x ae oR O RN RS Serer Lt) Seale ine ion EELS are Eee RP EL 3 Pe ET TCR Satalateaa o SUL t ey Sean Sere) Sy RTS ee CORNELL UNIVERSITY. THE Roswell P. Flower Library THE GIFT OF ROSWELL P. FLOWER FOR THE USE OF THEN. Y. STATE VETERINARY COLLEGE 1897 2787 MEDICAL MICROSCOPY. OERTEL. MEDICAL MICROSCOPY DESIGNED FOR STUDENTS IN LABORATORY WORK AND FOR PRACTITIONERS io” sa T. EOERTEL, M.D. PROFESSOR OF HISTOLOGY, PATHOLOGY, BACTERIOLOGY, AND CLINICAL MICROSCOPY, MEDICAL DEPARTMENT, UNIVERSITY OF GEORGIA With 131 Ittastrations, some of ahich are Colored PHILADELPHIA P. BLAKISTON’S SON & CO. IOI2 WALNUT STREET 1902 o RB a 8 bag" f No. 14 ble Copyright, 1902, By P. B. BLAKISTON’S SON & CO. PRESS OF WM. F. FELL & CO., 1220-24 SANSOM ST., PHILADELPHIA, PREFACE, In this day of multiplicity of medical books none should venture to afflict the medical public with a vol- ume which is not called for by some legitimate voice. Believing this, I have still brought forth the following work. Microscopy is a new and a growing science. Those of the profession who were graduated more than a decade ago for the most part received scanty instruction in lab- oratory work, and since that time much has been added to the technique of medical microscopy. There are many who are unable to avail themselves of a post-graduate laboratory course who desire to do such microscopic investigation as will be helpful to them in their daily practice. The more complete works on microscopy offer to such an one a bewildering mass of material from which he is unable to cull that which will be of service to himself. It is to the beginner in microscopy, and particularly to him who must work without the advantage of the personal guidance of a teacher, that the book may prove of value. : It will be noticed that usually only one method is given whereby to reach a certain result. This is the method which has proved the best for routine work and which is at the same time the most simple and the least liable to error. Claim of originality can be made only in the matter of presentation of the subject. The work is necessarily a Vv vi PREFACE. compilation from authorities upon the questions of which it treats. I am particularly indebted to Mr. Wm. Bausch for the use of cuts from his valuable little book, ‘‘ Manipulation of the Microscope,”’ and much of the material in the first section has been gleaned from the same source. Nearly all the text-books upon clinical microscopy, bacteriology, and pathology have been consulted and credit given in the text wherever direct quotation has been made. I wish to express my thanks to my friend, Mr. Wm. R. McKinley, to my assistants, Drs. Kellogg and Fargo, and to my wife for aid in proofreading and indexing the work, and to Messrs. P. Blakiston’s Son & Co. for their uniform courtesy and their skilful product of the bookmaker’s art. THE AUTHOR. AuGustTa, GEorcia, November 1, 1902. CONTENTS. THE MiGROSCOPE) jc.) oy op eh oo ese a eee eeeeaes The Compound Microscope,................ Parts of the Microscope,................... Choice of Microscope,..................... Manipulation of the Microscope,............ How to Look through a Microscope,........ PREPARATION OF TISSUE,..........00 0000000005 FEA LION oo ANS capes aheee vad Does Ia ohare a Dehydration; a. .ikeheres eyes new seg Ke Infiltration, 024% 00 ay pad beans Ped Raeecn yd PA MOGUMtins, oo ciate ts tue Ree het RAR The Paraffin Method,...................... Section Cutting; s.as:¢edsacd Rew Ad se eeans oe Cutting Paraffin Sections,.................. The Freezing Microtome,.................. Staining Celloidin Sections,............... Staining Paraffin Sections on the Slip,...... Staining Celloidin Sections,.. ............. Staining Paraffin Sections,................. BACTERIA 4 dccgd.g Hive HA AA ERR Ge DRAPER Baill 5 acs terien dee cid. Mees nb tr beeevaaie e heeete Growth of Bacteria,....................... BACTERIOLOGIC METHODS,...............0000005 List of Necessities for Bacteriologic Work,.. Preparation of Culture-media,.............. STAINING METHODS,.........0.00020000se eee eeee Recapitulationy acest aves eee eee rk PATHOGENIC BACTERIA,......000 00000 ccc ene eee Hydrophobia, Rabies,..................... Veasts,: nse ne vi vaiasedaee regents eeeaiyics MY COSES oo 5ise east a Se eee sik Se aaa Mea ACRE ACUHOMYCOSIS:,. ois6o sce aie ee Minded ee nek eee S SLUMORS) fe Sin nis Yesicisigs Pega So gee ee ae Reve oe Epithelial Tumors,.................0.00008 PAGE 35 35 39 40 43 45 46 48 49 50 51 » 56 vill CONTENTS. PAGE ULHE BROOD, aun y ie enaise a BR eae der AES ES ee ara DERE Lew 231 White Blood-corpuscles of Normal Blood,..................+. 236 Colorless Cells peculiar to Pathologic Conditions of the Blood,.. 240 POCHHIGQUE), eae cactcca os Git BES OAT AIRY Ried eobag dees 241 tal): ole tee gtr death oe We cha ELISE RUA bE tah MRE ata tee a 245 Number of Blood-corpuscles,.............. 00.002. c cee eee 248 Enumeration of Red Blood-corpuscles,..................00.. 249 Technique of Counting Red Blood-cells,...................00, 251 Enumeration of White Blood-cells,................0 002200005 254 VARIATIONS IN THE FORMED ELEMENTS OF THE BLOOD,........ een Lot Red Blood-corpuscles,...........00 00.00. eee 257 Increase in the Number of White Blood-corpuscles,.......... 259 Changes in the Blood of Leukemia Produced by Intercurrent DISEASES; c cease aware same yee ae OR RED Eee a tat 265 Bacteria: im the: Blood) es. gece igcvimsw bbs wawea Ga mawalenu vues 287 WURINE pote Fscce Sredue’, d dacs Eta aAWAe PAG LARGE ARLES aah weaeede day 291 Microscopic Examination of the Urine,...................... 292 AGA UIEL As 252-8 Sate ai tafe CMe Gr ty ele eee eat hi necit abe ante fac Re 305 Hémoglobinutia, onss25548 4248868 wondaldowwdaedeweuped ani 305 PYM. 5. arsedie incigctes adhd, b downs eee 1. WH at AND A ee ee teas 307 Usinary Casts c.0iois sake hase deanhae. ala aie aahone Hm pagcaagela Ginahs 313 Wermes: I the: Urine)... cue acaiseg waa ehae a hans basawenees 323 Other Foreign Bodies,..... His. aa2gidee tisouskicas sree ake eid 326 SY SPILES'y face aig snes Win staacn gies We Ree neuen sai Lecd tn Ridin Gelaes RCA aeemee 326 Vesical Calculus,.. 0.0.0.0 000 ccc e eee neeeee 327 Vesical Tuberculosis,..........00000 feces ee 327 CDH SEMEN, cas Sac ager mtey RAR tote A NEUE Rc CLR RE VA BOS 91 Arnold Steam Sterilizer,......0000.0 00.000. c eee eee en dehee 92 Stab Culture) nincexn ots oe ve onc ney iy sitlaraehesnigh Heémin;: Crystals; i jcaawedacd ect ee abe s taleg es Beet eee st 288 Filaria sanguinis hominis, ................ 000000 e ee eee ee 289 Uric Acid Crystals—(Greene), ...............: Paes dae Colored, 296 Some Deposits in Acid Fermentation of the Urine —(Landois), 297 Some Deposits from Ammoniacal Urine (Alkaline Fermentation). (DANES) yc pcg aA in Soe ER SA RAED SERRE RE MORE GORE 298 Calcium Oxalate, ‘‘Envelope” Crystals—(Greene), ........... 299 Forms of Crystals of Ammonio-magnesium Phosphate.— (PYSOM) sy occas 2x de anus situlade tn MR Raa ees eT ER aS BSE 300 Feathery Crystals of Triple Phosphate —(Tyson),............. 301 Calcium Phosphate and Calcium Sulphate Crystals —(Jakob),.. 302 Crystals of Cystin and of Oxalate of Lime,................... 303 Magnesium Phosphate Crystals.—(Gould), ................... 304 Leucin and Tyrosin Crystals—(Coplin),..............0.0000. 304 xii FIG. 117. 118. 119, 120. 121. 122, 123. 124, 125. 126. 127, 128. 129, 130. 131, LIST OF ILLUSTRATIONS. PAGE Pus in the Urine.—(Gould),..... 0... eens 307 Renal and Vaginal Epithelium.—(Greene), ............0--0000- 309 Various Forms of Renal Cells.—(Greene), .......0. 00 cee eee 310 Bladder Epithelium.—(Greene),..... 0. 311 Effect of Low Illumination, Hyaline Casts being Plainly Visible, 314 Effect of High Illumination, Hyaline Casts being Lost in the Flood of Light, and Only the Renal Cells Appear—(Greene), 315 Casts of Leukocytes and of Acid Sodic Urate Crystals—(Lan- DOCS) 5 sates Rats iocaiee 8 te Reece RO Ob evap BR WBA aa APs bl Sabo aceh Suse’ 316 Blood-cells and Blood-cast.—(Landots), ........ 0. cece eee .. 316 Granular Casts.—(Greene), 0... 0. ccc ce eee 317 Waxy Casts.—(Greene), 0... 0c cece ene 318 Waxy Casts and Fatty Casts.—(Greene),....... 00. cece eee 319 Seminal Elements, Some of which May be Found in the Urine — (COpln).: sata deiduss ante wales Rewek ees a6 ga nmen eames ee 330 Curschmann’s Spirals.—(Schmaus), 0.00... 000 ccc ccc eet 342 Cholesterin Crystals.—(Landots),.......00.0 0000 ccc cece 343 Trichina spiralis—(Reeves), 2.0.0... cece cece 349 ‘MEDICAL MICROSCOPY. THE MICROSCOPE. The word microscope is derived from the Greek prxpés, ‘small,’ and oxomos, “a spy.” Microscopes are of two forms—simple and compound. A simple microscope consists of a single lens or a combina- tion of lenses in close contact and united by a cement, and with this we view an object directly and see an image of it in an upright position. A compound microscope is composed of two lenses or systems of lenses, the lower, called the objective, forming an image which is again magnified by the upper lens or ocular. In the compound microscope the rays of light forming the image cross in their passage between the objective and ocular, so that we see a reversed picture of the object, and it appears upside down. Lenses.— When a ray of light passes from one transpar- ent medium to another of greater or less density, it is de- flected from its original course; this action is called re- fraction, and upon this physical quality is based the sci- ence of optics. If we view an object through a glass prism, it will be seen to be in a plane different from its real one, and the amount of deviation thus caused is dependent upon the 2—O 17 18 MEDICAL MICROSCOPY. density of the glass from which the prism is formed and also upon the angle of the sides of the prism. A ray of light passing through a prism is refracted when it enters the surface and again when it emerges (Fig. 1), and is always bent toward the base of the prism. Fie. 1. Fic. 2. If we place two prisms with their bases together, the effect upon a ray of light passing through each would be as shown in Fig. 2; and if the position of the prism is reversed, the rays of light would pass as shown in Fig. 3. Lenses are of two principal forms, and each of these is Fic. 3. in reality only a combination of prisms with curved sur- faces (Fig. 4). If the bases of the prisms are placed together, we have resulting a convex lens which will bring together or converge rays of light passing through it, while if the bases are placed outward, there results a concave THE MICROSCOPE. 19 lens which separates or diverges rays of light which pass through it. In Fig. 5 are shown the various forms of convex and concave lenses—double convex, plano-convex, convex- meniscus, and double-concave, plano-concave, concave- meniscus. Fic. 4. We have seen that parallel rays a, c’ passing through a convex lens are converged (Fig. 6). The point at which these rays meet and cross is called the focal point, or prin- cipal focus, and the distance from the center of the lens b to cis the focal distance. Fic. 5. The line d c represents the principal axis of the lens and the line a c’ its radius. Upon the radius or amount of curvature of the surface of a lens depends its power of con- vergence, so that as the radius becomes longer the mag- nifying power of the lens is decreased. 20 MEDICAL MICROSCOPY. Parallel rays impinging upon a concave lens are di- verged, and such lenses are called dispersing lenses (Fig. 7). As the rays are never united, we have no actual image, but only an apparent or virtual image, which lies upon the a Fic. 7. same side as the object from which the rays come. The point where this image is formed is called the virtual focus. We have also a virtual focus in a convex lens formed upon the same side of the lens as is the object viewed by the projection of the rays. THE MICROSCOPE. 21 The rays coming from the object are converged and im- pinge upon the eye (e, Fig. 8); and if the lines between c and e be sufficiently elongated, we find that they come together and form a virtual magnified image at a b. Spherical Aberration.—It has not been mentioned that these rays passing through the margin of a double convex Rte Pate. k= ~ Abeer" Fic. 8. C A a GF. & Z a 7 Fic. 9. lens are refracted more strongly than those passing through its center. Such is the case, however, and we get in consequence a blurred image when we view an object through the periphery of the lens, while in the center it is clear and well defined. This quality is called spherical aberration. 22 MEDICAL MICROSCOPY. In Fig. 9 we have a graphic illustration of this effect, and here it will be seen that the marginal rays e c being strongly refracted at e, meet at g, while the central rays are brought to a focus at fj. This defect can be partially remedied by employing lenses of different form, and is least in a lens in which the radii of the two surfaces differ as 1:6 and the surface of the shorter radius is placed toward the object. The most efficient method of decreasing spherical aber- ration is by the use of a diaphragm or stop, before or be- hind the lens, which consists of a disc having a circular opening and which cuts off the confusing marginal or aberrant rays and allows only the central rays to proceed and form the image. Chromatic Aberration. — A beam of light passing through a prism is broken up or dispersed into its primary constituents, so that we have formed the colors of the spectrum—violet, indigo, blue, green, yellow, orange, and red. As we have seen, every lens, of whatever form, is virtually a prism, and therefore we have in all lenses a greater or less degree of chromatic aberration manifested by color fringes about the periphery of the field and of objects therein. The violet end of the spectrum is more refrangible, and will be first brought to a focus, within the principal focus of the lens. In Fig. 10 we see that the ray a b, impinging upon the surface of the prism, divides, and that upon its emergence the violet ray v v’ is at some distance from the red ray r7’. This effect may be entirely neutralized by placing in the path of these rays a second prism of the same form and density as the first, with the base directed upward; the rays being now brought together, emerge from the second prism as white light. THE MICROSCOPE. 23 But the ray c d now takes the same direction as the ray a b, and as we must have converging rays in order to ob- tain a magnified image, it is not possible to correct chro- matic aberration in this manner. It may in a measure be corrected by combining two Fic. 10. lenses of different density, and consequently of different dispersive power, the one partially neutralizing the effect of the other and producing what is known as an achromatic lens. Such a form is shown in Fig. 11, the convex lens being of crown glass while the concave is of flint glass. This is the simplest form of corrected lenses and meets all ordinary requirements. An aplanatic lens is one in which the chro- matic and spherical aberrations are corrected. It must be remembered that opticians have not yet succeeded in producing a lens of perfect correction, but the higher grades approach per- fection in this respect. Fic. 11. In the compound microscope the objective is the lower lens or system of lenses and forms the first image, which is afterward again magnified by the eye- piece. Objectives are of two kinds—dry and immersion. In the dry, air is the medium intervening between the front 24 MEDICAL MICROSCOPY. of the bottom lens of the objective and the upper surface of the cover-glass. Optical difficulties increase with mag- nifying power, and therefore the higher power lenses are constructed with a view to filling up the space between the front lens of the objective and the cover-glass with a medium of the same refracting power as the crown glass of which the lens is constructed, for purposes to be ex- plained. Water and glycerin were formerly used, but it has been found that thickened cedar oil best meets the requirements, and such a fluid is known as homogeneous immersion fluid, or is, for the sake of brevity, generally called simply ol. It is of vital importance to remember that the highest efficiency can be obtained from a lens only when the oil used is of the same refractive and dispersive power as the hemisphere of the objective, and for this reason the oil furnished by the maker of the particular lens in question should alone be used. Naming of Objectives. Unfortunately until recently each maker has been a law unto himself in naming objectives, many having been des- ignated by arbitrary numbers and letters. In this coun- try there has long been a standard, and the European firms are gradually adopting it. According to this stan- dard, objectives have marked upon them a focal length which indicates their magnifying power. The value of an objective is arrived at by comparing it with a simple lens of equal magnifying power and marking the objective with the focal length of the simple lens. If the combined lenses of an objective give the magni- fying power equal to that of a simple lens of one inch focus, the objective is marked one inch, or if the image from the system of an objective equals in power that of a THE MICROSCOPE. 25 simple lens having the focus of ;/;, the objective is marked qe: As the magnifying power of a lens increases in ratio with a decrease in the focus, a 1 inch objective having an initial or real magnifying power of ten diameters, a #5 inch objective will magnify ten times this or 100 diameters, a qs inch will magnify twelve times this or 120. Powers.—Objectives range in power from 5 inch to ;4 inch. In ordinary work it is found inexpedient to use higher powers than #,, and most workers prefer a +4, inch, which gives sufficient magnification for bacteriologic and blood work. Angular Aperture.—In Fig. 12 the angle C D E represents the angular - aperture of the objective. It is there- fore the angle which is made by the most extreme rays which can enter cS M the lens from the point of focus. This is true only of objectives 4 eA where all the rays entering the front lens are utilized in forming an image, and does not hold good in those ob- a jectives in which the marginal rays Fie, 12. are not all of service, as would be the case if a diaphragm were employed behind the front lens. Objectives of the same power may differ in angle, while those of unequal power may have the same angle. De- finition is directly dependent upon angular aperture, for the reason that as light rays proceed from an object equally in every direction that lens which from a given surface will collect the most rays will show us the object more plainly. This is well illustrated in Fig. 13 and Fig. 14. Here we 26 MEDICAL MICROSCOPY. have two lenses of equal power and focus, a, b, c being the angle of aperture in each case. But the lens in Fig. 13 transmits more rays than that in Fig. 14, and will there- fore produce the more distinct image of the two, though the picture will be no larger than that of Fig. 14. Thus we find that lenses of the same angular aperture will produce an image of equal distinctness, though one be of much lower power than the other. Numerical aperture is the ratio between the focal length of the objective and the diameter of the emergent pencil of light from the back of the objective. The efficiency of an objective depends directly upon its numerical aperture, as was first pointed out by Professor Abbe. Upon objectives the term is indicated by the abbreviation N. A. Resolving Power.—By b € b ¢ this is meant the ability of an objective to bring a a to view minute details. Fic. 13. Fic. 14. If an objective is proper- ly corrected for spherical aberration, its power of definition or resolving power isin direct ratio to its numerical aperture. It goes without saying that this is the chief characteris- tic of a good objective, as our object is to see such struc- tures as we wish to examine not only enlarged, but with the greatest possible distinctness. By reference to the ‘‘ Aperture Table” published by the Royal Microscopical Society we will find that an objective of twice the aperture of another will resolve or show twice the number of fine lines. One with an aperture of 0.0 5 will THE MICROSCOPE. 27 resolve 4.821, and one with an aperture of 0.10 will resolve 9.641 lines to the inch. It will be found necessary when viewing fine structural details to use not only a lens of sufficient angular aperture to define the details, but also to magnify them so that the eye will be able to distinguish them. The number of ruled lines that the normal eye is capable of seeing unaided is about 200 to the inch. We must, therefore, use an objective which will have power enough to bring fine details up to at least this magnification, while at the same time its aperture is such as to enable us to see them distinctly, for we may have an image of much greater magnification with a lens of lower aperture which is indisinct or unresolved. We wish not to see an object as large as possible, but to see its details as clearly as pos- sible. The power of magnification may be increased in several ways; either by using an objective of higher power, by using an eye-piece of higher power, or by increasing the tube length. Suffice it to say that the first method is the preferable one, as by high eye-piecing we lose light and definition and magnify the defects of the initial image of the objective, and by increasing the tube length fall into the same and other difficulties. Penetration.— By this we understand the power to look down into the structure of an object, as in a piece of tissue to see several cells each lying in different planes, at the same time without refocusing. This quality is in inverse ratio to power and angular aperture. With a # inch ob- jective we can see much deeper into a tissue than we can with a 4 inch, and with lenses of the same power but of different angle we would find greater penetration in the one of the lower angle. Penetration is a quality which is of some importance to the worker in tissues as it enables 28 MEDICAL MICROSCOPY. him to see the relations of cells without undue and con- tinued focusing. One must not lose sight of the fact that after all resolution is more to be desired, and an objective of wide aperture should not be condemned because it is lacking in the quality of penetration. Chromatic Aberration.—We have seen that a prism of any angle will break up a pencil of light into its primary colors, thus forming the spectrum, and also that a lens of any form is a modified prism and that the correction of an objective for color is accomplished by combining glasses of proper density. It is not possible, however, entirely to reunite the light ray and produce white light. In the achromatic lenses only two colors, red and violet, are eliminated, the remain- ing rays forming green and purple, and this is known as the secondary spectrum, and we find it most apparent in objectives of high aperture and increased when high ocu- lars are employed. In testing an objective some form of diatome is gener- ally used; and if when using such the light from the mir- ror is made to fall upon it obliquely, a fringe of color will be noticed around its edges, yellow-green upon the side opposite to that from which the light comes, and violet upon the same side as that from which the light comes. For ordinary work this has little bearing upon the effi- ciency of the lens. When central light is employed, color fringes are obviated to a large extent, and in stained tissue sections we seldom see them. If the objective is wnder- corrected we will observe that with central light we obtain about our test object a blue color if the focus is slightly above the object, an orange color if we focus the lens on a point just beneath it. If the objective is over-corrected the reverse effect will be obtained. Oblique light will bring colors more clearly into view. THE MICROSCOPE. 29 The apochromatic objectives first made by Zeiss are corrected for three spectral colors and are more perfectly corrected for spherical aberration than the achromatic ob- jectives. It is also possible to obtain in these objectives a much higher magnification than in the achromatics of the same power, and consequently they are superior in resolving power. The uncorrected error which remains in these lenses is partly overcome by using an eye-piece correspondingly overcorrected, and these are called com- pensating oculars. It is a matter of regret that in the Zeiss apochromats one of the systems contains a lens composed of fluorite which frequently, in our climate, degenerates and renders the objective valueless. The Bausch & Lomb Optical Company now list a series of apochromats after the computation of Professor Hast- ings, of Yale, which they claim are equal in performance to any and are not subject to the above grave fault. For photomicrography apochromats are invaluable on account of their higher resolving power, flatness of field, and the fact that in them the visual and chemical rays are coincident; so that no allowance in focusing must be made. Flatness of Field. If we focus an objective upon a ruled stage micrometer, it will be observed that while the lines in the center of the picture are straight and distinct in outline, as they ap- proach the periphery they become more or less curved and blurred. If we now focus downward slightly, the central lines will become indistinct and those at the edges plainer and less curved. If our objective is well corrected for spherical aberra- tion, the marginal lines only will be affected, and with 30 MEDICAL MICROSCOPY. proper focusing will be shown with almost the same degree of clearness as the central lines originally were. If we are unable by adjusting the focus to obtain a sharp picture of the outer portion of the image, the objective may be con- sidered to be poorly corrected for spherical aberration, and this is a grave defect. It is not possible to entirely correct spherical aberration, and therefore the field of no objective is entirely flat. Spherical aberration is in inverse ratio to the angular aperture, and is increased with the power of the objective, and we should take these points into consideration before condemn- ing an objective. To obtain the best image possi- ble, the object must be brought to the center of the field. Working Distance. By this is meant the distance be- Fie. 15. tween the external surface of the front lens of an objective and the top of the object viewed. As in all objectives there is a slight projection of a metal mounting beyond the lens, and as generally a thin piece of glass is used to cover the object, the practical working distance is thus reduced. Broadly speaking, the working distance decreases with the increase in power and aperture, but the assertion does not hold entirely good, as the optician may, within limits, overcome the difficulties set by these conditions. In low powers the working distance is always quite sufficient for practical purposes, but in selecting the higher powers one should always pay close attention to securing a lens that will permit of the use of a cover-glass of medium thickness. The working distance of an objective is always THE MICROSCOPE. 