15 3.3° 3.45 4 4,15 4,30 4,45 5 5,15 5,3° 5,45 6 6,15 6,30 6,45 7 7,15 7,3° 7.45 8 8,15 8,30 8,45 9 9,15 9,3P 9,45 10 10,15 10,30 10,45 11 ",I5 ",30 u.45 12 12,15 12,30 12,45 13 13,15 x 3,3o '3,45 14 14,15 14,30 14,45 15 15,15 15,30 15,45 0.01745 '0.02181 0.02618 0.03054 0.03490^7 0.0392617,15 0.04362117,30 0.04798 17,45 0.05234 0.05669 0.06105 0.06540 0.06976 0.0741 1 0.07846 0.08281 0.08716 0.09150 0.09585 0.10019 0.10453 o. 10887 0.1 1320 0.1 1 754 0.12187 0.12620 o. 13053 0.13485 0.13917 0.14349 0.14781 0.152 12 0.15643 0.16074 o. 16505 o. 16935 0-17365 0.17794 0.18224 o. 18652 0.19081 0.19509,26,15 0.1993726,30 16°, l6°,i 5 16,30 16,45 18 18,15 18,30 18,45 19 19,15 19,30 19,45 20 20,15 20,30 20,45 21 21,15 21,30 2i,45 22 22,15 22,30 22,45 23 23,15 23,30 23,45 24 24,15 24,30 24,45 25 25,15 25.30 25,45 26 0.20364 0.20791 0.2 12 18 O.21644 0.22070 O.22495 0.22920 0-23345 O.23769 O.24I92 0.246I5 O.25O38 O.25460 0.25882 O.263O3 O.26724 O.27I44 26,45 27 27,15 27,30 27,45 28 28,15 28,30 28,45. 29 29,15 29,30 29,45 30 30,15 30,30 30,45 O.27564 'O.27983 O.28402 0.28820 O.29237 O.29654 O.3OO7I O.30486 O.3O9O2 O.3I316 O.3I73O O.32144 C32557 O.32969 0-3338I 0.33792 O.34202 O.34612 0-3502 0.35429 0.35837 O.36244 O.36650 O.37056 O.3746I O.37865 O.3826S O.3867I O.39073 0-39474 0-39875 0.40275 O.40674 O.4r072|39,I5 0.41469 39,30 3i°, 3i°,i5 31,30 3i,45 32 32,15 32,30 32,45 33 33,i5 33,3° 33,45 34 34,15 34,3o 34,45 35 35,15 35,30 35,45 36 36,15 36,30 36,45 37 37,15 37,3o 37,45 38 38,15 38,30 38,45 39 0.41866 0.42262 0.42657 0.43051 o-43445 0.43837 0.44229 0.44620 0.45010 o. 45399 0.45787 0.46175 0.46561 0.46947 o.4733 2 0.47716 0.48099 0.48481 0.48862 0.49242 0.49622 0.50000 0.50377 0.50754 0.51129 39,45 40 40,15 40,30 40,45 41 4i,i5 41,30 41,45 42 42,15 42,30 42,45 43 43,15 43,30 43,45 44 44,15 44,30 44,45 45 45,15 45,30 45,45 0.51504 0.51877 0.52250 0.52621 0.52992 0.53361 o.5373o 0.54097 0.54464 0.54829 o-55i94 o. 55 557 0.55919 0.56280 0.56641 0.57000 o-57358 o-577i5 0.58070 0.58425 0.58779 o-59'3i 0.59482 0.59832 0.60182 0.60529 0.60876 0.61222 0.61566 0,61909 0.62251 0.62592 0.62932 0.63271 0.63608 0.63944 0.64279 0.64612 0.64945 0.65276 0.65606 0-65935 0.66262 0.66588 0.66913 0.67237 0-67559 0.67880 0.68200 0.6S518 0.68835 0.69151 0.69466 0.69779 0.70091 0.70401 0.707 1 1 o.7ioi9J6o,i5 0.7132560,30 0.7163060,45 46 , 0.71934 46°, 15' 0.72236 46,30 0.72537 0.72837 0.73135 o.73432 0.73728 0.74022 o.743i4 0.74606 0.74896 0.75184 o.7547i o.75756 0.76041 0.76323 0.76604 0.76884 0.77162 o.77439 0.77715 0.77988 0.78261 0.78532 46,45 47 47,15 47,30 47,45 48 48,15 48,30 48,45 49 49,15 49,3o 49,45 50 50,15 50,30 50,45 51 5i,T5 5i,3o 51,45 52 52,15 52,30 52,45 53 53,15 53,30 53,45 54 54,15 54,30 54,45 55 55,15 55.30 55,45 56 56,15 56,30 56,45 57 57,15 57,3o 57,45 58 58,15 58,30 58,45 59 59^5 59,30 59,45 60 0.78801 o. 79069 0.79335 0.79600 0.79864 0.80125 0.80386 0.80644 o. 80902 0.81157 0.81412 0.81664 0.81915 0.82165 0.82413 0.82659 0.82904 0.83147 0.83389 0.83629 0.83867 0.84104 0.84339 0.84573 0.84805 0.85035 0.85264 0.85491 0.85717 0.85941 0.86163 0.86384 0.86603 0.86820 0.87236 0.8725075,45 61°, 0.87462 61°, 15' 0.87673 61,30 0.87882 0.88089 0.88295 0.88499 0.88701 0.88902 0.89101 0.89298 0.89493 0.89687 0.89879 0.90070 0.90259 0.90446 0.90631 0.90814 0.90996 0.91176 o.9i355 0.91531 0.91706 0.91879 0.92050 0.92220 0.92388 0.92554 0.92718 0.92881 0.93042 0.93201 o.93358 0.93514 0.93667 0.93819 093969 0.94 1 18 0.94264 0.94409 0.94552 0.94693 6.94832 0.94970 0.95106 0.95240 0.95372 0.95502 0.95630 o.95757 0.95882 0.96005 0.96126 0.96246 0.96363 0.96479 0.96593 0.96705 0.96815 0.96923 6i,45 62 62,15 62,30 62,45 63 63,15 63,30 63,45 64 64,15 64,30 64,45 65 65,15 65,30 65,45 66 66,15 66,30 66,45 67 67,15 67,30 67,45 68 68,15 68,30 68,45 69 69,15 69,30 69,45 70 70,15 70,30 7o,45 7i 7i,i5 71,30 71,45 72 72,15 72,30 72,45 73 73,i5 73,3o 73,45 74 74,1.5 74,30 74,45 75 75,15 75,3o 76°, 76°,i5 / 76,30 76,45 77 77,15 77,30 77.45 78 78,15 78,30 78,45 79 79,i5 79,30 79,45 80 80,15 80,30 80,45 81 81,15 81,30 8i,45 82 82,15 82,30 82,45 83 83,15 83,30 83,45 83 84,15 84,30 84,45 84 85,15 85,30 85,45 85 86,15 86,30 86,45 86 87,15 87,30 87,45 87 88,15 88,30 88,45 88 89,15 89,30 . 89,45 90 .- 1 - -, - - > : '---' : ' - -■■ ,:- i: ..■■ • . ; leSs , I. - ■ '<• I' , - ^ : :■ ■;■•. ii$S%H**iw&£h ..- >:;*''>- ' mm