31 considerably less than the focal distance of a simple lens of the same power. Eye-piece or Ocular. The eye-piece occupies the upper end of the tube and receives the rays which come from the objective and con- ducts them to the eye. The eye-piece serves to increase the magnification of the image which we see by so refract- ing the rays which it receives from the objective that they form an enlarged image within the tube at the virtual focus of the eye-piece. Eye-pieces are of two forms— a positive and negative. WO A negative eye-piece is one in which the focus is within the eye- piece at the diaphragm. A positive eye-piece is one in which the focus lies just below the lower or field lens. Negative eye-pieces are most com- monly used. Fig. 16 shows the con- struction of a negative eye-piece of the Continental type. The upper a Fic. 16. is called the eye lens and the lower e is called the field lens. Atb is the perforated disc or dia- phragm, and here the real image is formed. The image is magnified again by the eye lens, so that we see a virtual image. This is best seen by reference to Fig. 17, in which o g equals the objective and f/ the field lens of the eye-piece. Rays passing from the object ab through o g and f/ form an image at the diaphragm cd. The rays from this image pass on through the eye lens of the ocular and meet the eye, which perceives at ej a virtual image much larger than that at c d. ] Y K SK CK SN Ve B y y He VA y Y y y Z 32 MEDICAL MICROSCOPY. Fic. 17. THE MICROSCOPE. 33 By unscrewing the eye lens and placing on top of the diaphragm a small piece of oiled paper the image here formed may be seen. Compensating eye-pieces have already been spoken of in connection with apochromatic objectives. Mirror. The microscope is provided with a double mirror swing- ing beneath the stage. One side of the mirror is flat or plane, the other side is concave. The mirror is for the purpose of reflecting light through the object into the objective. In this case the object must be thin and trans- parent. If the object is opaque, it may be viewed by re- flected light thrown upon it from above by a mirror or other device. Substage Condenser. For the high powers it is not possible to obtain sufficient light by the use of the mirror alone, and the form of sub- stage condenser devised by Professor Abbe of the firm of Zeiss is in general use. This consists of a series of lenses held in a mounting which fits into the substage ring. The lenses collect the light from the mirror and focus it so that the cone of light falls upon the objective, this cone having approximately the same N. A. as the +4, objective in general use. By the use of the Abbe condenser we are able to better control the light, and while with low powers the condenser is not necessary, it is quite essential when we come to the use of objectives above the } inch, and is indispensable with a 7, inch objective. Two forms of Abbe condenser are made: one (Fig. 18) of two lenses, which has an angular aperture of 1.20; the other (Fig. 19) of three lenses, having an angular aperture of 1.42. The one of 1.20 aperture is generally used, it being 3-0 34 MEDICAL MICROSCOPY. cheaper and answering the purpose for ordinary work. An achromatic Abbe condenser of an aperture of 1.0 is also made, and may be used with advantage in photo- micrography. ‘To obtain the full angular aperture of the Abbe condenser it is necessary to use it as we would an immersion objective. A drop of cedar oil is placed upon the top of the condenser and the slide upon which the ob- ject is to be viewed is laid upon the stage and the con- Fic. 18. ol i Fre. 19. Optical part of condenser 1.42 Aperture denser moved toward it by means of the focusing device of the substage until the drop of oil comes in contact with the bottom of the glass slide. While this is necessary theoretically, the results ob- tained when using the condenser dry are so good as to make the use of the immersion fluid optional in ordinary work. It is troublesome to use it, and for this reason most workers prefer to use the condenser dry. THE MICROSCOPE, 35 THE COMPOUND MICROSCOPE. In the accompanying cut (Fig. 20) is shown the form of microscope known as the Continental, on account of this model having originated on the European continent. This differs from the American model in three important particulars: CONTINENTAL MODEL. AMERICAN MODEL, 1. Horseshoe-shaped base. 1. Tripod-shaped base. 2. Tube length 160.0 mm.(6.4 in.). 2. Tube length 216.0 mm. (8.5 in.). 3. Mirror bar fixed in optical axis. 3. Mirror bar may swing so as to - (This is modified upon some bring mirror above stage for stands, but there is no provision illumination of opaque objects for swinging the mirror above the stage.) Each model hasits stanch advocates. For medical and biologic use the Continental form is the one generally used on account of its compactness and general adaptability to such work. PARTS OF THE MICROSCOPE. The parts of the microscope as shown in the figure are: (A) Base.—In the Continental form, horseshoe-shaped, lead-loaded, and resting on three points. (B) Pillar.—An upright column firmly fastened to the base and supporting the remainder of the microscope. (C) Arm.—This includes the parts for adjustment by micrometer screw, rack, and pinion, and supports to the body. (D) Body.—The tube which carries the optical parts and the movements of which are actuated by the adjust- ments. (E) Nose=piece.—An attachable part which carries the 30 MEDICAL MICROSCOPY. Fic. 20 —TuHe Compounp MICROSCOPE. THE MICROSCOPE. 37 objectives; it may be double, triple, or quadruple. The triple is generally used. The nose-piece revolves, and one is thus enabled to turn into place either objective at will. The Society screw or universal screw for objectives and nose-pieces is now used by all microscope makers. ‘This is a thread of standard pitch and caliber established by the Royal Microscopical Society of London. The objective should be so fitted to the nose-piece that if an object be focused with the low power and the higher powers be then turned into place, they will be approxi- mately in focus, a turn of the fine adjustment being all that is necessary to give a clear image. ‘The objectives are then said to be par-focal. (F) Objective-—This is screwed into the nose-piece and contains the optical parts which make the first image. It is of the utmost importance that the objective be of good quality, as upon it depends in large part the perfec- tion of the final image. (G) Eye-piece or Ocular.—The optical part nearest the eye which receives the image from the objective and again magnifies it. Oculars should be par-jocal; that is, when one is re- moved and another of different magnifying power is in- serted in the tube, there should be no necessity for refocus- ing if the object was in focus with the first eye-piece. (H) Draw=tube.—An inner tube which slides up and down within the body and carries the eye-piece at its upper end. The lower end is generally fitted with a So- ciety screw into which an objective may be screwed if one of very low power is desirable, or which may be used to hold an amplifier or other apparatus. (1) Collar.—A ring united with the draw-tube. (J) Coarse Adjustment.—For moving the body quickly up and down in order to bring the objective into such rela- 38 MEDICAL MICROSCOPY. tion with the object that an image of the object will be formed. For this purpose most instruments are pro- vided with a rack attached to the body and a correspond- ing pinion fixed upon the arm. The coarse adjustment in the cheaper stands is obtained by means of a tube sliding in a collar attached to the arm. (K) Milled Heads.—Attached to the shank of the pinion these serve to regulate the coarse adjustment of the in- strument. (L) Fine Adjustment.—The fine adjustment is the vital point of the microscope, and should be perfect. By turning the milled screw-head a slow and accurate move- ment of the arm and parts thereto attached is obtained and the relation of the objective to the object so regulated that the sharpest possible image is secured. (M) Stage.—The flat projection from the arm upon which the object is placed. It may be square or round and revolving, and may also have mechanical parts for moving the object by rack and pinion. (N) Clips.—Devices for holding the object in place upon the stage. (P) Mirror Bar.—This holds the mirror below the stage and may be pivoted so as to permit lateral motion of the mirror. (O) Mirror.—Plane upon one side, concave upon the other. The plane side should always be used in connec- tion with the Abbe condenser, as by using the plane mirror more light and more perfect illumination are obtained. (Q) Substage.—That part below the stage carrying the substage ring and other devices. The form of the sub- stage may vary greatly. On the better microscopes it is provided with a rack and pinion adjustment for focusing the Abbe condenser. In many stands this movement is THE MICROSCOPE. 39 obtained by means of a rapid screw placed at the left of the substage. (S) Diaphragm.—The iris diaphragm has superseded all other forms. It consists of a number of metal leaves actuated by a lever, so that any desired diameter of open- ing may be obtained. (T) Joint for Inclination.—This is now provided in all of the higher class microscopes. It is not only a conve- nience, but in certain work a necessity, and one should never purchase a microscope that can only be used in the upright position. CHOICE OF MICROSCOPE. There are many manufacturers now making good microscopes. Among the best of these are the instru- ments turned out by Carl Zeiss, of Jena, E. Leitz, of Wetzler, Reichert, of Vienna, Germany; Powell & Leland and Ross in England; and in this country the Bausch & Lomb Optical Company, of Rochester. This latter firm now makes a microscope that leaves little to be desired. After an experience that the writer believes should entitle him to an opinion, he does not hesitate to say that the brasswork of the microscopes made by this firm are superior to that of stands of foreign manufacture, while the optical parts are certainly as good. For general medical work one should have a stand equipped with a substage, an Abbe condenser, a triple nose-piece, # and % dry and +, oil-immersion objectives, two inch and one inch eye-pieces. The objectives should be of medium angle. While a higher angular aperture will give the better definition, it will not have the penetra- tion, and will probably have a shorter working distance than an objective of the same power but of lower angle. 40 MEDICAL MICROSCOPY. On this account the objectives of medium angle are more satisfactory for general work. If one intends doing blood work or studying bacteria under the microscope, the +4, inch oil-immersion lens is essential. If unfamiliar with the microscope, the intending pur- chaser should always follow competent advice before selecting his instrument, as otherwise he may find too late that he has an instrument either of faulty construction or of such a model as to be unsuited to the work for which he desires it. MANIPULATION OF THE MICROSCOPE. One has said ‘‘ Experience is the best teacher,” and this is doubly true of learning the skilful manipulation of the microscope. ‘There are a few cardinal points that the beginner will do well to bear in mind, and which may be laid down for him, but it is only by that knowledge born of labor and time that he will become expert. Sources of Light.—Daylight is by far the preferable light for general work. One should, if possible, select north light, as this is the purest. The table should be near the window and should be of such a height as will allow one to use the microscope in the vertical position without undue effort. Method of Illumination. The beginner will do well first to take out the eye-piece, turn on the low power (2 obj.), open to the full extent the iris diaphragm, and then with the mirror bar in line with the body of the instrument tilt the mirror so that it will cast a bright beam of light into the objective, watching meanwhile through the open end of the draw-tube for this effect. THE MICROSCOPE. 41 When a good beam of light is obtained, the eye-piece should be replaced and the object placed upon the stage in the optical axis. After a little practice it will be found unnecessary to remove the eye-piece as above. If the reflection from a white cloud can be secured, it is the ideal light. Sunlight should be avoided, as except in expert hands tt is entirely unsatisfactory. Focusing.—There are two methods of focusing. Until one becomes familiar with the microscope the following one should be used. Selecting the low power (2 obj.),— for the object should always be first examined with the low power,—the coarse adjustment is used to lower the body gently until the point of the objective just misses touching the preparation. One must use care in order to avoid forcing the objective upon and perhaps through the preparation, thus spoiling the specimen and perhaps damaging the objective. Then while looking through the eye-piece the tube should be slowly racked up by the use of the coarse adjustment until an image of the object is seen. The moment the magnified image becomes clear and distinct, movement by the coarse adjustment should be discontinued. The fine adjustment is now used until the clearest possible image of the object is obtained. Never use the fine adjustment until an approximate focus shall have first been obtained by use of the coarse adjustment. Modifying the Light.—It will now be seen that the ob- ject is still not distinct and has a milky look. ‘This is because it is flooded with light and its finer details thus rendered imperceptible. This effect is easily overcome by closing the iris diaphragm until so much light is cut- off as may be deemed desirable. There can be no rule laid down for this, Each object 4—O 42 MEDICAL MICROSCOPY. is a law unto itself, and one must use his judgment as to its proper illumination. With the low power (3 obj.) one will need to close the diaphragm almost entirely, with the medium power (} obj.) more light will be needed, while with the high power (1 obj.) one will often find it advantageous to use the full aperture of the diaphragm. Always use the diaphragm wide open when viewing stained bacteria with the ;4, objective, as it renders them more distinct and the flood of light drowns out other ob- jects that may be present. While the above directions apply to stained objects such as are used in the examinations of tissues and bacte- ria, if one wishes to examine any clear fluid, such as urine for deposits, or desires to observe unstained bacteria, as in the hanging-drop, of course the light must be modified considerably, only enough being used to make objects clearly visible. If a flood of light be turned on such objects, they being almost transparent will be quite invisible. Care of the Microscope. The microscope should as far as possible be protected from dust, nor should it be allowed to stand in the hot sun, as this may affect the cement of the lenses. Before using the instrument it is well to wipe off the eye lens of the eye-piece and to see that the front of each ob- jective is clean. ‘‘Lens paper’’ for the purpose of clean- ing these parts may be bought from dealers in micro- scopic supplies, and is best adapted for this work, asa fresh piece may be used each time, obviating the possible dan- ger of scratching the lenses with cloths or chamois, which are sometimes recommended and are apt to be dusty and unsuitable, THE MICROSCOPE. 43 Never use alcohol in cleaning any part of the micro- scope. If balsam or cedar oil has gotten either on the lenses or lacquer of the stand, it may be quickly removed by the aid of a clean, soft cloth moistened with xylol, benzine, or benzole. After using the oil-immersion lens it should always be cleaned by the use of lens paper before the instrument is put away. If oil becomes dried upon the end of the lens, one of the above agents may be used to soften it. A glass bell-jar of sufficient size to cover a microscope may be procured and the instrument kept under this when not in use. One may also use a cardboard cone of home manufacture for the same purpose. It is wonderful how much hard usage a microscope may stand and still be in good working condition, but, as with any instrument of precision, it is better to take as good care of it as circum- stances will permit. If we keep our lenses free from dirt and scratches, we may expect better work from them. The sliding parts of the body and the bearings of the pinion may every few months be lubricated with a minute quantity of clock oil. The working parts should always be kept free from dust. When it is necessary to oil the instrument, first thoroughly clean the working parts with a cloth dampened with benzine or xylol. Never use alco- hol for this purpose. HOW TO LOOK THROUGH A MICROSCOPE. There is a way not to look through a microscope, and unfortunately this method is usually adopted by be- ginners. The proper way is as follows, and it is very important that one should from the beginning accustom himself to 44 MEDICAL MICROSCOPY. the rules laid down, for the sake of his comfort, for the increased facility with which he will work, and for the protection of his eyes. First: The light should be good and not too brilliant. This can be controlled by the use of the diaphragm and mirror. Second: The height of the table and chair should be such as will admit of looking into the microscope when it is vertical without undue bodily effort. The eye should come over the tube by simply inclining the head. There should be nothing about the neck that will when the head is bent act as a constricting band, as a high collar might do, for this will affect the circulation of the head and pro- duce congestion of the eye, and, if long continued, the evils that may arise from this cause. Third: Use both eyes open. ‘This habit is easily culti- vated. One at first experiences some difficulty in seeing the image in the microscope because of the confusion arising from the image seen with the other eye. A little practice will soon overcome this. Tilting the head to one side so that the nose cuts off the external image we wish to forget is one means of attaining the desired end. After a time the brain will take no account of the image cast upon the retina of the eye not applied to the microscope, and one may even train himself to the point where he can look in the microscope with one eye and draw the image there seen, watching the pencil point with the other eye. Fourth: Accommodation should be thoroughly relaxed, as if one were looking at a distant object, and the focusing be done not with the eye, but with the fine adjustment of the microscope. The eye should not grow more tired from microscopic work than from other use. One must remember that the ciliary muscle 7s a muscle, and is subject to the same laws as other muscles, and if it PREPARATION OF TISSUE. 45 is contracted to see that which is really out of focus, it must invariably grow tired in a short time andrelax. The image will then become blurred and the observer will be obliged to rest his eye before he can proceed. Fifth: When at work with the microscope always keep one hand on the fine adjustment, and while moving the object about keep up a slight to and fro movement of the fine adjustment. In this way one brings into focus objects lying in different planes which otherwise would not be seen and relieves the eye from an effort to focus upon the preparation which must necessarily be con- stantly occupying a different relation to the true focus of the objective. PREPARATION OF TISSUE. It is obvious that tissues must be prepared in some way before they can be studied under the microscope. It is important that we see them in as nearly a normal condition as possible. We must, therefore, obtain them as soon after death as possible, whether it be death of the whole or only part of the body, as in an operation for tumor. We must then use some chemical agent which will pre- vent the tissues undergoing minute changes due to de- composition or other causes. This step is known as fixation, and upon the successful fixing of a tissue depends its value for study. If for any reason fixation of the cells of a tissue has been poor, we cannot see it in the state in which it was during life, and may be misled in our deductions. 46 MEDICAL MICROSCOPY. FIXATION. As chemical agents will not in a short time penetrate deeply into the tissues, they must be cut in thin pieces so that the reagent may reach and kill the cells and coagulate their proteids as soon as possible. The size of the piece of tissue must depend upon the fixing agent used and the future method to be employed. It is better to err on the safe side and not cut the blocks of tissue larger than half an inch in one diameter and a quarter of an inch thick. The selection of the fixing agent depends also on the object in view and the method of staining to be used. For routine work one may use either of the following. Absolute Alcohol.—This is one of the best fixing agents. The tissues should be placed in it without having been previously washed in water. For a piece of tissue the above size, an ounce of the alcohol should be used. It is well to place in the vessel a quantity of cotton upon which the specimen may be laid, or in some other way to keep it in relation with the upper part of the fluid, for the reason that as the alcohol abstracts water from the tissues the lower portion of the alcohol becomes diluted, and we wish to keep the specimen in the stronger portion. After a few hours (six or eight) the alcohol should be changed. The tissues may remain in the second alcohol for a few days without damage. Synthetic Alcohol.—Recently there has been put on the market an alcohol made synthetically that is claimed by the manufacturers to be equal in every respect to the distilled absolute alcohol, and which in my hands has given very satisfactory results as a fixing agent, so that for routine work, I now use no other. PREPARATION OF TISSUE. 47 Commercial Alcohol.—This is supposed to be 95 per cent. strength, generally it is about 88 per cent. It is largely used as a fixing agent, and isa fairly goodone. It should be used in a little larger quantity than the absolute alcohol, but in the same way. ‘The strength of alcohol may be preserved or increased by the use of some agent having a strong affinity for water. If several lumps, as large as a walnut, of fresh, unslaked lime be dropped into a bottle of alcohol, the water present in the alcohol and that taken from the tissues in the process of fixation will be appropriated by the lime, which will gradually slake and crumble. Sulphate of copper which has been heated till the water in it has been driven off, when it will turn white, may be used instead of lime. As the copper salt takes up water, it will regain its blue color, when it may again be heated and used the second time. Formalin.—By formalin is meant a 40 per cent. aqueous solution of formaldehyde gas. It is sold under a variety of trade names. Formalin may be used diluted with water in from 2 to Io per cent. strengths of the original solution. It is the best fixing fluid for the eye. For general tissues it is not so good as some other fluids. It has the property of keeping tissues well indefinitely, whether the fluid be frequently changed or not. It does not bleach tissues to the extent that alcohol does, and is probably the best fluid at our disposal for the preservation of museum specimens. Miuller’s Fluid.—This fluid has both advantages and dis- advantages, but may be reckoned among the best fixing agents. Itis composed of the following ingredients: Potassium bichromate, ...............0005 2.5 Sodium sulphate, ........ 00... 00.0200 ee 1.0 Water, i-4o.t8e0% ss yard eee were e ange wets 100.0 It may be made in quantity, as it will keep indefinitely. 48 MEDICAL MICROSCOPY. Pieces of tissue fixed in Miiller’s fluid may be somewhat larger than those fixed in alcohol. Proportionately larger quantities of Miiller’s fluid must be used, and the fluid must be changed after the first few hours and upon each succeeding day during the first week, and after this as often as the fluid becomes cloudy. The tissues must remain in the fluid until they become quite firm. If they are not too large, this will be in from two to three weeks. They must then be washed in run- ning water for ten or twelve hours and preserved in 80 per cent. alcohol. The great disadvantage of Miiller’s fluid is the time occupied in fixing tissues by it. The results obtained are very good, and for some staining methods it is necessary to use it. DEHYDRATION. The water normally contained in tissues must, after fixation, be abstracted before the usual infiltration pro- cesses can be used. If absolute alcohol or synthetic alcohol has been em- ployed as a fixative and the tissue has been put from the first solution into a fresh solution of the alcohol, it should remain here for twelve hours, and then is ready for the next step. If 95 per cent. alcohol is used as a fixing fluid, for subsequent dehydration the tissue should either be finally transferred to absolute alcohol, or, if the celloidin method is to be used, put into the third fresh solution of 95 per cent. alcohol. If the fixing fluid has been of a watery nature, then the tissue must be run through at least three changes of 95 per cent. alcohol, remaining in each twelve hours or more, and if the paraffin method of embedding is to be used, finally put into absolute alcohol for twelve hours. PREPARATION OF TISSUE. 49 INFILTRATION. We must in some way cut thin sections of tissue and prepare them by staining before they are fit for examina- tion. Tissues are normally springy, and even when well hardened cannot be cut thin enough unless they have some sort of support. The first to be described is the celloidin method. Celloidin is refined collodion, and comes in granular form and also in shreds. Two solutions of celloidin are made and kept in stock in tightly stoppered bottles. The celloidin is dissolved in equal parts of sulphuric ether and absolute alcohol (95 per cent. will answer, but is not as good). Solution No. 1 should be of the consistency of thin syrup. Solution No. 2 should be of the consistency of thick syrup. The tissue should be put directly from the last alcohol into a solution of equal parts of alcohol and ether (95 per cent. alcohol may be used). Here it should remain for some time, preferably for twenty-four hours. It should then be transferred to celloidin solution No. 1, enough being used to cover the tissue well. A small wide-mouthed bottle is the most convenient receptacle, and of these, vas- elin bottles of various sizes are perhaps the cheapest and easiest obtained. In solution No. 1 the tissue should remain at least twenty-four hours, unless the pieces be very small. It may remain in this solution for several days with advantage. It should then be transferred to celloidin solution No. 2, where it should remain twenty- four hours or longer. 50 MEDICAL MICROSCOPY. MOUNTING. The tissue must now be mounted upon a stable support capable of being held firmly in the jaws of the microtome. Cork is largely used and is recommended in some text- books. On account of its springy nature cork is entirely unsuitable, and should never be used for this purpose. The author uses pieces of hard wood,—maple or black gum,—having a flat circular head 1} inches in diameter, 1 inch thick, and with a central stem 14 inches in length and 3 inches in diameter (Fig. 21). These mushroom-shaped pieces of wood have many advantages, and any carpenter will turn them out on his lathe at a nominal cost. The tissue, now thoroughly in- filtrated with celloidin, is removed from the celloidin solution No. 2 with a pair of forceps and placed upon the flat surface of the wooden support. Enough cel- loidin is poured around it to form a ring of this substance, which when hardened will serve to hold the tissue firmly in place upon the support. Within a few moments it will be found that the evaporation of alcohol and ether from the celloidin has caused the part exposed to the air to become firm, so that there is a thick skin formed, sufficient to keep the still fluid portion below in place. The block of wood with its tissue should now be transferred to a jar of 80 per cent. alcohol. The block should be placed in the alcohol tissue-side down and allowed to remain twelve hours. The alcohol will harden the celloidin both around and in the tissue, Fic. 2 21. PREPARATION OF TISSUE. 51 giving the whole mass quite a firm consistency, which will readily admit of thin sections being cut from it. The specimen may be left in the 80 per cent. alcohol indefin- itely. If one is in a hurry to secure a section, chloroform may be used as a hardening agent for the celloidin. Chloroform will harden celloidin in about half an hour. The speci- men may then be preserved in 80 per cent. alcohol. THE PARAFFIN METHOD. Paraffin may be used as an embedding material, and for certain work is better than celloidin. Paraffin is especially valuable for making serial sections, as in embryologic work. With it large and thinner sec- tions may be cut. Paraffin embedding is generally used for cytologic work on account of the thinness and uniform- ity with which sections may be made. It has the addi- tional advantage of preserving the block of tissue indef- initely. The paraffin should thoroughly infiltrate the tissue, so that it will universally support it. The preparation of tissue for the paraffin method is the same as for the celloidin method, with one exception— absolute alcohol must be used in the last steps of dehydra- tion. As absolute alcohol is expensive, it is the author’s practice to use two or three changes of 95 per cent. alcohol in dehydrating tissue fixed in aqueous fluid, and to finish dehydration in one or more changes of absolute alcohol. If possible, the tissue should remain in each of the various alcohols for at least twelve hours. With pieces of tissue no larger than 1 cm. square the process may be somewhat hastened. The tissue is now transferred to a small jar or wide- mouthed bottle containing chloroform. If thoroughly de- 52 MEDICAL MICROSCOPY. hydrated, the tissue will float in the chloroform. If not dehydrated, the tissue will not float or will scarcely do so, and there will be given off from it a milky cloud which will be very perceptible in the clear chloroform. If this milkiness is observed, the tissue must at once be put into fresh absolute alcohol and allowed to remain there at least twelve hours. It may then be tried again in chloroform, and will probably be found to be dehydrated. If the above directions are disregarded and tissue which is not thoroughly dehydrated is carried through the par- affin, it will be found that it is entirely unfit for sectioning and study. One should therefore ‘‘make haste slowly.” Infiltration with Paraffin. Paraffin is solid, and it is necessary to reduce it to a liquid state before it will enter the tissue. To do this we must resort to heat. This is unfortunate, because too high a degree of heat may produce changes in the tissues, and we must have a special apparatus in which we can melt the paraffin. In the catalogues of the dealers are found descriptions of the various kinds of paraffin baths. The writer much prefers the ordinary water-bath (Fig. 22). Not only is it more cleanly, but it provides a more uniform heat and may be used for other purposes. A thermostat must be used in connection with it. The melting-point of paraffin varies; in winter a soft paraffin may be used, while in summer it is necessary to use a hard paraffin. A paraffin having a melting-point of 54° C. will generally answer for ordinary work. When one wishes to do the fine sectioning required in cytologic research or in cutting small embryos, the melt- ing-point of the paraffin must be chosen with reference to the room-temperature at which the sections are to be cut. Two paraffins should be used. PREPARATION OF TISSUE. 53 Paraffin No. 1 should have a melting-point of about 44°C. A quantity of this soft paraffin is cut into small pieces and placed in a suitable receptacle—an ordinary teacup answers for this purpose—and melted in the water- bath. The block of tissue is transferred from the chloro- form to the paraffin and allowed to remain in the water- bath twelve hours. It is then transferred to the melted paraffin No. 2. Paraffin No. 2 is pure paraffin having a melting-point of, say, 54° C. Tissue should remain in No. 2 in the water-bath twelve hours, and then is ready for embedding. Embedding in Paraf- fin.—The block of tis- sue is now thoroughly impregnated with the harder paraffin. It must next be embedded in a mass of paraffin which will act as a support to the margins of the tissue and serve to fasten it to the wooden block. The tissue is removed from the melted paraffin with a pair of forceps and placed upon a piece of glass, that part which it is desired to be cut first being placed in contact with the glass. The tissue is then surrounded by a wall of some substance that will act as a mould. A piece of paper may be bent to answer this purpose, or brass or lead piping of various diameters cut in short pieces answers very well. Moulds made of rectangular pieces of type metal are 54 MEDICAL MICROSCOPY. perhaps the best, as with these we secure a square block, and the size of the block may be varied by adjustment of the sides of the mould. Such embedding boxes may be bought in the shops (Fig. 23). The mould is now filled with melted paraffin No. 2 and the whole cooled as rapidly as possible to prevent crystallization of the paraffin. This may be accomplished by placing the glass plate in cold water, being careful not to allow the water to run over the mould until the surface of the paraffin has cooled. When the paraffin block is thoroughly hardened, it is removed from the mould, and may be cut immediately or at a convenient season. Mounting the Paraffin Block. When it is desired to section the tissue, the block of paraffin is fastened to a piece of wood that may be clamped in the jaws of the microtome. This is best accomplished by heating the blade of a case- knife in the flame of a bunsen burner or spirit-lamp until it will melt the paraffin. The heated knife-blade is placed flat upon the wooden support and the bottom of the paraffin cast brought in contact with the knife-blade immediately over the block. When the paraffin begins to melt, the knife is slipped quickly from beneath it and the cast and support pressed firmly together. Ina few minutes the paraffin will have cooled and the cast is firmly fixed to the support. The mushroom-shaped pieces of wood spoken of in con- nection with the celloidin method will be found equally adapted to paraffin work. Itis often found advantageous to place the tissue eccentrically upon the block, as this Fic. 23.— PARAFFIN EMBEDDING Box. PREPARATION OF TISSUE. 55 facilitates the proper adjustment of the paraffin cast in its relation to the knife-edge. TABULATION OF STEPS IN CELLOIDIN METHOD. 1. Fixation Absolute alcohol or synthetic alcohol or 95 per cent. alco- hol, or formalin in 10 per cent. strength, or Miiller’s fluid. 2. Dehydration.—If fixed in absolute alcohol, into the fresh absolute alcohol for twelve hours. If fixed in 95 per cent. alcohol, into: (a) 95 per cent. alcohol No. 2 for twelve hours. (6) 95 per cent. alcohol No. 3 for twelve hours. If fixed in watery solution, into: (a) 95 per cent. alcohol No. 1 for twelve hours. (6) 95 per cent. alcohol No. 2 for twelve hours. (c) 95 per cent. alcohol No. 3 for twelve hours; or, better, in absolute alcohol for twelve hours. 3. Ether and Alcohol.—In ether and alcohol equal parts for twelve hours. 4. Thin Celloidin.—Celloidin consistency thin syrup twenty-four hours, or longer. 5. Thick Celloidin.—Celloidin consistency thick syrup twenty-four hours or longer. 6. Mount.—On wooden support. 7. Alcohol.—Harden celloidin and preserve in 80 per cent. alcohol. (Chloroform may be used.) May be cut after twelve hours. 8. Sectioning. TABULATION OF STEPS IN PARAFFIN METHOD. , 1. Fixation.—Absolute alcohol or synthetic alcohol or 95 per cent. alco- hol or formalin 10 per cent. or Miiller’s fluid. 2. Dehydration.—Must be thorough. If fixed in absolute alcohol: (a) Absolute alcohol No. 2 twelve hours. (b) Absolute alcohol No. 3 twelve hours. If fixed in 95 per cent. alcohol: (a) 95 per cent. alcohol No. 2 twelve hours. (b) 95 per cent. alcohol No. 3 twelve hours. (c) Absolute alcohol twelve hours. If fixed in aqueous solution: (a) 95 per cent. alcohol No. 1 twelve hours. (b) 95 per cent. alcohol No. 2 twelve hours. (c) 95 per cent. alcohol No. 3 twelve hours, (d) Absolute alcohol twelve hours. 56 MEDICAL MICROSCOPY. 3. Chloroform.—For twelve hours. Tissues should float and not turn chloroform milky. Failure in this indicates in- sufficient dehydration. 4. Infiltration.—Paraffin No. 1: Soft paraffin. Melting-point of paraffin 44°C, ‘Tissue kept in melted solution in water- bath twelve hours. Paraffin No. 2: Hard paraffin, melting at 54° C. in water-bath at temperature just sufficient to keep melted twelve hours. 6. Embedding.—On glass plate in mould. 7. Mounting.—On wooden support ; adhesion secured by melting bottom of cast. 8. Sectioning. SECTION CUTTING. The microtome is an instrument designed to cut pieces of tissue thin enough for microscopic examination. There are a variety of forms of microtomes upon the market, but two chief principles are employed: the one, in the Thomer model, in which the tissue is fed upward by being advanced on an inclined plane; and the other, in the Schanze model, in which the tissue is fed upward by a micrometer screw. In either model the knife is carried on a sliding block and propelled by the hand. - Each form of instrument has its merits, but mechanic- ally the Schanze model is the more perfect instrument. It is also capable of much more rapid manipulation. In this country the Bausch & Lomb Optical Company, of Rochester, makes a very good line of microtomes, and from their catalogue one may make a selection according to the work one desires to do and the money one has to expend. The student microtome (Fig. 24) is a very serviceable instrument for individual work. It is unsatisfactory for large sections; and if much sectioning is contemplated, a larger instrument should be secured. 57 58 . MEDICAL MICROSCOPY. It is not possible to get one microtome that will meet every requirement. Serial sectioning of small embryos of other objects can best be done upon the microtome designed especially for the work. The best microtome of the class is the Minot. It is quite expensive, however, and the Ryder microtome, manufactured by Zentmeyer, of Philadelphia (Fig. 25), will be found to be a very efficient instrument, not only for serial sectioning, but for general paraffin work. It is Iie, Dey small, compact, and cheap, a very serviceable model and deserving of general use. Unfortunately, it is not well made. Care of the Microtome.—While the microtome is not a very delicate instrument, it will do better work if properly caredfor. It should be kept covered when not in use, and the sliding parts should always be cleaned before using by wiping them with a cloth. They should then be oiled with a drop or two of paraffin oil, which has proved more PREPARATION OF TISSUE. 59 satisfactory in the writer’s hands than any of the lubri- cants generally used, because it does not gum and harden. The micrometer screw should also be frequently cleaned and oiled. The Knife.—One who possesses a good microtome knife is fortunate. It must be of just the proper temper or it is a failure, and it does not seem possible for the man- ufacturers to with certainty make two tempered alike. At any rate, the purchase of a microtome knife is a lottery. The size of the knife must correspond to the size of the microtome upon which it is to be used. The larger the knife, the better the results it should give, as there will be less spring to cause deviation in the thickness of the sec- tions. Care of the Knife. One cannot be too careful of his microtome knife. Its edge must be perfect or it will not cut thin sections. It must never be left upon the microtome when work is done. For the time, remove the knife, thoroughly cleanse, and put it in its case. If paraffin is caked upon it, use a solvent—xylol or chloroform—to remove it. Never touch the edge with any hard substance. Sharpening.—One must learn the art of sharpening a microtome knife by practice. At first he will succeed but poorly. Two hones should be kept for the purpose— a yellow Belgian water-hone and a slate water-hone. The first is used only occasionally, when the edge is very dull or has become rounded or nicked. In other words, this stone has a sharp tooth and is only serviceable when we wish to cut away metal rapidly. The blue hone cuts very slowly and leaves a polished, fine edge. The lower surface of the knife is made flat or very slightly concave, and when the knife is on the micro- 60 MEDICAL MICROSCOPY. tome should lie nearly on a level with the plane of the ways upon which the carrying block slides, there being a slight upward tilt of the back of the knife so that the blade does not touch the block of tissue except on the cutting- edge. The upper surface of the blade is slightly concave. In sharpening the knife the bottom surface should be laid flat against the stone. The upper surface is sharpened quite differently, being held so that the edge will be worn away at an angle of about 15 degrees. Fig. 26 is the cross-section of a knife as it should appear when properly sharpened. In honing, the knife should always be drawn from the heel to the point, the sharpening being done largely from the upper, beveled surface, the lower surface needing only a few occasional strokes with the blade flat upon the stone. Many workers use finally a strop backed by a flat piece of hard wood so that the edge of the knife will not be turned by the sagging of the leather. For some years the writer has discarded the strop. With a good knife and correct honing it is not necessary. A microtome knife, when properly sharpened, should cut a hair held in the hand, as in testing a razor. Sectioning Tissues Mounted in Celloidin. In cutting celloidin sections the knife and the tissue should be kept flooded with 80 per cent. alcohol. One PREPARATION OF TISSUE. 61 method of keeping the knife wet is to mop the alcohol on it with a large camel’s-hair brush between each stroke. This is clumsy and unsatisfactory. A better way is by means of an inverted flask of alcohol suspended over the knife. The mouth of the flask is closed with a perforated rubber stopper with a piece of glass tubing passing through it. Over the outer end of the glass tubing is passed a small piece of soft-rubber tubing of such length as nearly to reach the knife. A pinch-cock controlled by a screw clamping the rubber tube completes the device. With this simple apparatus one may exactly regulate the flow of alcohol to the desired quantity. The alcohol should be allowed to drop in such a way that the surface of the specimen and of the knife are kept wet with it. A little practice will soon determine the quantity necessary to insure good sections. In cutting celloidin sections the knife should be set so that it will have the longest possible sweep. In other words, all of the edge of the knife should be used in the stroke. The tissue should generally be placed so that the knife will strike the tougher portion first. As an illustration, in cutting a piece of tumor having a capsule the capsule should be set toward the knife-edge. As the sections are cut they must be transferred to 80 per cent. alcohol. After a few have collected upon the knife they may be removed by sliding them off the edge of the knife on to the finger placed beneath the blade and in contact with it. Some use a camel’s-hair brush for this purpose. Trained fingers are more satisfactory in every way. The sections may be indefinitely preserved in 75 per cent. or 80 percent. alcohol. In stronger alcohol they are apt after a time to lose their staining qualities. 62 MEDICAL MICROSCOPY. CUTTING PARAFFIN SECTIONS. Tissue embedded in paraffin is always cut dry. After the block has been fastened to the support the redundancy of paraffin should be trimmed away from around the upper part of the tissue with a knife, leaving enough paraffin to act as a support to the edges. It is not necessary in general work to have the knife at any fixed angle to the tissue. The slant at which it works best is the one to be secured, and this can be obtained only by trial and varies with the tissue to be cut. If the temperature of the room is just right, the sections when made will remain comparatively flat. If the paraf- fin is too soft, the sections will crumple and stick together; if too hard, the sections will curl. The temperature may be regulated in some degree by opening or shutting a nearby window or by placing a lump of ice in the vicinity of the microtome. In spite of all we can do, the sections will sometimes be refractory. The section flattener devised by the late Dr. James E. Reeves is a useful adjunct. It consists of a wire frame clamped to the knife and holding a piece of stiff paper in such a way that the sections as they are cut slide under it and are kept from curling. A piece of paper held in place by the hand answers the same purpose. The paper should lie in a plane with the surface of the knife and almost in contact with it. As sections are cut they should be removed from the knife and laid on clean paper, or, if the temperature of the room necessitates it, in dishes of cool water. Paraffin sections cannot be handled with the fingers. A pair of fine-pointed forceps is useful for this purpose. Paraffin sections cannot be preserved unmounted, but the em- PREPARATION OF TISSUE. 63 u bedded block of tissue may be kept for years and sections cut from it when desired. Mounting Paraffin Sections. The sections must now be attached to glass slips pre- paratory to being stained. It is necessary to use some cement to fasten the sections in place. A 0.5 per cent. solution of the best gelatin in a 2 per cent. watery solu- tion of carbolic acid is quite satisfactory. The slips should be well cleaned and free from dust. With a pipette a quantity of the ‘fixative’ is put ona slip, a section is lifted with the forceps and laid upon the solution which must be in amount sufficient to float it, and the slip is warmed either in an oven made for this pur- pose or by passing it a few times through the flame of a bunsen burner. As the liquid becomes warm the section flattens out perfectly if it has not been allowed to roll up tight when cut. ‘The section is placed in the middle of the slip and the fixative drained off; when cool, the section is lightly blotted with bibulous paper and the slip set aside to dry overnight. Generally the tissue is well attached to the glass by this method. In order to insure success, the writer is in the habit of submitting the specimen to the action of formalin, which renders the gelatin insoluble. The slips with the sections attached are put in an ordinary slide-box, a few drops of formalin are poured into the bottom of the box, and the cover is closed. When the gelatin water has thoroughly dried, staining may be proceeded with. 64. MEDICAL MICROSCOPY. t THE FREEZING MICROTOME. Various forms of freezing microtomes may be bought, and in most of these the freezing agent is ice and salt. Carbonic acid gas is also used, and is preferable to ice be- cause of the cleanliness, more rapid action, and from the fact that it is always ready. Very good sections may be made by the freezing method, and when time is limited, as in the diagnosis of tumors during operations, it is valuable. It is not appli- cable to general work. If tissues have been fixed in alcoholic solution, they must first be washed for several hours in order to remove the alcohol, else they cannot be readily frozen. Tissues, whether fresh or hardened, should be put on the freezing block and covered with a thick solution of mucilage. Do not freeze too hard or the knife may suffer damage. As the sections are cut they should be transferred to a dish of water, or, if the tissue is wnhardened, toa 5 per cent. aqueous solution of formalin. After remaining in forma- lin for five minutes they will be hardened sufficiently to bear handling, and may be stained and mounted for ex- amination after the manner of celloidin sections. Thickness of Sections.—In general pathologic work the mistake of cutting sections too thin is very often made. While we must have our sections thin enough to freely transmit light, we get a better idea of the relation that cells have to each other if they are moderately thick. In cytology, and frequently in bacteriologic research, it is necessary to have sections as thin as possible. One must use his judgment in this matter and cut his sections ac- cording to the end in view. The beginner will do well to purchase a few preparations as a standard with which to compare his work. PREPARATION OF TISSUE. 65 STAINING CELLOIDIN SECTIONS. Stains are used for the purpose of bringing out the structural details of tissues. Almost every known dye has been used in some staining method, and the mass of material in books on technique from which one must cull that which is serviceable is very confusing to the beginner. For general work, the hematoxylin and eosin method is unsurpassed. It is simple, effective, and permanent. There are numerous formule for hematoxylin stain, and each has its advocates, and perhaps all are good. A standard formula is the following: BOuHMER’S HEMATOXYLIN. Hematoxylin crystals, ..................... 1.0 AICORG! 9. ails doencyd sha Sennen e ota eee meee 10.0 Potash alii ps icaiana palinw oye he ek RESO 10.0 Water cwuwecladcue naan law owen ow ileus ee 200.0 Dissolve the hematoxylin crystals in alcohol, making solution A. Dissolve the alum in the water, making solution B. Add Ato B, drop by drop. Place the mix- ture in an unstoppered bottle in a strong light to ripen. It will gradually turn dark and will be fit for use in about ten days. Filter and add a few small lumps of gum camphor to keep moulds from growing in the stain. Most of the camphor will remain undissolved. Keep in a well-stoppered bottle. The staining qualities increase with age. The stain works best undiluted and may be used over again any number of times. Hematoxylin is a nuclear stain; that is, it stains the nuclei of cells and for the most part leaves the protoplasm of the cells unstained. Nuclei stained by hematoxylin should be a deep purple color. If they are red or brown, 6—O 66 MEDICAL MICROSCOPY. the stain is not good or they have not been washed long enough. 1. Time of Staining.—One must use his judgment as to the length of time to stain a given section. Newly made stain does not work so quickly or with such intensity as old stain, and there is much variation in the affinity of tissues for stain. With a well-ripened stain the process should take from one to three minutes. Practice will soon make one expert in this matter. 2. Staining.—Hematoxylin stain must always be fil- tered before using. One should have at hand a small glass funnel kept for this purpose alone. The same filter- paper may be used many times. Sections in 80 per cent. alcohol should first be put in a dish of clear water. Sufficient stain to cover the section well is filtered into a suitable dish or Syracuse watch-glass; the section is transferred from the water directly to the stain and im- mersed there for the space of time deemed necessary. Sections are best handled with a blunt, curved needle, such as may be bought of the dealers. A common saucer with straight sides is the best dish for fluids used in the following manipulations. 3. Washing.—Transfer the section to a saucer of clean water and by gentle manipulation of the curved needle wash it free from stain. When it is freed from super- fluous stain, transfer to a saucer of fresh water for five minutes or longer. If one is not in a hurry, it is better to leave the section in the second water for several hours or even overnight. This long washing serves to extract the stain from those parts of the cell which have not a special affinity for it and gives us “differentiation,” so that a clear, sharp picture results. 4. Dehydration.—The water must now be gradually PREPARATION OF TISSUE. 67 extracted from the section in order that it may be cleared and mounted. This is accomplished by putting the section first in an alcoholic solution which is weak (70 per cent.) for five minutes, and then into a stronger alcohol (95 per cent.) for five minutes, and finally into a second strong alcohol (95 per cent.). 5. Counterstaining. — Eosin stains the protoplasm of the cell, connective tissue, red blood-cells, and tissues which have undergone some of the degenerative changes. It is a contrast stain and tissue renders the section more intelligible to the beginner. Many advise staining with aqueous eosin before dehydration, but alcohol being a good solvent of eosin much of the stain is lost in the latter process, and one is never quite certain how deep his sec- tion will be colored when mounted. By using a } of 1 per cent. solution of eosin in absolute alcohol (95 per cent. will answer) after dehydration one may exactly regulate the degree of staining. Two min- utes in this solution is generally sufficient. Overstaining with eosin makes a muddy section and obscures fine detail. 6. Clearing.—In order that the section may readily transmit light and to extract the alcohol from it some clearing agent must be used. The essential oils—oil of cloves, origanum, turpentine, and others—are used for. the purpose, and in some staining methods it is essential that a particular oil be employed. The best clearing fluid for general use is carbol-xylol, and it is surprising that it is so little used. It clears sections dehydrated with 95 per cent. alcohol, while with some of the oils it is necessary to use absolute alcohol, it does not dissolve the celloidin, and the pene- tration is much more rapid than with any other agent. It is also free from the disagreeable odor of clove oil and 68 MEDICAL MICROSCOPY. creosote, and being very liquid and volatile is easily cleaned from the slide. These qualities and the fact that it may be used with most staining methods, and clears the section quite as well as other fluids used, should make it more popular than itnowis. ‘The formula is as follows: The xylol is very volatile, and, as the fluid is repeatedly used, this factor gradually evaporates and the percentage of carbolic acid becomes greater. When this occurs fresh xylol should be added in such quantity as is considered necessary. The section is put from the alcoholic eosin into a saucer of carbol-xylol, where it at first floats on the surface but within a few seconds slowly sinks. Two minutes is ample time to allow for clearing with this mixture; one minute being in most instances quite sufficient. 7. Mounting.—The section is now ready to be put upon the glass slip. Metal lifters are generally used for this pur- pose, but a piece of cigarette paper is far better. A strip of paper a little wider than the section to be handled is used. It should be laid against the straight side of the saucer with the lower end projecting into the fluid for a distance slightly exceeding the diameter of the section. The latter is now floated on to the paper by manipula- tion with the curved needle. The paper with the section clinging to it is withdrawn by sliding it up the slanting side of the saucer, the surplus clearing fluid drained off, the paper inverted, and the section brought into contact with the center of a clean glass slip. While the paper is PREPARATION OF TISSUE. 69 still covering the section, blot the latter with bibulous paper. The paper is now gently lifted, the section remaining on the slip. A drop of Canada balsam dissolved in xylol is put upon the section and the whole covered with a clean cover-glass of proper size. The cover-glass should be tightly pressed down upon the section with the butt of the forceps. ‘The slide, as the finished specimen is called, is now ready for inspection with the microscope. STAINING PARAFFIN SECTIONS ON THE SLIP. The sections being mounted on pieces of glass 1 X 3 inches, it is necessary in order to bring them in contact with the various reagents to have some form of dish that will admit of the slips being stood on end in the fluid. The Stender dishes (Fig. 27) or the Naples staining jar (Fig. 28) are adapted to this purpose. The paraffin must first be gotten rid of. This is accom- plished by immersing the slip in a dish of xylol for ten minutes or more. When it is removed, the surplus re- agent should with a towel be wiped from the under surface of the slip and from around the section, care being taken 79 MEDICAL MICROSCOPY. not to touch the latter. The slip is then placed in 95 per cent. alcohol for five minutes to take out the xylol, then in 75 per cent. alcohol for one minute, and then in water for one minute. The remainder of the staining process is identical with that described for staining the celloidin sections except that Stender dishes must be substituted for saucers. The slips should be wiped free from each reagent before being put into the next in order to avoid contamination of solutions. The finished sizdes may be placed in the drying oven for several days for the purpose of hardening the balsam. STAINING CELLOIDIN SECTIONS. The sections are presumed to be taken from 80 per cent. alcohol. Water—two minutes. Hematoxylin—one to three minutes. Water to wash off stain. . Water, clean, to differentiate, fifteen minutes or longer. Alcohol (70 per cent.) five minutes. Alcohol (95 per cent.) five minutes. Alcohol (95 per cent.) five minutes. Alcoholic eosin (4 of 1 per cent.) two minutes. Carbol-xylol, two minutes. Balsam. Cover-glass. BON H = roo MIAN = STAINING PARAFFIN SECTIONS. Staining on the slide. 1. Xylol ten minutes. 2. Alcohol (95 per cent.) five minutes, BACTERIA. 71 Alcohol (75 per cent.) one minute. Water one minute. Hematoxylin one to three minutes. Water to wash off stain. Water, clean, to differentiate, fifteen minutes or longer. 8. Alcohol (70 per cent.) five minutes. 9. Alcohol (95 per cent.) five minutes. 10. Alcohol (95 per cent.) five minutes. 11. Alcoholic eosin (4 of 1 per cent.) two minutes. 12. Carbol-xylol, two minutes. 13. Balsam. 14. Cover-glass. Deep dishes in which the slides may be immersed in the fluid must be used. OG eS BACTERIA. For a long time it was thought that bacteria belonged to the animal kingdom. They are now known to be vege- table. The term bacteria is applied to those minute and simple forms that occupy the lowest place in the vegetable kingdom. Bacteria are classified according to their form into round bacteria or cocci ,* rod-shaped bacteria or bacilli, and spiral bacteria or spirilla. The first group is further divided into single forms, monococci; double forms, diplococci; those in which four organisms are grouped, forming a square, tetracocci; and sarcine, the latter occurring in bale-like masses. * Pronounced kok’-sz. 72 MEDICAL MICROSCOPY. Single cocci occurring in bunches are called staphylo- cocci (from oragudy, ‘a grape’’), because in the tissues they grow in clusters or masses, like a bunch of grapes. Diplococci a 2 > Tetracocci ne) Be 2 Spider cell ws ? * 27 Bacilli in chains Diplococcus Vibrios (spirill pirilla) With ge ( capsules eee 2254 Monococcus No- é ae Comma bacilli Centrally situated o spores = é Spirochetze Clostridia forms = aca Knobbed bacteria with SK terminal spores Fic. 29.—Forms oF Bacrerta.—(From Schenk.) Other cocci occur frequently in chains, and they are caled streptococci. The variation in the arrangement of cocci is due to their BACTERIA. 73 method of development. All cocci multiply by fission or division. The diplococci after the division of the single cell remain for a time in apposition. Tetracocci are formed by the splitting up into four parts of a single coccus, and the sar- cine by the division of a single coccus in a number of directions. BACILLI. Any micro-organism having one diameter exceeding another is called a bacillus. Some of the bacilli are so nearly round as almost to deserve classification among the cocci. Bacilli multiply by division into pairs, this process be- ing called fission. If bacilli exist in an end-to-end forma- tion or chain, they are called strepto=bacilli. Many bacilli form spores. Spores are minute globular bodies formed within the bacilli by a condensation of their protoplasm. ‘The spores may be situated in the middle or end of the rod. Some bacteria may contain several spores. Spores may be likened to the seeds of the higher plants, and under favorable conditions each spore pro- duces one bacterium. Spirilla are rod forms that have a spiral shape. Some spirilla are long, narrow, and wavy, while others are short and thick. ‘The spirillum of cholera is of the latter type. Multiplication of spirilla usually takes place by fission, but spores have been observed in a few species. Bacteria are divided into two great groups, those that live normally in the living body of animals or plants being called parasites, while those that live in dead matter are known as saprophytes. Some parasites can grow only in living matter; these are called obligate parasites, while those that may grow 74 MEDICAL MICROSCOPY. either upon the living body or upon inert material are called facultative parasites. In other words, these para- sites may become saprophytes. Motility.—Some bacteria have the power of motion, and can be seen, when studied under the microscope in the hanging drop, moving across the field, often with consid- erable rapidity. The movements are sometimes erratic or they may be quite direct or they may be dancing. The activity of motile bacteria must not be confounded with the motion that all small particles of matter in sus- pension may assume, known as the Brownian movement. In the Brownian movement the particles dance to and fro Fic. 30.—HANGING-DROP PREPARATION. but do not change their relations to each other nor move from the field of vision. Bacteria move through the agency of small thread-like prolongations of their protoplasm. ‘The tail-like ap- pendages are called flagella, their number differing in varieties of motile organisms. A bacterium may have a single flagellum at one end or there may be several at one end, or each end may contain tufts of flagella, or the or- ganism may have them projecting from it in every direc- tion, in which case it presents a mossy appearance. Fla- gella are found on many species of bacilli and spirilla or vibrios, but only a few varieties of cocci possess them. It is impossible to see flagella in unstained bacteria, neither do they stain by the ordinary methods. In order to see them we must use special technique, which will be given in detail later (see p. 114). BACTERIA. 75 GROWTH OF BACTERIA. There are five varying conditions necessary to the growth of bacteria: food, moisture, temperature, light, and atmosphere. Food.—Bacteria perform the great function of breaking up organic compounds into simpler elements. We find in nature that bacteria are responsible for all decay, whether of animal or vegetable bodies. We know also that the growth of one variety may be retarded or acceler- ated and its characteristics changed by the presence of other varieties in the same soil. In the laboratory it is necessary to have some food of definite composition in which to grow bacteria for experi- mental purposes. A number of such foods have been devised and are in common use, and these are known as culture-media. These culture-media are not ideal foods, but most bacteria grow fairly well upon at least some one of them. When planted on culture-media, we find that bacteria produce a chemical substance that retards and finally stops their growth. The nutrient portion of the medium is not all used up, and yet the growth of the organisms may be limited to a small area. Generally the limit of growth is reached in forty-eight hours. The retarding chemical substance or enzymes produced by one variety of bacteria are generally unfavorable to the growth of another variety. Moisture.—Bacteria may exist for a time in the dried state, but they cannot grow without a proper amount of moisture. Spores will live much longer in the dried state than will the bacteria themselves, but the amount of resistance to drying depends largely on the variety of the organism. 76 MEDICAL MICROSCOPY. Anthrax spores in the dried state retain their vitality for several years. Temperature is an important factor in bacteriologic growth. ‘The temperature at which a bacterium will grow best is called the optimum temperature. ‘The temperature above which an organism will not grow is called the maximum temperature, and the temperature below which it will not grow is called the minimum temperature. Naturally, the optimum temperature for parasites is the temperature of the body they are parasitic to. In the case of bacteria inhabiting animals, the body-tempera- ture of 37° C. is the optimum temperature. A few organ- isms grow best at a very high temperature, 60° or 70° C., and these are called thermophilic bacteria. An organism may not be killed by being subjected to a temperature below its minimum or above its maximum temperature. Many organisms will resist freezing tem- perature for a long time, and will grow as soon as the proper conditions are again established. Other bacteria will resist high degrees of heat, and this is partially true of the spore-bearing organisms. A temperature above the maximum temperature of an organism does not necessa- rily kill it, but it stops its growth. Pathogenic bacteria grow at a degree of heat just below their maximum temperature, but lose their virulence; and those bacteria which produce color under normal con- ditions fail to do so if grown at a temperature near their maximum temperature. Light is nature’s greatest germicide. Sunlight kills bacteria in even a few hours’ time, and strong electric light has a similar effect. Even diffuse daylight kills bacteria if they are subjected to its influence for a considerable time. The less bacteria are protected by surrounding influences, the quicker will light affect them. Dried BACTERIA. 77 anthrax spores are killed in an hour and a half by direct sunlight; but if the spores are moist, they are not killed so quickly. Atmosphere.—Most bacteria grow only in an atmos- phere of oxygen, such are called obligate aerobes; while others will not grow in the presence of oxygen, and these are called obligate anaerobes. A third class may grow either in the presence of oxygen or without it, and these are called facultative anaerobes. For cultivating anaerobes special methods are resorted to, they being generally grown in an atmosphere of hy- drogen. Chromogenic Bacteria——Many bacteria have the prop- erty, under favorable conditions, of producing color in the course of their growth. The pigment in some varieties is within the bacterium, and in other varieties is produced outside of the organism and may even vividly stain the culture-medium at a considerable distance from the area of bacteriologic growth. The pigments of the chromogenic bacteria are generally soluble in alcohol, chloroform, or ether, and insoluble in water. The more common colors thus produced are reds, yellows, and violets. The common pus organism Bacillus pyocyaneus imparts a vivid blue color to the medium in which it grows. This may be abstracted by chloroform in the form of pyocyanin, which is crystallizable. Pathogenic Bacteria.—Certain forms of parasitic bacte- ria are capable of producing disease or pathologic condi- tions of the body, and to these the term pathogenic has been applied. The pathogenic organisms are the ones of most interest to the physician, and the scope of this book does not admit of other than these being considered. 78 MEDICAL MICROSCOPY. BACTERIOLOGIC METHODS. For the study of bacteria one must have certain facili- tiesat hand. A small outlay will equip a personal labora- tory moderately well. Dependent upon the amount of work one expects to do, the larger pieces of apparatus may be simple or more capacious and costly. It is quite possible for one to dispense with an incubator provided he wishes to grow only a few organisms for clinical purposes. For instance, the culture-tube may be placed near a stove, where a temperature approximating that of the body will be maintained, or it may be carried in an inner pocket. Fic, 31.—TEstT-TUBE ‘ ‘ BASKET. Most organisms grow fairly well at room-temperature. As a suggestion to him who wishes to fit up a laboratory for individual investigation on a limited scale, the follow- ing list of essentials is given. Much more may be added with advantage, and, on the other hand, some of the arti- cles are not absolutely necessary, but will materially facilitate the work. LIST OF NECESSITIES FOR BACTERIOLOGIC WORK. Scales and metric weights up to 25 gm. 1 glass cylindric graduate 1000 c.c. 1 glass cylindric graduate 100 c.c. 1 glass funnel 170 mm. in diameter. 2 glass funnels 120 mm. in diameter. 2 pounds glass tubing for pipettes. BACTERIOLOGIC METHODS. 79 6 glass rods for stirring. 2 platinum needles. 100 filter-paper discs 24 mm. in diameter. to sheets bibulous paper. I gross test-tubes 150 X 15 mm. 3 Koch’s flasks 1000 c.c. 6 Koch’s flasks 500 c.c. Cotton-wool for plugging tubes. 2 wire baskets. 6 Petri dishes 100 mm. 4 gross glass slips. 4 oz. glass covers No. 2 squares. 100 labels. 2 hollow-ground slips. 1 hot-air sterilizer, with thermostat, thermometer, and burner. 1 Arnold sterilizer or autoclave, with burner and tubing complete. 1 incubator, with thermostat, thermometer, burner, and tubing complete. 1 jar of beef extract. Litmus paper (red and blue). 1 bottle peptone (Witte’s). 6 fermentation tubes. 1 Cornet forceps. 1 cover-glass forceps. 2 test-tube brushes. DRY STAINS AND CHEMICALS. 30 gm. methylene-blue. 30 gm. gentian-violet. 30 gm. eosin-yellow. 30 gm. fuchsin. 10 gm. Bismarck-brown. 80 MEDICAL MICROSCOPY. 50 gm. carbolic acid crystals. 10 gm. iodin crystals. 20 gm. potassium iodid. 50 c.c. glacial acetic acid. 30 c.c. Canada balsam. 50. gm. potassium hydrate. 50 gm. pyrogallic acid. 1o gm. sodium hydrate. 10 gm. litmus. PREPARATION OF CULTURE-MEDIA. In making culture-media absolute cleanliness must be observed throughout the entire procedure. Every possi- ble source of contamination must be avoided when once the medium hasbeen made. Each time that it is exposed to any infection it must be sterilized. Never allow that portion of the cotton plug that enters the Koch flask (Fig. 35) to touch any object that will contaminate it. If it should be thoughtlessly laid upon the table while the medium is being poured from the flask, it should be re- placed in the flask and the whole sterilized at once. There are many ways in which contamination of media may take place, and one can only learn by experience to be bacteriologically ‘‘clean’’ in his manipulations. The following directions are made simple and explicit for the sake of the beginner, each step being laid down Ssertatim, Bouillon consists of: BGC RtLACKS, tapas ua ware sakes eee eee ey 3 gm. Peptone (Witte’s),................0.000. 10 gm. Sodium CHO ji sh sceskdlige esas ae auee aoe ak 5 gm. WE OR airs yeh 216! Ak is Sod the etek ant edeseoke Bodaea 1000 c.c. BACTERIOLOGIC METHODS. 81 Put the water in a clean agateware saucepan and heat by means of a radial burner or on the stove. The solid constituents should be weighed out upon a piece of paper and stirred with a glass rod into the water after it has reached the boiling-point. The paper should be thrown into the water, in order that any beef that clings to it may not be lost, and as soon as the beef has dissolved the paper is fished out with the stirring-rod. Fic. 33.—FERMEN- TATION TUBE, Fic. 32.—Dr. DuN- st UI HAM’S THERMO- Fic. 34.—PETRI REGULATOR. DIsH. The mixture is gently boiled for five minutes. It must now be made neutral or very slightly alkaline. For this purpose two solutions are kept on hand—a 5 per cent. solution of sodium hydrate and a 5 per cent. solution of hydrochloric acid. One strip of red and one strip of blue litmus paper are 7—O 82 MEDICAL MICROSCOPY. placed side by side on a piece of clean filter-paper and with the glass stirring-rod a drop of bouillon is put on each of the strips. If the bouillon is found to be acid, as is usually the case, a few drops of sodium hydrate are added to the mixture, it is vigorously stirred, and again tested, sufficient soda solution being added to make the bouillon neutral or, better, very slightly alkaline, so that it will turn the red litmus paper a faint blue color. If this stage is inadvertently passed, a few drops of the hydrochloric acid solution are used. Should the bouillon when origi- nally tested be alkaline, it may be neces- sary to neutralize with the acid solution. The variation of the reaction of the bouillon depends upon the meat extract. At times we may obtain an extract that will be quite acid, and again one will make a bouillon of marked alkalinity. The bouillon must be allowed to cool y inorder that the meat salts may be pre- ==" _—s cipitated and be filtered through filter- Fic. ou paper, folded in the usual way and pre- viously wetted with water and pressed into the stem of the funnel. Sufficient water must be added to bring the mixture to the original quantity of 1000 ¢.c. If it is desired to preserve the medium in bulk, it must now be placed in a Koch’s flask, the mouth of the flask closed with a large and light plug of cotton-wool, and the whole sterilized. The bouillon may also be drawn into test-tubes and sterilized for immediate use. Bouillon should be of a clear amber color. Frequently bouillon is modified by the addition of one of the sugars, so that we may have a glucose bouillon, lactose bouillon, and sacchrose bouillon. In each case BACTERIOLOGIC METHODS. 83 I per cent. of sugar is added to the formula for plain bouillon. Glycerin bouillon consists of plain bouillon to which has been added, after filtration, 5 to 8 per cent. of a good qual- ity of glycerin. Nutrient Gelatin. Beet extracts .wsteii 100. They present as rounded, smooth, or irregularly lobulated bodies, firm or elastic to the touch. Not infrequently they are associated with mucous tis- sue (chondromyxomata), bone (osteochondromata), or sar- comatous elements (chondrosarcoma). They may undergo mucoid or cystic degeneration, or become calcified or bony. Microscopically they present, to greater or less extent, the appearance of normal carti- TUMORS. 209 lage; or if associated with other tissues, islands of carti- lage may here and there be seen. The cartilage cells vary greatly in size and number, and may or may not have capsules. When pure, the chon- dromata are benign. Osteoma. An osteoma is a neoplasm consisting of bony tissue. Fic. 79.—CHONDROMA. X 100. Osteomata occur usually in connection with some portion of the skeleton as outgrowths (exostoses), and in the periosteum of bones, the tendons, and very rarely in the lungs, meninges, and skin. In connection with sarcomatous tissue they are fre- 18—O 210 MEDICAL MICROSCOPY. quent in the long bones and periosteum of the bones of the extremities and jaws, forming osteosarcomata. Pure osteomata present microscopically the typical structure of normal bone and are benign growths. Glioma. A simple glioma is a neoplasm which has sprung from the neuroglia or nerve connective-tissue cells of the cen- Fic, 80.—NEUROGLIOMA GANGLIONARE. XX 160. tral nervous structures. The gliomata follow the type of the neuroglia cells, and in section are seen to consist of a mass of cells with round or oval nuclei and possessing numerous protoplasmic processes whose delicate extremi- ties interlace in a most complex manner, forming a rich network. ‘These tumors may be very vascular and give TUMORS. 211 rise to severe hemorrhage. They may also become sar- comatous through proliferation of the cells of the perivas- cular connective tissue. Those round-celled tumors of the eyeball which for- merly were classed as gliomata are more properly round- celled sarcomata. They arise from the granular layer of the retina and usually run a very malignant course. The true gliomata are essentially benign. A neuroglioma ganglionare consists of glia cells, large ganglionic nerve-cells, and nerve-fibers. These neoplasms are probably congenital. Sarcoma. A sarcoma may be said to be a malignant neoplasm arising from connective tissue, the cells being embryonic in type, without definite fotm of arrangement and bound together by a scanty intercellular matrix. They may or may not be encapsulated. The sarcomata always spring from previously existing connective tissue of some kind; the softer connective tissues occurring normally through- out the body or from the specialized connective tissues, bone, and cartilage. As connective tissue exists in one or another form in every portion of the animal body, it fol- lows that sarcomata must have a wide field of origin. They occur more frequently in the dense structures, fascie, periosteum, skin, and bone, than in the internal organs, though they develop as primary growths in the uterus, kidney, liver, lungs, testes, and ovaries. The varieties differ widely in the form of cells and amount of intercellular substance, and in the number and size of the blood-vessels. The soft or medullary sarcomata are rich in cells, while the intercellular substance is scanty. The harder varie- ties are poor in cells and the intercellular substance is 212 MEDICAL MICROSCOPY. composed of a dense fibrous tissue. These latter are fibrosarcomata. The blood-vessels in sarcomata are usually atypical and poorly organized. Many of the smaller vessels may be composed of the cells of the tumor arranged in a more or less definite manner around the blood-channel, which has Fic, 81.—LARGE ROUND-CELLED Sarcoma.—(Thayer.) the usual endothelial lining. In some sarcomata well- formed blood-vessels exist. The sarcomata appear to be devoid of lymph-vessels. Degenerative changes are common in sarcomata, chief among these being fatty degeneration, necrosis, hemor- rhage, gangrene, liquefaction, and ulceration. TUMORS. 213 Sarcomata are always malignant to a greater or less degree, though the encapsulated forms may be very slow in growth, and the fibroid changes may be such that the neoplasm becomes almost a pure fibroma. But even tumors of such a character form metastases which may prove of a malignant nature, or they may themselves take on a rapid and destructive growth. According to the form of cell predominating, sarcomata are divided into: Small round-celled sarcoma. Large round-celled sarcoma. Small spindle-celled sarcoma. Large spindle-celled sarcoma. Mixed sarcoma. Small round-celled sarcoma is, as the name implies, com- posed chiefly of small round cells which have a large nu- cleus and a relatively small amount of protoplasm. The cells are held together by a scanty hyaline or fibrillated intercellular substance. The blood-vessels may be very numerous and vary in degree of organization, though generally they are little more than canals in the tumor-substance around which the 5... 9) tympyosar- tumor cells are more or less radially coma.—(Gould.) arranged. The so-called lymphosarcoma belongs to the small round-celled variety, but differs from the above in that it has a well-formed reticulated ground-substance of con- nective tissue, many of the cells of which are branching. In this meshwork lie the tumor cells proper, and these may closely follow the type of cell found in the normal lymphatic gland. These latter tumors usually spring 214 MEDICAL MICROSCOPY. from the various lymphatic tissues, but may occur in other situations. The small round-celled sarcomata of either type are among the most malignant of neoplasms, as evinced by their rapid growth and frequent formation of metastases. Large round-celled sarcomata differ from the small-celled variety in that they are composed of large cells having Fic. 83.—SPINDLE-CELLED SARCOMA. XX 360. oval bladder-like nuclei and a large amount of protoplasm. They lie in a reticulum composed of branching connective- tissue cells with long anastomosing processes. Some of the tumor cells possess more than one nucleus. The vessels are usually poorly developed. These tumors are usually not so malignant as the small-celled sarcomata. Spindle-celled sarcomata are divided into the large and TUMORS. 215 small varieties, the one differing from the other only in the size of the cell, as either may be highly malignant or may follow a chronic or almost a benign course. They are composed of long spindle cells having large elongated nuclei and delicate protoplasmic prolongations at either end. Stellate cells may also be found. The intercellular substance may be quite prominent or Fic, 84.—PERITHELIAL ANGIOSARCOMA. X 55. so scanty as to be difficult of demonstration. The tumor cells frequently lie in bundles which may follow the course of the blood-vessels or arbitrarily cross each other in a complex manner. Again, the cells may lie in a confused way entirely without perceptible motive. The fact that these growths may sometimes be confounded with leiomyomata has already been pointed out. 216 MEDICAL MICROSCOPY. Mixed sarcomata are those in which there are both round and spindle cells in about equal amount. Endotheliomata are those sarcomata which have sprung from endothelial cells. Such are not infrequently found arising from the lining of the blood- or lymph-vessels, or from the serous membranes of the body, pleura, and meninges. They form flattened growths which have all Fic. 85.—ALVEOLAR Sarcoma. X 100. the characteristics of malignancy. Frequently the ar- rangement of the cells may be in tubular form, so that the glandular type of carcinoma is closely simulated. In such cases the origin of the tumor determines its true nature. The cells are frequently large and epithelioid in character. Angvosarcomata are sarcomata growing from the walls of blood-vessels. The vessels may be surrounded by TUMORS. 217 dense deposits of cells forming columns, with dilated vessels asacore. ‘The vessels may be very numerous and follow a complex course. To such growths the name plexiform angiosarcoma has been given. They have also been called perithelial angiosarcoma. Alveolar sarcomata have a highly organized reticulum consisting of bands of connective tissue forming alveoli Fic. 86.—ENDOTHELIOMA OF PLEURA.— (Ziegler.) a, Fibrous stroma. 6, Cords of endothelial ce'ls. in which lie nests of sarcoma cells. The peculiar struc- ture is probably formed by the tumor cells crowding in and pushing apart the normal connective tissue of the part. Melanotic sarcomata may be of either variety described, but contain melanin or pigment in varying quantity. They are always extremely malignant. Their most frequent origin is the skin, pigmented moulds, or the 218 MEDICAL MICROSCOPY. choroid of theeye. The pigment-granules are usually of a light yellow color. Giant-celled sarcomata are frequently found arising from bone or periosteum. They are usually of the spindle- celled variety, but in addition have many large cells con- taining numerous nuclei. These nuclei may be quite separate and clearly outlined, or may be fused together in Fic. 87.—MELANOTIC SARCOMA. X55. a poorly defined mass which may be to one side of the cell or may occupy its center. Because of the similarity of these cells to the myeloplaques of bone-marrow the tumor has received the name of myeloid sarcoma. Such tumors are frequently slow in growth and do not form metastases, and therefore do little damage. On the other hand, they may be very malignant. Not infrequently sarcomatous tissue is combined with a TUMORS. 219 varying quantity of some other tissue of the connective- tissue group. Thus, we have myxosarcoma, chondrosar- coma, osteosarcoma, and a variety of other combinations. aN STA teD ue Ww e Bae Fic. 88.—GIANT-CELLED Sarcoma.—(Coplin.) a, b, c, d, e, Giant cells. Secondary changes, softening, liquefaction necrosis, or calcification may take place in the sarcomata of either variety: EPITHELIAL TUMORS. The tumors about to be described differ from others in that they are derived from the epiblast and hypoblast, and are classed as epithelial growths. The tumor cells proper are of the epithelial type of cells, 220 MEDICAL MICROSCOPY. and they are separated by a connective-tissue framework of greater or less amount. ‘he epithelial cells of the body normally go to make up those structures which are pro- tective,—skin and mucous membranes,—and the secret- ing parts or glands. We have seen that tumors follow more or less closely the normal structures from which they are developed, and so it is not surprising that we find the epithelial neo- plasms made up of those tumors which are of a glandular type or which are similar in structure to the mucous mem- branes or are composed of cells arranged in a manner sim- ulating the stratified epithelium of the skin. Papillomata. These neoplasms are outgrowths from the skin or mu- cous membranes. ‘They usually arise from previously Fic. 89.—PapiILLoma.—(Schmaus.) existing papilla, and are by some authors considered hypoblastic growths rather than true neoplasms, or fibro- mata with an epithelial covering. TUMORS. 221 They consist of a central core of connective tissue, blood- vessels, nerves, and lymphatics, with a tunic of epithelial cells of the type normal to the part from whence the papil- loma arises. The common skin wart, the soft venereal wart, ‘and the villous wart of the bladder are common examples. The papillomata are benign growths. They differ from the epitheliomata in that they grow up and not down into the tissues from which they arise, and present none of the other characteristics of malignancy. They may produce death through mechanical means, hemorrhage, or obstruction in the respiratory or genito- urinary passages. They may also take on a malignant ’ growth, becoming true epitheliomata. Adenomata. The adenomata are neoplasms which spring from and closely follow the type of some normal gland structure. They occur usually as round or pear-shaped tumors of comparatively slow growth, in the skin, where they arise from the sweat and sebaceous glands; in the mammary glands, uterus, liver, kidney, stomach, rectum, and other situations. They are comparatively rare growths, and when pure are benign. It is, however, difficult and at times impossible to sepa- rate them, on the one hand, from simple inflammatory hyperplasia of a glandular structure, and, on the other, from the carcinomata. In making a diagnosis the verdict must be based upon the microscopic findings, together with the history and clinical symptoms of the individual case. An adenoma differs from the normal gland structure in that it does not possess all of the anatomic elements, nor can it perform the physiologic functions of the gland from which it sprang and the type of which it follows. Ducts, if present, are abortive and end ina blind pouch. 222 MEDICAL MICROSCOPY. Two chief forms of adenomata are recognized, one hav- ing a complicated arrangement of acini lined with col- umnar epithelium supported by a basement of connective tissue, the whole resembling a normal racemose gland. ‘There may even be imperfectly developed excretory ducts. Such a growth is called an alveolar adenoma. Fic, 90.—SIMPLE ADENOMA.—(Coplin.) A simpler form is the tubular adenoma. As the name implies, this is made up of irregular, dilated tubules, com- posed of a connective-tissue framework, often quite dense, the spaces in which are lined by columnar epithelium. When either of the above by rapid growth of the epithe- lium forms finger-like or branching processes which pro- TUMORS. 223 ject into the lumen of the dilatations, it is known as a paptlliferous adenoma. Dependent upon the amount of connective tissue, an adenoma may be soft or hard. When the epithelial cells of an adenoma develop a ten- dency to break out of the bounds set by the connective tissue upon which they rest, and to form new and inde- pendent nests of cells, the tumor may become destructive and form metastases, in which case it is called an adeno- carcinoma. Another form of adenocarcinoma consists of tubular structures similar to the original adenoma, malignancy being manifested by rapid growth, invasion of neighbor- ing tissues, and metastases to more distant parts. The growth may retain its adenomatous type or may eventually become a true carcinoma. Such neoplasms are not un- common in the stomach. The adenomata are subject to secondary changes. They may become cystic, myxomatous, or even calcareous. Carcinomata. A carcinoma is a malignant neoplasm arising from epi- thelial structures and following the type of tissue from which it springs. There are two great classes of epithelium, that which covers surfaces—skin, mucous membranes—and that which forms the secreting portions of glandular organs— mammary gland, liver, kidney, etc. The first of these is composed of flat cells, usually in the stratified form. The second is composed of polyhedral cells in tubular or other glandular arrangement. It naturally follows that there are two great types of car- cinoma—those arising from surface epithelium of the squamous type which follow this type of tissue and are 224 MEDICAL MICROSCOPY. composed of flat cells, and those having their origin in glands and following the glandular arrangement of tissue more or less typically. Epithelioma.—These neoplasms, as has been pointed out, arise from surfaces covered with stratified squamous epithelium. They are composed of columns of epithelial cells which penetrate the surrounding structures and Tic, 91—EpimTHELioma, X 100. which frequently form central whorls or nests of cells, the so-called epithelial pearls. These nests are composed of concentrically arranged horny epithelium and represent an effort of the abnor- mally proliferating cells to conform to the normal type of stratified squamous epithelium. It is safe to class the epitheliomata among the malig- TUMORS. 225 nant neoplasms, as they frequently grow with astonishing rapidity and cause great destruction of neighboring tis- sues and form metastases in distant parts. They are prone to degenerative processes, and frequently become infected with pyogenic organisms, resulting in ulceration and abscess. Glandular Carcinoma.—In this form of cancer the tumor Fic, 92.—ADENOCARCINOMA OF STOMACH.— (Ztegler.) a, Mucosa. 0b, Submucosa. c, Muscularis. d, Serosa. e, New growth proceeding from mucosa and infiltrating other layers. is composed of a connective tissue in which lie groups of epithelial cells arranged in a manner more or less typical of normal glandular tissue. The connective tissue does not penetrate between the individual cells, as in sarcoma, but surrounds groups of epithelial cells, so that if these epithelial aggregations 19—O 226 MEDICAL MICROSCOPY. should be shaken out, a sponge-like mass of connective tissue would remain. Adenocarcinoma, as has been shown, is but a step re- moved from true adenoma, and consists of a neoplastic tissue of distinctively glandular type the epithelial ele- ments of which have a tendency to rapid proliferation, so Fic. 93.—MELANOTIC CARCINOMA. XX 100. that they pile up one upon another, forming in some in- stances alveoli lined with two or more layers of cells. They also push out bud-like projections of epithelial cells into the neighboring tissues. , In the more common form of glandular carcinoma the alveoli are entirely filled by epithelial cells of a polyhedral type, so that no central lumen exists. TUMORS. 227 These growths are frequent in the mammary gland, liver, pancreas, intestines, and other glands. Clinically they are divided into medullary or soft, simple, and scirrhous cancer. In the soft varieties there is a pre- ponderance of the epithelial cells over the connective tissue, while in the hard or scirrhous cancers the opposite Fic, 94.—MEDULLARY CARCINOMA. X 360. condition prevails. The simple carcinoma is composed of about equal amounts of these two factors. This classifi- cation is of little value, as the same tumor may show all of the above varieties of structure in different parts. Carcinoma myxomatoides is a form in which the connec- tive-tissue framework has undergone mucoid changes. Giant-celled carcinoma has large epithelial cells, many of 228 MEDICAL MICROSCOPY. which may be dropsical. True giant cells with several nuclei may also be present. Melanocarcinoma may be of any of the preceding forms, and is distinguished by the presence of pigment granules within the stroma and epithelial cells. This form is al- ways very malignant. Fic, 95.—ScIRRHOUS CARCINOMA. X 100. Epithelial Cystomata. From the simple retention cyst these tumors are dis- tinguished by there being a distinct new-formation of tis- sue. They consist of one or more cavities containing a fluid or mucoid substance. The walls of the cavities are composed of connective tissue lined with epithelium, fre- quently of the columnar variety. They cannot be sharply differentiated from either the TUMORS. 229 adenomata or carcinomata. Usually they are benign, but they may at any time take on a malignant character. Papillary cystomata are distinguished by prolongations from the walls of the cysts, consisting of a connective- tissue core covered with one or more layers of epithelial cells. A rapidly growing tumor of this type constitutes the papillary cystocarcinoma, From such growths metas- Fic. 96.—GIANT-CELLED CARCINOMA, X 360. tases may occur that exhibit the characteristics of ordi- nary glandular carcinoma. Teratomata. A teratoid tumor is one of complex composition, and “consists, at least in part, of tissues which do not nor- 230 MEDICAL MICROSCOPY. mally occur at the site where the tumor is found” (Ziegler). The dermoid cyst belongs to this class. The rhabdo- myomata of the kidney may also be cited as an example, Fic. 97.—SIMPLE CARCINOMA. uw, Fibrous trabeculer. 6, Large atypical epithelia of polygonal outline, showing mitotic figures —(Zezgler.) and rarely there are retroperitoneal neoplasms presenting structures similar to those of the gastro-intestinal tract. The teratomata are usually benign, but may become sarcomatous. They are supposed to be congenital. THE BLOOD. 231 THE BLOOD. Histology of the Blood. The blood consists of a fluid plasma in which float red and colorless blood-corpuscles and blood-platelets. The normal blood-plasma is a thin, colorless fluid of alkaline reaction. The red blood-cells of normal adult human blood are bi- concave discs about 7.5 » in diameter. They consist of semifluid protoplasm and a coloring-matter, the hemo- globin, upon which the staining quality of the cell directly depends. The question as to whether they have a definite cell- wall still remains unsettled. Probably there is such a structure, or it may be that the appearance of one is given by the condensation of the protoplasm of the cell about its periphery. The red cells are capable of great mobility and in fresh blood or in the capillary vessels, as seen in the web of a frog’s foot, they readily assume various shapes, accommodating themselves to the caliber of the channel, or are indented by contact with neighboring cells. They immediately assume their normal form when the pressure is removed. Frequently they collect in coin-like masses or rouleaux. The non-nucleated, biconcave form of red blood-cor- puscle is found in all mammals with the exception of the camel, in which the red blood-cells are oval, but contain no nucleus. Oval nucleated red corpuscles are normally in the blood of all fowls, reptiles, and fishes, with the exception of the Cyclostomata, in which the cells are round, discoidal, and nucleated. The largest of all red blood-corpuscles are those of the 232 MEDICAL MICROSCOPY. Amphiuma, which are about 0.046 mm. wide and 0.075 mm. long, and may be seen by the unaided eye. Development of the Red Blood-cells. In early fetal life the development of the red blood- corpuscles is thoroughly determined, while their origin in the latter part of fetal existence and during adult life is still a matter of uncertainty. The red cells first appear in early embryonic life as isolated groups of cells in the so-called ‘“‘islands of Pan- der’’ or vascular areas. They are of mesodermal origin and are formed simultaneously with the first blood-vessels and from the same group of cells. At a later stage the liver and lymphatic glands contribute largely toward the manufacture of red blood-cells. After the formation of the bones the red marrow plays an important part in this function, and is considered by most authors to be the ex- clusive site of the manufacture of the red blood-cell during extra-uterine life, though by some it is held that under pathologic conditions the spleen may, as it were, return to a fetal condition and again take up the labor of pro- ducing red blood-corpuscles. Varieties of Red Blood-corpuscles. The normal-red blood-cell or erythrocyte of adult life has been described. There are, however, in embryonic life and pathologic conditions other forms of red blood- corpuscles that now deserve our attention. They are classified according to size into: NUCLEATED. NON-NUCLEATED. Microblasts. Microcytes. Normoblasts. Normocytes (Erythrocytes). Megaloblasts. Megalocytes. THE BLOOD. 233 All varieties of red blood-cells at first contain a nucleus which at a later stage disappears entirely or is demon- strable only in part and with difficulty. ‘The nucleus in most cases probably disintegrates and fades in situ, though Ehrlich and Lazarus contend that this is not the case with the nucleus of the normoblast, but that it is ex- truded from the cell entire. As free nuclei of red blood- cells may frequently be met with, this view is evidently, at least in part, correct. From the above tabulation it will be seen that when the red blood-cell loses its nucleus it takes the termination cyte, so that upon the extrusion of the nucleus of a normo- blast it becomes a normocyte (erythrocyte) or normal red blood-corpuscle of adult life. Maicroblasts.—These are red blood-cells that are con- siderably smaller than the erythrocyte and which measure from 1 4 to 4 in diameter. The nucleus is single and stains very deeply with all nuclear dyes. It may be proportionally very large, there being only a small rim of protoplasm present. They probably arise from division of degenerated or immature mother-cells, and are not uncommon in perni- cious anemia and splenic myelogenous leukemia. They are also frequent in the fetal blood. Microcytes are small non-nucleated red blood-cells de- rived either from the microblasts or by the contraction of erythrocytes due to loss of hemoglobin or the fragmenta- tion of degenerating red cells of normal size. They are common in blood that is undergoing severe and rapid de- generative changes. Usually they are deficient in hemo- globin, and therefore do not take up dyes readily and ap- pear pale and thin. Normoblasts are the progenitors of the erythrocytes, and are therefore about 7.5 » in diameter. The nucleus 20—O 234 MEDICAL MICROSCOPY. is usually single, occupies about one-third of the corpuscle, and stains more deeply than the nucleus of any other cell. The protoplasm is rich in hemoglobin, and therefore takes the dye with avidity, being deeper in color than in the erythrocytes. Normoblasts are found in fetal life, and even in small quantity for some time after birth in normal blood. Normocytes (erythrocytes) have been already described; they constitute the normal red blood-corpuscle of adult life. Megaloblasts. These are nucleated red blood-cells and are from 10 to 20 w in diameter. The larger forms are sometimes called gigantoblasts. They differ from normoblasts in several important particulars. Besides their large size, the nucleus is frequently multiple, lobulated, or stellate, or may show various stages of mitosis, usually of an im- perfect character. Generally there is to be distinguished about the nucleus a clear zone which evidently is devoid of hemoglobin and therefore does not take the stain. The nucleus is deeply though often irregularly stained, and may vary in color in different parts owing to degen- erative changes. ‘The protoplasm usually stains deeply, but may show a loss of hemoglobin and corresponding paleness, and at times there may be granular fragments of the nucleus in the protoplasm. When found in consid- erable numbers, they always indicate pernicious anemia of the primary type, but a few may occur in secondary anemias. They are also present in leukemia, often in large amount. Megalocytes are derived from the megaloblasts by de- generation of the nucleus of the latter. They are from 10 to 20 # in diameter and may show either an excess THE BLOOD. 235 or a deficiency of hemoglobin. At times they may un- doubtedly be formed by the swelling of the normocytes due to altered conditions of the blood-plasma, as may be seen in cases of severe malaria. Usually they are accom- panied by megaloblasts, and are therefore chiefly found in those conditions in which the latter obtain. Blood=plates. The origin and physiology of these bodies is not yet determined. They occur in normal blood in considerable numbers as oval or spheroidal bodies from 1 yp to 3 y» in diameter, and stain faintly with methylene-blue and other basic dyes. They have a marked tendency to form in clumps, and may then appear as a mass the individual components of which it is not possible to make out. The author has recently observed them in specimens stained by Goldhorn’s method apparently lying within the red blood-corpuscle, though possibly this is an arti- fact. In such cases, however, the most careful focusing fails to show that they lie at a level different from that occupied by the red blood-cell, nor is its characteristic coloration seen through them. With the above stain they appear, when discrete, as round bodies, having a clear-cut outline of colorless protoplasm (?) in which isa recticular structure of a brownish-red color. The stain of this retic- ulum is exactly similar to the chromatin of the malarial parasite as dyed by the Goldhorn method. This would seem to indicate that they are derived from nuclear ele- ments, as has been frequently contended by various ob- servers who consider them to be the results of fragmenta- tion of the nuclei of degenerating leukocytes. Blood-dust.—This name was given by H. F. Miiller to granular colorless particles first observed by him in fresh blood. ‘They are highly refractile and have a rapid danc- 236 MEDICAL MICROSCOPY. ing (Brownian) movement. Their significance is not known. WHITE BLOOD-CORPUSCLES OF NORMAL BLOOD. The classification of the leukocytes is based upon their morphology and their reaction to certain staining agents of complex nature, for it is found that the protoplasm of some forms of white blood-cells contains granules differing in their action toward dyes from the granules of other leukocytes which, except in this respect, are identical. Three forms of granules are distinguishable: 1, Basophile, .............. Staining with purely basic dyes—methyl- ene-blue, hematoxylin, thionin, dahlia, etc. 2. Oxyphile or Acidophile, ..Staining with purely acid dyes—eosin, fuchsin, etc. 3. Neutrophile, ............ Staining with combinations of basic and acid dyes, forming a new specific dye. Ebrlich’s tri-acid, Jenner’s stain, etc. These latter combinations stain also one or both of the other varieties of granules. Ehrlich’s tri-acid stain is the one generally employed for the purpose of differentiating the white blood-cells. This has the property of staining two kinds of granules, the neutrophilic and the oxyphilic, but does not stain the basophile granules. In order to demonstrate these latter we must use either a purely basic dye or a combination of pigments, such as Jenner’s stain furnishes, which has the property of stain- ing each of the three kinds of granules. Lymphocytes. These cells constitute about 22 to 25 per cent. of the white blood-corpuscles of normal blood. ‘They are about THE BLOOD. 237 the size of a red blood-corpuscle and possess a central round nucleus the reticulated structure of which is often visible in well-stained specimens. The protoplasm is of comparatively small amount and is strongly basophilic, and in many instances is more deeply stained than the nucleus itself. The protoplasm is reticulated, as may be seen by its unequal staining properties in different parts. Frequently portions of the protoplasm may bud off and become separated from the mother-cell and float free in the blood. At times larger forms of lymphocytes are to be found. Their recognition is easy on account of the strongly basophilic protoplasm and the absence of proper granulations. Occasionally may be seen lymphocytes having notched or double nuclei. The lymphocytes stain more deeply than other cells found in the blood with the exception of normoblasts, from which they are readily distinguished. Large Mononuclear Leukocytes.—In normal blood these are found in small numbers, forming only about one per cent. of the total amount of white blood-corpuscles. They are quite unique and easily recognizable on account of their large size, two or three times that of the erythrocytes, their large, pale, oval nucleus, and faintly basophilic protoplasm, which is devoid of proper granules. The protoplasm is finely reticular and abundant, stains less deeply than the nucleus, and in it the nucleus is generally excentrically placed. Except in deeply stained specimens it is easy for the beginner to overlook these cells, on account of their uni- versal paleness, which contrasts so greatly with the char- acter of the other forms of leukocytes. Nucleoli are some- times demonstrable. Transitional Forms.—These are similar to and represent a later stage of the development of the large mononuclear 238 MEDICAL MICROSCOPY. leukocyte. They differ from them in that the nucleus stains slightly deeper and shows a distinct notch in one side, giving it a horseshoe shape. ‘There may be a few small neutrophilic granules in the protoplasm. Together with the large mononuclear forms, these constitute from 2 to 4 per cent. of the white blood-cells of normal blood. Polynuclear Leukocytes.—These may be considered the most important of the white blood-cells, constituting as they do 70 to 72 per cent. of the total amount of these ele- ments in normal blood. They are usually about twice the size of the erythrocyte. The nuclei show a great variety of forms, being frequently Z-, S-, or E-shaped, the various portions connected by delicate threads (polymor- phonuclear), or there may be several distinct nuclei, the latter fact having led Ehrlich to give them the above not strictly descriptive name. The nucleus is colored deeply with all nuclear stains and exhibits a coarse recticulum. The protoplasm is finely reticulated, slightly basophilic, and contains numerous neutrophilic granules, of varying size but usually quite fine. Eosinophiles. These are polynuclear cells about the size of a=poly- nuclear leukocyte, though they may be somewhat smaller. The nucleus is coarsely reticular and stains less deeply with nuclear stains than does that of the polynuclear leukocyte. Their distinguishing feature is the large deeply staining oxyphilic granules of the protoplasm. With eosin these appear as numerous shot-like bodies of a deep red color, while with Ehrlich’s tri-acid stain they are a rich copper color. In fresh blood on the warm stage, or even at room- temperature, they are exceedingly active, often traveling THE BLOOD. 239 by ameboid movement entirely across the field, teh streaming motion of the granules being distinctly visible. In fresh preparations the beginner often mistakes them for the malarial parasite—an error to be guarded against. They constitute about 2 to 4 per cent. of the white cells. TABULATION OF CELLS ACCORDING TO STAINING REAC- TION. 1. Basophile Cells. Lymphocytes. Protoplasm strongly basophilic. Large mononuclear leukocytes. Protoplasm faintly basophilic. Mast Cells. Granules strongly basophilic. 2. Oxyphile Cells. Polynuclear Eosinophiles. Granules oxyphilic. Eosinophilic Myelocytes. Granules oxyphilic. 3. Neutrophile Cells. Polynuclear Leukocytes. Granules neutrophilic Myelocytes. Granules neutrophilic. Nature of the Granules. As to the exact structure and significance of the gran- ules of the white blood-corpuscle there has been considera- ble controversy. According to recent views, ‘‘they are a secretory product, and represent the specific function of the cell’”’ (Ewing). It has also of late been shown that the basophilic cells are possessed of a reticulate structure which is continuous with the spongioplasm of the nucleus. Where the lines of this fine network connect with each other there are found nodal thickenings. It has been demonstrated that the granulations of the cells are connected with this reticulum by fine lines, and they are therefore integral parts of it. Nucleoli may also be demonstrated in the nuclei of the leukocytes. Mast Cells. These must be considered as rather accidental in nor- mal blood, where they are rarely found; according to 240 MEDICAL MICROSCOPY. Ehrlich, they are present in the proportion of 0.5 per cent. of white cells. In size and inthe form of the nucleus they are very ir- regular, but from other cells they are sharply defined by large granules in the protoplasm which stain deeply with basic dyes. With certain stains, notably thionin and polychrome methylene-blue, these granules do not show the pure color of the dye, but stain metachromatically. The nuclei do not stain deeply, and it must be remem- bered that with Ehrlich’s tri-acid stain the granules are unstained, so that the protoplasm of the cell remains clear. COLORLESS CELLS PECULIAR TO PATHO- LOGIC CONDITIONS OF THE BLOOD. Myelocytes. These vary in size from about that of an erythrocyte to several times this diameter. They have a large, round or oval, pale-staining nucleus and a relatively large amount of protoplasm, and may be easily distinguished from the large mononuclear leukocyte, which they otherwise re- semble, by the numerous neutrophilic granulations of their protoplasm. They are never present in normal blood. In myelo- genic leukemia they may occur in enormous numbers, and they have been observed in smaller quantities in various infectious diseases, in poisoning by mercury, in the anemia of infantile pseudoleukemia, and in diphtheria and pneu- monia. Eosinophilic Myelocytes. In myelogenic leukemia and infantile pseudoleukemia mononuclear cells with oxyphilic granulations of the pro- THE BLOOD. 241 toplasm are frequent, but they rarely occur in other con- ditions. Small Neutrophile Pseudo-lymphocytes. Ehrlich describes these cells, which he first found in a case of hemorrhagic smallpox, and considers them as pro- ducts of division of the polynuclear cells. They are about the size of a lymphocyte and present a small amount of protoplasm studded with neutrophile granules. TECHNIQUE. So delicate a structure as a blood-cell requires faultless technique if we would see it in its normal state. The beginner must first perfect himself in the various methods, using his own or other normal blood, before he ventures to draw conclusions from blood derived from presumably diseased subjects. Not one but many pre- parations should be made and studied, so that he will become thoroughly familiar with the normal histology of the blood and with those artifacts that in spite of all cau- tion occur in preparations made by the most experienced workers. The study of the blood is so fascinating that such a labor is light. Except in the case of malaria it is rarely advisable to study blood in the fresh, unstained condition. A descrip- tion of the method of making fresh preparations is given in the section on malaria. For most purposes we resort to thin spreads of blood that have been stained by some dye. A close attention to minute details is necessary to insure success. It is quite necessary for the student to recognize this in the beginning, as it will save him vexatious failures. 242 MEDICAL MICROSCOPY. Making the Smear. Many text-books recommend the use of cover-glasses for this purpose. The author much prefers to use the glass slip, for the reasons that it is easier to handle, the smear is more apt to be successful, it can be carried through the various manipulations with greater ease and certainty, and it is better not to use balsam and a cover-glass upon the finished product, as the preparation is apt to fade, — while it keeps perfectly for years when left uncovered. The slips must be absolutely clean in order to insure thin Fic. 98.—How To MAKE A BLOOD-SMEAR. and even smears. It is well to first clean them with alcohol and a clean cloth, rubbing them vigorously. They should be again briskly rubbed with a clean cloth imme- diately before use, for the double purpose of removing any dust that may have settled on them and of warming the glass by the friction, as this facilitates the spreading of the blood. 5 The lobe of the ear is the best place from which to ob- tain the drop of blood. In order to rid the skin of grease and dirt and free epithelial scales the part should be cleaned with a clean cloth, soap and water. It is entirely THE BLOOD. 243 unnecessary to use alcohol and ether, as is frequently rec- ommended. For the purpose of making the puncture a small lance, such as is sold for the purpose, or a thin-bladed tenotome should be used. The instrument should be very sharp, as it will give less pain to the patient and produce a better flow of blood. After use it should be washed and wiped clean. Experience shows that it is not necessary to ster- _‘ilize it except in cases known to be septic. The lobe of Fic. 99.—How Not To Make A BLOOD-SMEAR. the ear is held between the thumb and finger of one hand and strong pressure gradually made upon it for a moment and the point of the lance then thrust with a quick move- ment a sufficient depth into the tissue. In nervous pa- tients and in children by following this method it will be found that frequently the puncture may be made without the knowledge of the patient. The puncture should be deep enough to allow the blood to flow spontaneously or with a gentle “milking” pres- sure. The first few drops are to be wiped away. 244 MEDICAL MICROSCOPY. A cleaned slip is laid upon a convenient table; another, held by the end between the thumb and finger of the right hand, is to be used to make the smear. Milk out a fresh drop of blood; bring the free end of the slip in contact with it so that a small drop will cling to its edge; quickly transfer this to the slip upon the table by allowing the end of the slip containing the blood-drop to come in contact with its surface a short distance from its further end. The upper slip is now dragged along the surface of the lower one, the blood-drop gradually escaping beneath it and being distributed in a thin, even smear. In this latter part of the manipulation the slip must not be held at too great an angle. ‘The proper angle is best determined by allowing the free end of the slip to lie upon the tip of the index-finger, which is then drawn along the surface of the table, the weight of the slip being sufficient to carry it along at the same time. A much better result is thus obtained than if the slip is grasped firmly between the thumb and finger, as a steady movement is then im- possible and an unequal smear results. Caution: The drop of blood must be small; a large drop means a thick, useless smear. The smear is allowed to dry in the air, which it does very quickly, and the slip may now be wrapped in paper and carried to the laboratory. Fixation. If Ehrlich’s tri-acid stain is to be employed, it is neces- sary to fix the blood by heat. This is best accomplished by the use of the hot-air sterilizer, or some other form of oven in which a high degree of dry heat can be obtained. The author secures the best results by putting the smears in the oven when it is cold and gradually raising the temperature to 125° C., at which point it is kept from THE BLOOD. 245 ten to fifteen minutes. The smears are now removed and cooled before applying the stain. Fixation by heat may be accomplished by passing the slip repeatedly through the bunsen flame until it is too hot to be borne in contact with the hand. This degree of heat must be kept up for about five minutes. After experience it is possible to obtain fairly uniform results in this way, but the method is at best a makeshift and unreliable. For methylene-blue, eosin, and other stains it is not necessary to use heat as a fixing agent. Either of the following methods is good: 1. Immerse the slide for at least fifteen seconds in methyl alcohol. 2. Wash in water. MopIFICATION OF FUTCHER’S METHOD. 1. Formalin (full strength), 95-per cent, alcohol, 244) OQ} PO] O@ Po Leon Se Q @Q © AS) Soo Ot qh oO a eS Bo Q vi bb op oh OQ 9 @ ge Q © |9 © Q a Q Q ® a: ap} 6 2 Pp? be Pg 5 Pao eh a" @ aI 39 3] 2° @ Pb So Glo” a SO OO] Oo 6 S 1a © D AK) og P19 9 ® 4 (o¢4 elo © 2 & © © Fic. 103.—PLAan oF CoUNTING ERYTHROCYTES. order to avoid error one must count as in the square under observation all those upon the left-hand and bottom lines, leaving those on the lines to the right, which will by this method be counted in the next square. It is well to make a rough ruling on paper of lines forming a block of 25 squares and as each square is counted the result can be set down in the corresponding square of the diagram. At least four of the blocks of 25 squares must be 254 MEDICAL MICROSCOPY. counted, and the result is more accurate if twice this num- ber be enumerated. Computation. If now we have as a result of counting the cells in 100 of the small squares a total of 1250 red cells, we will obtain the number of red cells in a cubic millimeter of blood by multiplying the product of the count by 4, for we have counted one-fourth of the surface of the square milli- meter; again by 10, because the cell is only 4; mm. deep; and again by 100, on account of the blood having been diluted with 100 parts of fluid. We have, therefore, 1250 X 4 X 10 X 100 = 5,000,000 per cubic millimeter. As 4 X 10 X 100 = 4000, when we have counted 100 of the small squares the multiplier is 4000; if 200 squares, 2000; and if 400 squares, 1000. ENUMERATION OF WHITE BLOOD-CELLS. The normal number of white blood-cells being so small, it is usually necessary for this purpose to have a special pipette, which allows of a dilution of 1: 10 or 1: 20. In order to count the leukocytes we must get rid of the red blood-cells, as otherwise these would obscure the white cells. This is accomplished by using as a diluent a solu- tion of 3 per cent. acetic acid slightly tinted with gentian- violet, which rapidly decolorizes all of the red blood-cor- puscles and also renders the leukocytes more plain by staining them a pale violet color. Otherwise than above, the technique of preparation of the drop is the same as that for the red-colored corpuscle. The lower power can be used for counting leukocytes, and the contents of each of the small squares of the square millimeter should be enumerated. THE BLOOD. 255 If the dilution has been 1: 10, the sum total is multi- plied by roo, the result being the number of leukocytes per cubic millimeter. Care of the Hemocytometer. The greatest cleanliness must be observed in order to keep the apparatus in proper condition. After using, the cell and cover should be washed with water—not ether or alcohol, as these will dissolve the Fic. 104.—GoweErs’ HEMOGLOBINOMETER. cement with which the cell is attached to the slide—and dried with a clean soft cloth. The pipette is cleaned by first sucking water into it, then with alcohol, and lastly with pure ether. This must always be done immediately after use. It is well occasion- ally to clean the pipette with pure nitric acid followed with alcohol and ether. Estimation of Hemoglobin. Various instruments have been invented for the purpose of determining the percentage of hemoglobin, none of 256 MEDICAL MICROSCOPY. which is entirely satisfactory. Only the simple modifica- tions of Gowers’ hemoglobinometer will be described, as this is largely used, and for clinical purposes answers fairly well, as a slight error is of little consequence. When the percentage of hemoglobin is known to be very low, twice the quantity of blood should be em- ployed and a corresponding division of the result made. The apparatus is exceedingly simple. It consists of a tube containing a standard solution of the tint of normal blood diluted with 100 parts of water, a second tube graduated to 120 parts, and a capillary pipette with which to measure 20 c.c. of blood. Technique. The tubes are placed side by side in the stand provided for them and a few drops of distilled water are placed in the tube in which the dilution is to be made. The blood is obtained from a free puncture in the lobe of the ear or finger, the part having previously been cleaned with soap and water. The pipette is filled to the 20 c.c. mark and the contents are quickly discharged into the graduated tube. The pipette is several times filled with distilled water and emptied into the graduated tube. The blood and water are mixed by agitation, and more water added drop by drop until the color of the mixture exactly corresponds with that of the standard tint tube. The percentage of hemoglobin corresponds to the height upon the scale reached by the fluid, the reading being taken from the middle of the concave surface caused by capillary attraction. VARIATIONS IN FORMED BLOOD-ELEMENTS, 257 VARIATIONS IN THE FORMED ELE- MENTS OF THE BLOOD. RED BLOOD-CORPUSCLES. Polycythemia.—An increase in the number of red blood- cells occurs in new-born infants, upon a removal from a low country to a high altitude, in those conditions which decrease the amount of blood-plasma,—diarrhea and watery exudates in general,—after the administration of certain drugs, and, as has been mentioned, after exercise and cold baths. Oligocythemia.—This term is used to denote a decrease in number of the red blood-cells. Such a condition is fre- quently met with, notably in the anemias, where in the pernicious form they may fall as low as 360,000 to the cubic millimeter, in chlorosis, after hemorrhage, in carci- noma, etc. Poikilocytosis.—Variations in size and form are indi- cated by this term. The condition is always present in pernicious anemia and is frequent in secondary anemia from any cause. At times it is scarcely possible to find a corpuscle of normal shape. They are distorted into every conceivable form, but perhaps most often assume a club-like shape with the handle curved and pointed, others are crescentic, many of the cells are much smaller than normal, and all are more or less faded in appearance, owing to the diminished amount of hemo- globin. The condition once seen is easily again recognized. Care must be exercised not to confound it with the distor- tion of the corpuscles caused by faulty technique in mak- ing the spread. In such a case it will be observed that all of the altered cells are elongated in one direction, their 22—O 258 MEDICAL MICROSCOPY. long diameters lying in the same axis, which is not the case in poikilocytosis. Microcythemia.—In anemias, toxic conditions, and after severe burns this condition of the red blood-corpuscles may be observed. It consists in the presence of large numbers of microcytes. It has been ascribed by Graber to the rapid abstraction of fluid from the red blood-cells and a consequent rapid shrinking of these elements. The condition may appear and disappear with equal suddenness. Clinically it is of little known importance, as its cause and significance are not understood. Polychromatophilic Degeneration. Ehrlich has pointed out that in certain conditions many of the red blood-cells have the property after hematoxylin and eosin of staining in an irregular way, there being examples varying from a bluish-red to a violet or even a deep blue. He considers this a form of degeneration. A number of other observers take issue with him on this question, believing that these are young cells in process of development. The subject is still swb judice, and therefore of no practical value at present. Granular Degeneration.—Grawitz first described as a degenerative process the peculiar basophilic granulations that may be noted in the red blood-cells of pernicious anemia, carcinoma, severe malaria, and other diseases. After methylene-blue and eosin they appear as dots or rods of a deep blue color lying within the red blood-cells, while with those dyes that affect chromatin they stain as does this substance. For the latter reason they have been thought to be the remains of nuclear degeneration. While the nature of the granules is not understood, their presence must be taken as indicating a destructive process of the red cells. VARIATIONS IN FORMED BLOOD-ELEMENTS. 259 Loss of Staining Reaction. Upon the amount of hemoglobin present in the red blood-cell its staining power directly depends. As Ehr- lich has shown, the general appearance of a stained prepa- ration gives a fair index of the quantity of hemoglobin present, and in the examination of a smear this quality of taking the stain should always be noted. Deficiency of hemoglobin is shown by the corpuscles being pale and their centers devoid of color. In severe anemias the hemoglobin may be so low that only shadowy outlines of the cells can be seen. In such cases poikilo- cytosis is usually marked. Caution: Faulty technique will produce a vacuolation of the cells which must be differentiated from the above. The borders of the vacuoles are sharply defined, and to the practised eye the artifact is patent. INCREASE IN THE NUMBER OF WHITE BLOOD-CORPUSCLES. The term leukocytosis was formerly used to designate an increase in number of any or all the forms of white blood-corpuscles. It has been found necessary, as our knowledge of the blood increased, to formulate more specific terms that indicate the kind of white blood-cell the number of which is increased. The increase may be either relative or actual. We have therefore: Lymphocytosis: increase in the number of lymphocytes. Leukocytosis: : “ os polynuclear leukocytes. Eosinophilia : es = oe eosinophiles. Mixed leukocytosis: f = ie several kinds of white blood-corpuscles. Hypoleukocytosis (leukopenia) indicates a decrease below the normal of the number of white blood-corpuscles. Hyperleukocytosis is sometimes used to denote increase of white blood- cells, but the term is too cumbersome and is not much employed. 260 MEDICAL MICROSCOPY. Physiologic Increase in the Number of White Blood-cells. As has been mentioned, this may occur under a variety of conditions, during digestion, after severe muscular exercise, following a cold bath, for-the first few days after birth, during pregnancy, and upon the administration of certain drugs—turpentine, camphor, etc. Pathologic Increase in the Number of White Blood=cells. Such an increase is found accompanying a variety of pathologic conditions, and often is of the greatest clinical significance. Aside from the special diseases in which it is a primary factor, the inflammatory type is perhaps the most important. Leukocytosis (Inflammatory).— Probably the chief source in adult life of the polynuclear cells is the red bone- marrow, but there has been much controversy upon this point, nor is it yet satisfactorily elucidated. In nearly all of the acute infectious diseases there is a distinct leukocytosis. In this category are included diphtheria, pneumonia, acute articular rheumatism, paro- titis, scarlatina, smallpox, and septic conditions from whatever cause. A notable exception to this exists in the case of uncom- plicated enteric fever and measles. In these diseases there is a marked decrease of the white blood-corpuscles, chiefly of the polynuclear variety. Usually the greater the leukocytosis, the more serious is the disease and the less favorable the prognosis. A leukocytosis accompanying enteric fever is considered an indication of some complication of grave nature. The increase of the polynuclear neutrophiles may be very considerable. In pneumonia Cabot found an average increase of 24,000 of these elements. According to Simon, there is in scarlatina a leukocy- VARIATIONS IN FORMED BLOOD-ELEMENTS. 261 tosis reaching to from 10,000 to 25,000 above the normal upon the second or third day after the appearance of the eruption. The leukocytosis in carcinoma, and more particularly in sarcoma, has long been noted. Lymphocytosis.—As the lymphocytes originate in the general lymphatic system, it follows that in conditions affecting the lymphatic glands and channels there will be a deviation from the normal number of lymphocytes in the blood. Such a deviation may be either relative or absolute. It may be broadly stated that whenever there is a hyperplasia of the lymphatic glands, there will be a corre- sponding increase in the number of lymphocytes in the peripheral circulation. Thus, Ewing mentions a case of enteric fever in which there was a lymphocytosis of marked degree and where there were found postmortem greatly enlarged mesenteric glands. So also there is in malignant lymphoma not complicating the lymphatic channels a uniform lympho- cytosis. In case the lymph-streams are blocked, Ehrlich be- lieves the decrease in the number of lymphocytes is due to their inability to gain entrance to the blood-stream. In whooping-cough lymphocytosis is often marked. Experimentally it may be induced by the injection of tuberculin and pilocarpin. Eosinophilia.—The normal number of polynuclear eosinophiles is always greatly increased in myelogenic leukemia, and may occur in many other conditions. Brown and Thayer first pointed out the eosinophilia accompanying trichinosis. In four cases reported by them the eosinophiles present reached not lower than 48 per cent. of the total number of white blood-corpuscles. 262 MEDICAL MICROSCOPY. The condition may also occur in various skin affec- tions, gout, gonorrhea, and the eruptions of syphilis. In cases of infection by anchylostoma and other intestinal parasites eosinophilia may be extreme, but is not con- stant. Leukemia.—This is a general term denoting an enor- mous increase in the number of white blood-cells depen- dent upon definite pathologic conditions of the formative centers of these cells. There is usually a decrease in the number of red blood- cells and in the percentage of hemoglobin. The propor- tion of white corpuscles to red may in severe cases reach the astonishing figure of 1: 3. By some authors it has been claimed that there exist three distinct clinical forms of leukemia: lymphatic, with general hyperplasia of the lymph-nodes; splenic, with enlargement of the spleen; and myelogenic, with corre- sponding hyperplasia and activity of the red bone- marrow. Ehrlich contends that it is not possible to distinguish as distinct the first two forms, and classes them together as lymphatic leukemia. Lymphatic Leukemia. It is now universally admitted that the lymphocytes of the blood are identical wih the fixed lymphoid cells of the lymph-nodes, of whatever variety. It needs, therefore, only a hyperplastic condition of these structures, together with a free access for the cells manufactured, to furnish to the blood-stream an excess of these elements. Such a condition has been noted in cases of malignant lymphoma, —the so-called lymphosarcoma,—in which the number of lymphocytes may be greatly increased. In pertussis dur- ing the convulsive stage there may also be a distinct VARIATIONS IN FORMED BLOOD-ELEMENTS. 263 lymphocytosis, perhaps to four times the normal number of these cells. In true lymphatic leukemia there are, however, distin- guishing features that allow a positive diagnosis to be made with certainty. Diagnosts.—The blood should conform to the follow- ing conditions: 1. A constant excess of lymphocytes and large mono- nuclear leukocytes amounting to about 150,000 per cubic millimeter and to 80 to 90 per cent. of the white blood-corpuscles. 2. A total absence or the presence in only small numbers of eosinophiles, polynuclear leukocytes, mast-cells, myelocytes, and nucleated red cells. 3. A decrease in the number of red blood-cells, sometimes the number falling as low as 2,500,000 per cubic millimeter. 4. Usually a decrease in hemoglobin. The internal organs, particularly the spleen, liver, and kidneys, may be greatly enlarged by the infiltration of white blood-cells, which may occur in large aggregations, appearing as white patches upon section, and as nodular masses when lying near the surface. The author has seen one case of lymphatic leukemia in which the splenic tumor weighed over 4000 grams. The lymphatic structures were uniformly greatly enlarged. Ehrlich recognizes two types: Acute lymphatic leukemia, which pursues a rapid course, presents a small splenic tumor, and is characterized by a tendency to the formation of petechie and hemorrhage. Chronic lymphatic leukemia, in which the course of the disease may extend over years, the spleen being often very considerably enlarged. The diagnosis between these two forms cannot be made 264 MEDICAL MICROSCOPY. from the microscopic appearance of the blood alone, as in both the proportions and general characteristics of the cells are identical. Myelogenic Leukemia. In the range of hematology there is no condition that presents a more typical microscopic picture than the blood of myelogenic leukemia. The distinctive feature of the disease is the presence in the circulating blood of large numbers of mononuclear neutrophile cells (myelocytes) which normally are found only in the red bone-marrow. There is also a great abso- lute increase of eosinophiles, both of the polynuclear and of the mononuclear types (eosinophilic myelocytes), and mast-cells are often present in large numbers, while atypi- cal polynuclear neutrophiles are frequent, presenting as dwarfed and distorted forms. Nucleated red blood-cells are a constant factor and may occur in large numbers. They consist largely of normo- blasts, though in some cases megaloblasts are frequent and microblasts may be found without difficulty. Diagnosts.—The essential characteristics of the blood in myelogenic leukemia are considered by Ehrlich to be as follows: “1, That, in addition to the polynuclear cells, their earlier stages, the mononuclear neutrophiles, likewise circu- late in the blood. 2. That all three types of granulated cells—the neutro- phile, eosinophile, and mast-cells—participate in the increase of the white blood-corpuscles. 3. That atypical cell forms—e. g., dwarfed forms of all kinds of white blood-corpuscles—and mitotic figures appear. VARIATIONS IN FORMED BLOOD-ELEMENTS. 265 4. That the blood always contains nucleated red blood- corpuscles, often in great numbers.” In addition, we may remark that the relative number of myelocytes usually amounts to 20 to 60 per cent., and that the total number of erythrocytes is decreased and ‘there is usually a diminution in the amount of hemoglobin. CHANGES IN THE BLOOD OF LEUKEMIA PRO- DUCED BY INTERCURRENT DISEASES. A reduction in the proportion of leukocytes or myelo- cytes may be occasioned when there is superimposed some form of infectious disease. This particularly is true in an intercurrent pneumonia or diphtheria, when temporarily the blood may give the picture of an acute inflammatory leukocytosis. In early stages of the disease a positive diagnosis may not be possible. Pseudo-leukemia (Hodgkin’s Disease). In the progressive primary enlargement of the spleen and lymphatic glands known as Hodgkin’s disease the blood fails to show the increase in white corpuscles char- acteristic of leukemia, and this condition cannot therefore be classed as a blood disease. Any blood changes occurring, e. g., anemia and diminu- tion of hemoglobin, must be considered as purely secondary. The true lymphosarcomata are so closely allied to the above disease that a clear distinction between them is not at present possible. A pseudoleukemia of benign type may be transformed into’a malignant proliferation of the most pronounced form, there being numerous metastases, invasion of neighboring tissues, and cachexia, while microscopic ex- 266 MEDICAL MICROSCOPY. amination of the tissues shows an undoubted sarcomatous structure. Pernicious Anemia. This term is used to designate a progressive pathologic condition characterized by marked changes in the formed elements of the blood—oligocythemia, poikilocytosis— and the presence of megaloblasts and normoblasts and a proportionate increase in the amount of hemoglobin. There occurs then a progressive decrease in the number of blood-cells with a relative increase in hemoglobin and nitrogen and also in the size of the red blood-corpuscles. This is believed by Ehrlich to be an effort on the part of nature to compensate for the loss of surface-area of the red cells. The disease is sometimes due to the presence of intes- tinal parasites, but more often it apparently is idiopathic in origin and distinct unto itself. Women are more fre- quently affected with this disease than men. As exciting causes may be mentioned malaria, tuber- culosis, diseases affecting the gastric mucosa, cancer of the stomach, persistent diarrhea, and pregnancy. Changes in the Blood.—The quantity of blood is greatly decreased, so that it appears thin and watery, and it may be difficult to obtain a drop for examination, a deeper puncture than usual being necessary. The hemoglobin, while it is relatively increased in the individual blood-corpuscles, is actually reduced, so that in the later stages of the disease it may fall as low as 10 per cent. Usually the percentage of hemoglobin is between 20 and 40 (Ewing), and it is frequently greater than occurs in secondary anemias from various causes. The number of red cells may fall very low, in fatal cases VARIATIONS IN FORMED BLOOD-ELEMENTS. 267 usually about 1,000,000 per cubic millimeter, and cases have been observed in which they reached the low number of about 200,000 per cubic millimeter. The size of the red cells is greatly increased, so that many of them measure as much as 18 y in diameter, while the average, as determined by Lazarus, is 11 # to 13 pv. According to Ewing, at least 33 per cent. of the red cells should show the increase in size in order to make the diagnosis of pernicious anemia sure. Megaloblasts constitute the majority of nucleated red cells present. They may occur in large numbers or be found only after careful search. Microblasts and normoblasts are conspicuous by their absence, being rarely found in the blood in pernicious anemia. Mitotic figures in megaloblasts may be ob- served, and Ewing asserts that “the presence of three or more unequal asters in one gigantoblast, as occasionally found in severe cases, is one of the most significant signs within the range of blood analysis.” The same author lays down the following rules to be observed in determining the diagnosis of suspected cases: “The diagnosis may rest upon the presence of: numer- ous megaloblasts and megalocytes with increased Hb; 33 per cent. of megalocytes with increased Hb; an excess of megaloblasts over normoblasts, a single gigantoblast or megaloblast in pathologic mitosis. The diagnosis cannot vest on an extreme reduction of red cells. The diagnosis may require the complete summation of all clinical and morphologic data, as well as observation on the course of the disease, or even the microscopic examination of the marrow.” Secondary Anemia.—Diminution of the number of red blood-cells and of the hemoglobin is of frequent occurrence 268 MEDICAL MICROSCOPY. after severe hemorrhage and following tuberculosis, chronic nephritis, and various infectious diseases. The changes described as occurring in pernicious anemia are absent; that is, the blood does not contain megaloblasts, the hemoglobin is not relatively increased, and the red bone-marrow fails to show the hyperplastic changes always present in pernicious anemia. Chlorosis. This term is applied to a primary anemia peculiar to young females, rarely existing in the male. It is charac- terized by a deficiency of hemoglobin without a corre- sponding diminution of red blood-cells. From progressive anemia it differs in the above respects, and also in that while nucleated red cells may occur in small numbers the presence of megaloblasts in greater proportion than normoblasts is absent; in fact, the pres- ence of megaloblasts in the blood of chlorosis is denied by some authors. Blood-plates are much increased in number and the coagulability of the blood is greater than normal. The female sex and the age of puberty seem to be im- portant factors, while heredity and tuberculosis also play a part in the etiology of the disease. Microscopic Appearance of the Blood.—The red blood- cells are slightly decreased in number, though rarely the count may fall to about 2,000,000 per cubic millimeter. Poikilocytosis and oval forms are frequent in the severer forms and the cells are notably pale, the centers frequently being quite clear on account of deficiency in hemoglobin. In cases of moderate severity the percentage of hemo- globin is usually about 40, while in severe cases it may fall much below this mark. Leukocytosis.—Usually the number of white blood- cells remains unchanged. VARIATIONS IN FORMED BLOOD-ELEMENTS, 269 Malaria. The specific cause of malaria was discovered in 1880 by Laveran, who observed in the blood of malarial pa- tients a crescentic parasite, which was in his honor called Laveramia malarte. The malarial organism belongs to the Protozoa, the lowest class of animal forms. It is endowed with the power of ameboid movement and possesses a nucleus. Multiplication takes place by division of the adult forms. The question whether there are several distinct vari- eties of malarial parasite, or only one species differing widely in its morphology and producing diverse phenom- ena under varying conditions, is one that has claimed a great deal of attention. It must still be considered un- settled. By most of the German and American authori- ties the theory of plurality of species is considered es- tablished. The theory that there is but one form of malarial organism is strongly maintained by Laveran and many of the Italian observers. The author prefers to accept the classification of the malarial parasite into three distinct forms—the tertian Hemamoeba vivax, the quartan Hemameceba malarie, and the estivo-autumnal Hemameeba falcipavum. Morphology and Biology.—Appearance in Unstained Preparations: After segmentation of the adult parasite the subdivisions or spores first adhere to the red blood-cor- puscles and soon after gain entrance to them, the subse- quent development being endoglobular. In the first stage the young malarial parasite is seen in fresh, unstained blood as a small, spheroidal, hyaline body within the red blood-cell, in which it is usually excentrically placed. It measures about 1 » in diameter, and therefore occupies about one-tenth part of the erythrocyte. It is at first not sharply differentiated from the sur- 270 MEDICAL MICROSCOPY. rounding greenish-yellow protoplasm of the blood-cell, but merges with it so gradually that no distinct line of de- marcation can be seen. Its position in the corpuscles may change, but the activity displayed by the older forms is usually not present. Within a few hours after the chill a notable increase in the size of the organism has taken place, and usually it has become ring-shaped, there being a central portion which shows the color characteristic of the normal proto- plasm of the red blood-corpuscle. Active motility may now be observed provided the specimen is freshly prepared and the temperature of the room is not too low or a warm stage is employed. The organism is seen to rapidly change its shape, sending out finger-like projections of its substance (pseudopodia), which may be as rapidly withdrawn, to be again shot forth in another direction. The whole organism may change its position in the red blood-cell, this being accomplished by the advancement of a pseudopod and the subsequent withdrawal of the remainder of the organism into the projected portion. The organism may frequently be seen to change its rela- tion to the surface of the blood-cell, the pseudopodia dip- ping down to a lower plane, thus apparently disproving the assertion of some observers that at this stage the or- ganism is simply attached to the corpuscle and not within its substance. As the parasite increases in size fine pigment-granules appear, their number increasing with the age of the organ- ism. ‘The pigment is of a dark brown color and frequently occurs in rod-shaped masses and in cubes, varying with the age and kind of organism. Great activity of the pigment is usually present. The particles exhibit a rapid dancing motion, forcibly re- VARIATIONS IN FORMED BLOOD-ELEMENTS. 271 minding one of Brownian movement, with which, how- ever, it is not identical, being due to the protoplasmic activity of the organism. Presegmenting Forms.—As the malarial organism ap- proaches maturity it occupies the greater part of the red blood-corpuscle and assumes a spherical shape, the pig- ment becoming at the same time less motile, and finally arranging itself more or less peripherally and ceasing its movements entirely. These presegmenting forms occur from six to eight hours before the chill, and a few may even be found within an hour or two after the chill, these representing organisms that have not kept pace with the uniform development of the majority. Segmentation.—Two or three hours prior to the chill segmenting forms begin to appear, becoming more and more numerous as the time of the paroxysm approaches, and being most abundant just before it begins. Asis the case with the presegmenting forms, the segmenting bodies may be found in small numbers sometimes as long as several hours after the chill has taken place. These adult forms consist of a number of perfect spores arranged in the form of a rosette, so that the outline of the organism is regularly lobulated, there being around the whole structure a thin zone of pale yellow color, the re- mains of the red blood-corpuscles. The organism itself is highly refractile and exhibits a faint rosy tint; the line of demarcation between the com- ponent spores may be quite indistinct except at the edges or there may be a radial striation that is quite easily made out. Each of the spores is seen to contain a small dot marking the site of the nucleus. The pigment is usually arranged centrally in an irregular mass. The organism now bursts from the inclosing blood- 272 MEDICAL MICROSCOPY. corpuscle, the individual spores are set free and probably soon attach themselves to the red blood-corpuscles to begin afresh the cycle of existence while the pigment either remains free in the blood-plasma or more probably is taken up by the leukocytes. Multiple infection of a single red blood-cell is frequently observed in all forms of malaria, but more particularly in the estivo-autumnal type. Flagellated Bodies.—Extracorpuscular bodies with long projections of their protoplasm (flagella) may occasion- ally be noted, and may be produced artificially by the methods to be mentioned (see Technique). They proba- bly represent degenerative forms and are incapable of re- production. The flagella are often so active that they can scarcely be seen. Within the radius of their activity the blood- plasma is set in violent motion and pigment-granules or red blood-corpuscles drifting within their reach are dashed aside by the whip-like motion of the delicate processes. As the organism becomes less active the flagella are seen to be several times longer than the diameter of the body, and frequently they possess a club-like extremity. Crescents and ovoids are found only in estivo-autumnal infection, and will be considered under that head. Their detection in fresh blood is a matter of ease on account of their peculiar shape and the presence of a considerable amount of pigment, usually centrally located and ar- ranged in masses. Organisms tn the Peripheral Blood.—The malarial para- site may be present in the internal organs and bone- marrow in enormous numbers, and at the same time so few may appear in the peripheral circulation as to be diffi- cult of demonstration. This fact should not be lost sight of. Care and perse- VARIATIONS IN FORMED BLOOD-ELEMENTS. 273 verance will enable the competent observer to find the malarial parasite in blood from the peripheral circulation in any case of malaria with an infection of sufficient in- tensity to give rise to clinical manifestations. Leukocytes in Malaria.—The phagocytic action of the white blood-cells is in malaria of particular interest and value. The fact that leukocytes are present, containing the characteristic pigment-granules and bars or blocks formed by the malarial organism during its development, is, of itself, sufficient data upon which to base a positive diagnosis. This fact is of great aid to the clinician in cases where the organisms are not present in quantity in the peripheral circulation or where the administration of quinin has reduced or obliterated the infection. In fresh preparations the process of phagocytosis may often be actually seen. Sometimes scarcely a leukocyte can be found that does not contain a recently engulfed malarial parasite which may still be alive with its pig- ment-granules in active motion. The author has several times observed the process, which is interesting in the extreme. The leukocyte by ameboid movement slowly approaches the malarial para- site until it is, as it were, within striking distance of it. A pseudopod is quite rapidly thrown out until it touches the parasite, the pigment-granules of which seem to be immediately set in more active motion, as if the para- site realized its danger. The leukocyte, on the other hand, quickly withdraws the pseudopod and retreats a short distance. It gives the impression of having received a rebuff and of fortifying itself anew for the attack. Again it advances and projects a pseudopod, which is again withdrawn, but with less rapidity than in the first instance, nor does the leukocyte remove itself so far from its prey as in the first retreat. 23—O 274 MEDICAL MICROSCOPY. Each time that it advances it seems to do so with more assurance, and the pseudopod remains longer in contact with the malarial organism, until finally it is no longer withdrawn and the white blood-cell literally crawls around the parasite. After having engulfed its prey the leuko- cyte may exhibit sufficient energy to travel out of the field of vision unless the slide be moved. This phagocytic action has been observed in several cases of estivo-autumnal type exhibiting large numbers of advanced extracorpuscular forms. From the action of the leukocyte it would seem that the malarial organism pos- sesses some power of resistance which must be gradually overcome by the leukocyte before inclosure can take place. Technique.—Before essaying the study of the malarial parasite one must thoroughly familiarize himself with the appearances of normal blood in both the fresh unstained and stained specimen, so that he will have become ac- quainted with those artifacts that continually arise with even the most careful technique. It goes without saying that he must have a thorough knowledge of the normal elements of the blood and their behavior toward the various staining methods employed. Continued and careful observation is the only qualifica- tion possible. Wet Preparations.—Though the technique is simple in execution, the beginner is apt to fail to make proper prep- arations of fresh blood. A little experience will soon remedy his defects. Careful attention to details is essen- tial. The lobe of the patient’s ear is cleansed with soap and water and wiped dry with a clean towel or piece of gauze. A puncture is made with the point of a small sharp- pointed tenatome or other suitable instrument, so that VARIATIONS IN FORMED BLOOD-ELEMENTS. 275 with slight pressure a drop of blood will flow. If the lobe is held between the thumb and finger and submitted to considerable pressure, the slight prick necessary will scarcely be felt by the patient. The first drop is wiped away, a cover-glass held in the forceps is touched to the succeeding drop and is quickly placed ‘“‘butter-side down” on a glass slip. The blood should spread quickly and evenly between the two sur- faces. This action may be facilitated if necessary by gently tapping the edge of the cover-glass with the point of the forceps. Pressure upon the cover-glass must never be made. Both the cover-glass and slip must be previously scrupulously cleaned. This is best accomplished by immersing them in alcohol and wiping them with a clean piece of gauze. Immediately before the preparation is to be made they should be vigorously rubbed with a clean cloth. If the slip is submitted to this friction until it is warmed, the blood spreads the better. The slightest piece of dust upon the cover or slip is fatal to a good re- sult. The smallest amount of acid will ruin the prepara- tion. Glass that has been cleaned in an acid bath must therefore be carefully washed with soap and water. After the slips and covers have been cleaned by ordinary methods, it is not amiss to pass them several times through the bunsen flame in order to remove any grease that may remain. Examination of Fresh Blood Preparations.—The fresh spread should be examined as soon as possible, as changes in the blood may soon occur. If the preparation is not to be viewed immediately, it is well to seal the edge of the cover with oil or vaselin to prevent evaporation. A drop of immersion oil having been put upon the cover-glass, examination with the j4, objective is pro- 276 MEDICAL MICROSCOPY. ceeded with. The specimen should, if properly made, exhibit, at least in certain portions, a thin stratum of blood-cells lying flat and separated slightly one from the other. If the cells have clumped or have formed rou- leaux, it signifies that too large a drop of blood has been used. Usually the spread is thickest at the edges or upon one side. It is sometimes advisable to search these thicker por- tions of the preparations, for we may there find the larger pigmented organisms after hunting in vain for them else- where. The fresh preparation is of advantage when we wish to study: 1. Ameboid and pigmentary movements. 2. Flagellated bodies. 3. Phagocytosis. Flagellated Forms.—In order to observe these bodies it is necessary to liberate the adult parasite from the re- straining bounds of the red blood-cell. The escape of the organism may be facilitated by adding to the fresh prepa- ration a small drop of water. This should be placed upon the slide at the edge of the cover-glass, under which it will be drawn by capillary attraction. Ewing recommends that fresh smears be placed imme- diately in a moist chamber (and the latter quickly sealed) for from ten to twenty minutes. Dried Blood Preparations.—The technique for making and fixing smears is fully described in the section on blood (p. 242). Stains.—Eosin and methylene-blue form one of the most satisfactory methods for diagnostic purposes. Two solu- tions are required: (1) Saturated alcoholic eosin; (2) saturated aqueous methylene-blue. The smear, having been fixed in methyl alcohol or VARIATIONS IN FORMED BLOOD-ELEMENTS. 277 formalin and alcohol, is stained with the eosin solution for ten seconds, washed in water and stained for thirty seconds with the methylene-blue, washed, blotted, dried, and mounted in Canada balsam or examined without a cover-glass, a drop of immersion oil being placed directly upon the stained blood. By this method the malarial organism and all nuclei of white blood-cells are stained blue, the erythrocytes being a bright red. Carbol-thionin.—This is a most serviceable stain, for the reason that the ring forms are sharply brought out by it, while with the methylene-blue they may stain faintly. The stain is made as follows, after the formula of Fut- cher and Lazear: Saturated solution of thionin in 50 per cent. AlEOHO 3 os. ee dee dane ad Soa a See ae 20 cc. 2 per cent. equcous carbolic acid solution, ..100 c.c. The mixture must be at least one week old, and im- proves withage. After fixation with alcohol and formalin or methyl alcohol, stain thirty to sixty seconds. Wash, dry, and examine either direct or after mounting in Can- ada balsam. The above stains suffice for the easy identification of the organism, but neither of them furnishes a differential stain of the chromatin of the nucleus. In order to accom- plish this purpose, which is desirable for critical study of the malarial parasite, recourse must be had to some one of the stains in which polychrome methylene-blue is em- ployed. Goldhorn has recently formulated an excellent stain for this purpose. It may be purchased from any of the larger dealers in microscopic supplies. The method of application is as follows: The smears must be freshly made. Fix in methyl alcohol fifteen seconds. 278 MEDICAL MICROSCOPY. Wash in water. Stain seven to thirty seconds in 75 of 1 per cent. aqueous eosin solution. Wash in water. Stain thirty to sixty seconds in Goldhorn’s methyl- ene-blue. Wash in water. Dry, without the use of blotting-paper or heat, by agitation of the slide in the air. Besides staining the chromatin reddish, the body of the parasite is stained blue, nuclei of leukocytes blue, and the granular degeneration of infected red blood-cor- puscles is beautifully shown. Advantages of stained specimens are: 1. Easy detection of existing parasites. 2. Structural details of parasite are brought out (with Goldhorn stain). : For routine work stained specimens are far superior to the fresh preparation. They are particularly recom- mended to the beginner in malarial parasitology. Tertian Parasite.—The life-cycle of this form of the malarial parasite is about forty-eight hours. It develops in well-marked groups, the individuals of which mature about the same time. An infection with such a group of tertian parasites results in a rise of tem- perature with a chill at the period of sporulation, so that we find the paroxysm appearing upon every third day. This is followed by a rapid fall of temperature, which usually becomes subnormal, the parasites at this stage being in the young, hyaline form. There follows a period of quiescence, the temperature remaining about normal the second day. The organisms will now be found in the ring form with beginning pigmentation. Upon the succeeding day they VARIATIONS IN FORMED BLOOD-ELEMENTS. 279 again reach maturity and produce a sudden exacerbation of temperature. Not infrequently there may be present two groups of tertian parasites (double infection). For some unknown reason when this is the case the groups usually mature about twenty-four hours apart. It will readily be seen that this would bring about a paroxysm every day—a quotidian fever. Microscopic examination of the blood about the time of chill would reveal one set of organisms half-grown, the other full-grown, with presegmenting forms and rosettes. It may even happen that more than two groups of ter- tian organisms are present in the blood of the same indi- vidual, in which case an irregular temperature curve would result. The tertian parasite in stained preparations shows the following characteristics; it is presumed that a method (as Goldhorn’s) that stains the chromatin has been used : The youngest forms are identical with the recently divided portions of the segmenting bodies. They are nearly spherical, about 1 » in diameter, the outer portion stains with methylene-blue and incloses a globular red nucleus which is surrounded by an achromatic belt— “the milky zone’”’ of Gautier. Within a few hours after the paroxysm these forms have been replaced by more or less characteristic rings which may reach 4 » in diameter. These may already contain a few fine pigment-granules of a reddish-brown color. ‘The center of the ring is occupied by the substance of its corpuscular host. “The chromatin is usually found within the ring, some- times lying in an isolated position in the center, but very often the chromatin is found outside the ring, connected by a very fine thread of protoplasm” (Ewing). 280 MEDICAL MICROSCOPY. The parasite continues to increase in size, pigment- granules multiply, the chromatin is subdivided into small granules, ten or more in number, which lie in a group sur- rounded by a “milky zone.’”’ The organism at this stage is very variable in form, and may stretch out long threads in various directions, at times spanning the entire red blood-cell. About ten hours before the chill the parasite seems to have reached its full size. It stains quite homogeneously, pigment-granules and bars are present in large numbers throughout the body of the organism, and the larger ‘‘milky zone”’ is occupied by many small granular masses of chromatin. Presegmenting bodies now begin to be seen. With methylene-blue and eosin these forms are readily distin- guishable on account of their capacity for taking the stain deeply and presenting a reticulated appearance. A chromatin stain discloses numerous coarse granules of chromatin scattered in groups throughout the body of the parasite, each group being surrounded by a “ milky zone.’’ Segmenting Forms.—Gradual fusion of the chromatin granules takes place until there are formed fifteen to twenty (Thayer) chromatin masses, each surrounded by a ‘““milky zone’’ representing the nucleus of a new segment. This pigment collects in a mass, usually in the center of the organism, the peculiar striated appearance of the rosette is clearly visible, and the lobulated rim of the or- ganism is surrounded by a thin border of the degenerated red corpuscle. Changes in the Infected Red Blood-cell.—In the tertian form of infection the erythrocyte attacked by the spore soon shows granular degeneration. As the parasite grows, the red blood-corpuscle increases in size and be- comes progressively paler. This is due partly to the de- VARIATIONS IN FORMED BLOOD-ELEMENTS. 281 generative process and partly to the fact that the hemo- globin, upon which its staining qualities depend, is being rapidly converted into melanin. Toward the latter part of the cycle of the parasite the red blood-cell may have increased to nearly twice its normal size. The Quartan Parasite—The cycle of development of the quartan organism lasts about seventy-two hours. In an infection by a single group of this form of the parasite there would therefore be a rise of temperature with a chill upon every fourth day. An infection by two groups of quartan organisms would produce a paroxysm upon two succeeding days, with an intermission of one day, while an infection by three groups of the quartan parasite maturing upon successive days would result in a quotidian type of fever. The quartan form of malaria is comparatively rare in America, so far as is known from present observation. Morphology of the Quartan Parasite.—Stained specimens of the younger forms are apparently identical with those of the tertian type. After a few hours it is not difficult to distinguish the one from the other. The quartan organism remains small, more rotund, and much more refractive in fresh prepara- tions than the tertian. The pigment-granules are fewer in number, larger, and darker, the movement of the para- site is at all times slower than that of the tertian, and the pigment is sluggish or nearly stationary. Presegmenting Forms.—These are frequently character- ized by a radial or star-shaped arrangement of the pig- ment, and the reticulation is coarser. Segmenting Forms.—On account of the slower develop- ment of the quartan parasite rosettes are more frequent in the peripheral circulation than in either of the other forms of malaria. 24—O 282 MEDICAL MICROSCOPY. They present only from 6 to 12 segments, and are con- sequently much smaller than those of the other varieties. The infected red blood-cell in quartan malaria is quite different in its appearance from that of tertian. Instead of increasing in size and becoming paler, it is diminished, and in fresh preparations of a peculiar brassy hue. Nor are its staining qualities so notably affected. The Estivo-autumnal Parasite.—Certain irregular forms of malarial fever, occurring chiefly in the late summer or autumn, present a form of parasite differing essentially in many particulars from the ones already described. The paroxysms may occur with regularity about forty-eight hours apart, but frequently vary considerably, so that we may have a typical quotidian range of temperature with chill every twenty-four hours. Paroxysms may, indeed, occur at intervals ranging all the way between the two extremes. In the more serious types of this form of malaria the paroxysms are usually tertian, while the milder forms are prone to follow with greater or less regularity the shorter interval. There is also a notable difference in the time the paroxysms of estivo-autumnal fever persist; they may last as long as thirty-six hours, and are commonly of twenty-four hours’ duration, while in the fever due to the tertian and quartan organisms the paroxysms are of much shorter duration, rarely exceeding ten or twelve hours. Again, in the re- mittent types the paroxysms may merge one with the other, so that the temperature does not at any time reach the normal point. It is a peculiar fact that while the parasites may exist in myriads in the internal organs, notably the spleen and bone-marrow, they may not be demonstrable in the peripheral circulation after the first few hours of their existence. Neither do they form well-defined groups of VARIATIONS IN FORMED BLOOD-ELEMENTS. 283 organisms of about the same age and stage of develop- ment. Besides, they present morphologic characteristics peculiar to themselves. On account of the variations in the clinical mani- festations of infections by the estivo-autumnal para- site, and because of real or fancied deviations in form, Marchiafava, Bignami, Celli, and others have contended that there are at least two distinct species of this variety, the one producing the milder tertian, the other the more serious quotidian and continued fevers. While there may be reasonable ground for this belief, the majority of observers are inclined to the opinion that there is but one form, which is capable of produc- ing those diverse manifestations on account of varied environment and other reasons at present not under- stood. ‘To this opinion we will adhere, without entering into a discussion of the merits of either side of the case. Morphology and Biology.—Except that they are some- what smaller, the earliest forms of the estivo-autumnal parasites are not distinguishable from those of the tertian and quartan forms. At an early stage they form rings possessing a marked thickening upon one side, and are therefore distinguished as ‘‘signet-ring’’ forms; these stain particularly well with carbol-thionin. According to Ewing, they nearly always present two nuclear bodies staining with methylene-blue and lying either close together or opposite to each other. The ring forms gradually increase in size until they are 3 » to 4 win diameter at the end of about twenty-four hours, the thicker portion of the ring increases in size, and fine pigment-granules begin to appear. Presegmenting bodies now begin to be seen which resemble those of the 284 MEDICAL MICROSCOPY. tertian organism, except that they are of smaller size, often being not more than half the diameter of a normal red blood-corpuscle. Segmenting Forms.—These are similar to those of the tertian parasite, but according to most observers differ from them in the number of spores formed, there being from 6 to 12, though in cases observed by Ewing nearly all of the rosettes showed from 18 to 20 spores. Crescents and Ovoids.—Besides the forms above de- scribed in estivo-autumnal infections there appear after a few days bodies of ovoid or crescentic shape. They oc- cupy red blood-cells or have clinging to them the remains of the original host and contain pigment bars which may be centrally grouped in an irregular star-shaped figure and motionless in fresh preparations, or may present con- siderable activity and be scattered throughout the or- ganism. The author has upon several occasions observed the active pigment of a crescent withdraw itself from the extremities of the body and assume the characteristic central arrangement. That the ovoids may become converted into crescents, and the latter under proper con- ditions frequently revert to the oval form, is well estab- lished. The genesis of the bodies is still a matter of doubt, despite much study and contention regarding it. Chromatin granules in the crescents have been demon- strated by a number of observers. They usually appear in the center of the body, among the pigment staves. Ovoids and crescents persist in the peripheral blood for weeks after the other forms have disappeared and the patient has apparently recovered. In one such case ob- served by the author there was a relapse after six weeks, following apparent recovery, the patient succumbing to the second attack. VARIATIONS IN FORMED BLOOD-ELEMENTS. 285 The question as to whether or not the ovoids and cres- cents influence the febrile movements or partake in the process of perpetuation of species is as yet not satisfac- torily answered. The fact that these forms contain numerous chromatin granules would incline one to the be- lief that they are not devoid of the reproductive faculty under favorable conditions. Each of these forms may become extracorpuscular and flagellated. Extracorpuscular Bodies.—In fresh preparations that have been allowed to stand for several hours large, swollen, hyaline bodies of spherical form may frequently be observed. They possess pigment-granules which dis- play considerable activity, but with the proper stains show little or no chromatin. They are probably the re- sults of changed environment, and do not properly belong to the normal cycle of development of the parasite. The Red Blood-corpuscle.—The changes occurring in the erythrocyte infected by the estivo-autumnal organism are even more marked than those occurring in tertian and quartan infections. The red cell increases in size at an early stage, and may become wrinkled and crenated, while in fresh blood it assumes a greenish-yellow hue. Granular degeneration and diminished staining reaction are prominent and early manifestations. Multiple in- fection of a single red cell is more frequent in the estivo- autumnal than in other varieties. The red cells in which a crescent has developed rapidly lose their hemoglobin in the vicinity of the organism, until finally only the outer border of the “‘bib’’ will respond to the stain. This results in the appearance, so frequently seen in connection with these bodies, of a faint, red line, either smooth or irregular in outline, connecting the horns of the crescent and bellying out from its concave 286 sides. MEDICAL MICROSCOPY. which it usually hugs closely. Diseases frequently confounded with malaria are en- teric fever, miliary tuberculosis, and septic infection. The microscope usually affords a ready means of diagnosis between these maladies. At times a similar line borders the convex edge, TABULATION OF DISTINGUISHING FEATURES OF VARIETIES OF MALARIAL PARASITES. TERTIAN. QUARTAN. EsTIVO-AUTUMNAL, 1 Life-cycle about forty-eight hours. 2. Ring forms, nu- cleus not stained by methylene- blue. 3. Ring forms ir- regular and thick. 4, Presegmenting forms large and fill the red blood- cell. 5. Rosettes 15 to 20 or more spores. 6, Pigment - gran- ules fine, numer- ous and active. Central arrange- ment in rosettes. 7. Infected red blood-cell grows larger and be- comes pale. 8. All forms in per- ipheral circula- tion. 9. Common, disease mild in charac- ter. Rapidly _— disap- pear after quinin. 10. 10. 1. Life-cycle about | 1. seventy-two hours. 2. Ring forms, nu-; 2. cleus not stained by methylene- blue. 3. Ring forms simi-| 3. lar to tertian but smaller. 4, Presegmenting 4. forms smaller than tertian. 5. Rosettes 6 to 12| 5, spores. 6. Pigment coarse] 6. and sluggish. Stellate arrange- ment in rosettes. 7. Infected blood-cell be- comes smaller and brassy. 8. All forms in per-| 8. ipheral circula- tion. 9. Rare, disease | 9. mild in charac- ter, Rapidly disap- pear after quinin. red| 7. 10. 11. Life-cycle twenty-four to seventy-two hours (uncertain). Ring forms, nucleus stained by methylene- blue. Ring forms more deli- cate and “‘signet’’ por- tion more marked. Presegmenting forms smaller than tertian. Rosettes 6 to 12 spores. Pigment-granules usu- ally coarse and slug- gish. Central arrange- ment in rosettes. Infected red blood-cell becomes smaller, bras- sy, and rapidly loses its hemoglobin. Late forms frequently absent from peripheral circulation. Common in tropics and southern United States. Disease frequently fatal. Quinin not always effi- cient, Ovoids and crescents occur. VARIATIONS IN FORMED BLOOD-ELEMENTS. 287 BACTERIA IN THE BLOOD. In cases of septicemia and pyemia various micro- organisms may be found in the blood-stream, often in considerable numbers. It is frequently not possible, or at best difficult, to demonstrate the bacteria by micro- scopic examination alone. We must therefore resort to cultural methods. Agar slants or plates are to be inoculated with about 1 c.c. each of blood taken from a vein of the patient’s arm with a hypodermic syringe. The field of operation must be rendered surgically clean in the usual manner and the needle sterilized by boiling. The vein may be rendered conspicuous by a ligature or by compression with the finger at a point above that chosen for insertion of the needle. The operation is simple and painless, and no evil results follow if cleanliness is observed. The inoculated medium is incubated and further procedures carried on as indi- cated in the section on bacteriology. In glanders and anthrax infections it is usually easy to demonstrate the bacteria by direct microscopic examination. Smears made on the slide (see p. 242) are prepared as follows: Fix in methyl alcohol thirty seconds. Saturated alcoholic eosin ten seconds. Wash. Methylene-blue (saturated aqueous solution) thirty seconds. 5. Wash. 6. Dry and examine with a ;, objective. Lipemia.—Fat is found in the blood in cases of fracture of the long bones with laceration of the tissues (embolic BW NHN HW 288 MEDICAL MICROSCOPY. lipemia), and occasionally in severe cases of diabetes, chronic alcoholism, nephritis, malaria, and other dis- eases. \lethod.—Make spreads upon the glass slip, which has been cleaned with ether and alcohol, and when they have dried add a drop of 1 per cent. osmic acid and a cover- glass. Examine with the } objective. If fat is present, the droplets will be stained black by the osmic acid. The lobe of the ear should be cleaned with alcohol and ether before making the puncture in order to cleanse the surface of any fat that may be present. Ether added to a blood-slide containing particles of fat will dissolve them. This may be used as a confirmatory method. Micro-chemical Test for Blood.—Dried blood treated in the following manner will produce small, dark brown, rhomboidal, discrete crystals of melanin, which may also Fic. 105.—HEMIN CRYSTALS. occur in irregular masses, the free ends of the projecting crystals being cut obliquely. Method.— 1. ‘A minute quantity of dried blood and a small crystal of sodium chlorid are placed on a glass slide. VARIATIONS IN FORMED BLOOD-ELEMENTS. 289 2. The two are powdered and mixed. 3. A cover-glass is added. 4. A drop of glacial acetic acid is allowed to run under- neath the cover-glass. 5. The slide is held over a low bunsen flame until the glacial acetic acid begins to bubble. 6. Set aside to cool. 7. Examine with the 4 dry objective. Under certain rare conditions the crystals may fail to form. This method may be exceedingly valuable in medicolegal cases. Filaria sanguinis hominis. This is the larval stage of a filiform worm which in- habits the lymph-vessels, Filaria Bancrofti. There area © % ® Oo © : go @a\\ .S —* ~ {tf Ys 2) fon Fic. 106.—FILARIA SANGUINIS HOMINIS. variety of species which are similar in their habits and produce the same lesions. Similar organisms occur in the blood of birds. The filaria is much more plentiful in the peripheral blood at night, as was first pointed out by Manson. 290 MEDICAL MICROSCOPY. ‘It is 0.007 mm. to o.oorr mm. in breadth and 0.27 to 0.34 mm. long. It has a short, rounded head with a tongue-like appendage, and a long pointed tail. From the hinder extremity a ribbon-like mass projects, and when viewed with the high power of the microscope this, like the cephalic appendage, is seen to be at the end of a closed sac in which the animal can coil or extend itself. This envelope is entirely structureless, but the contained parasite is seen under a very high power to be transversely striated and very granular” (von Jaksch). The filiara is common in some tropical countries and is not infrequently seen in the blood of patients dwelling along the lower Atlantic coast of the United States. In the blood it produces no changes, and even when present in large numbers may be unsuspected. The lesions caused by it are mechanical in origin, being due to blocking of lymph-vessels or perforation of blood-capil- laries, followed by inflammatory changes, chyluria, or hematuria. Distoma haematobium. According to von Jaksch, this parasite is found only in the greater cavities of the body, and is therefore rarely seen microscopically during the life of the patient. It is common along the north and east coasts of Africa and has been observed in South America. The female is 16 to 20 mm. and the male 12 to 14 mm. in length, the latter being thicker than the former and each possessing oval abdominal suckers anteriorly. The eggs are 0.12 mm. long and 0.04 mm. in width and exhibit a spike-like projection from one side. They cause ulceration of mucous surfaces and may give rise to severe diarrhea or- hematuria. URINE. 291 URINE. The urine is the excretion of the kidneys. The normal quantity voided in twenty-four hours may vary within wide limits, dependent upon the amount of liquids con- sumed, amount of skin excretion, and other well-known factors. For the healthy male it may be said to be about 1500 ¢.c., while in the female it is somewhat less. Normal urine which has just been passed is a clear, or faintly cloudy, pale yellow fluid having an acid reaction and a specific gravity of 1.015 to 1.025, according to the total quantity passed in twenty-four hours. Naturally, if the quantity of water is decreased the per- centage of solids is relatively higher and there is a corre- sponding increase in the specific gravity. Ifthe quantity of water be increased above the normal, or if there is a de- crease of the solid factors of the urine without a corre- sponding diminution of the fluid portion, the specific gravity will be lowered. On account of the fact that the constituents of the urine are not relatively constant at all hours of the day or night, one should always demand that a specimen submitted for examination be taken from a mixed collection of the total urine for twenty-four hours. The amount of solids in each 1000 c.c. of urine can be roughly estimated by multiplying the last two figures of the specific gravity by 2.33. The result equals the num- ber of grams of solids in 1000 c.c. of urine. The average normal specific gravity being 1020, it follows that 20 x 2.33 equals 46.60 grams of solids to each rooo c.c. of urine. If, then, we wish to determine the total amount of solids passed in twenty-four hours, knowing the total 292 MEDICAL MICROSCOPY. quantity of urine in a given case to be 1500 c.c., the equation would be: 46.60 X 1500 Ton8 = 69.9 grams of solids. If the first examination of urine from a suspected case of renal disease is productive of negative results only, this is not sufficient ground upon which to base a positive opinion, as in some forms of Bright’s disease the urine may for days be devoid of both albumin and casts. A second or even a third specimen should in doubtful cases always be required. MICROSCOPIC EXAMINATION OF THE URINE. Every urine on standing shows a precipitate of varying character and amount. The following table, after Simon, shows the various chemical precipitations that take place in normal urine, dependent upon the reaction of the urine: Reaction Acid: Uric acid. Urate of sodium. Oxalate of calcium. Primary calcium phosphate. Ammonio-magnesium phosphate. Reaction Alkaline (referable to fixed alkalies) : Secondary calcium phosphate. Tricalcium phosphate. Calcium carbonate. Ammonio-magnesium phosphate. Reaction Alkaline (referable to ammonia): Ammonium urate, Ammonio-magnesium phosphate. Tricalcium phosphate. Calcium carbonate. Besides these substances, urine in pathologic conditions may contain other substances to be described. URINE. 293 Sedimentation.—This may be accomplished by allowing the urine to stand, either in a clean bottle or in a conical glass. The addition of a small amount of chloroform will prevent the formation of the putrefactive bacteria and the consequent destruction of organic elements present. After the lapse of twelve to twenty-four hours the sedi- ment that will have gathered is first to be inspected macroscopically, and some simple chemical tests may re- veal the nature of the deposit present, though microscopic investigation should always follow. If it is light in color and flocculent, it probably consists of mucin containing a few white blood-cells and epithelium from the lower genito-urinary tract. Uric acid will give a granular, reddish-brown sediment, sometimes as large as grains of sand. The particles will form upon the sides as well as the bottom of the container. Urates in an amorphous form frequently occur as a heavy, pink deposit. The urine will be acid, and upon heating in a test-tube the precipitate will rapidly dis- appear, returning when the specimen is cooled. Earthy phosphates, carbonates, and alkaline urates may form in alkaline urine as a heavy, white, flocculent deposit, or be precipitated by heat. If the specimen is acidified with nitric or acetic acid, they rapidly disappear. Pus forms a white, viscid deposit, that resists the action of heat and acids and is rendered ropy or mucilaginous upon agitation with a piece of caustic soda. Blood in quantity renders the urine pink or reddish- brown and is easily detected with the microscope. Owing to the fact that the unorganized sediments are usually of little clinical importance, the centrifuge is a useful instrument, as by its aid we can gain the organized elements from fresh urine without loss of time and before they become affected by micro-organisms, and are also 2904 MEDICAL MICROSCOPY. enabled to determine whether bacteria exist in some por- tion of the urinary tract, normal urine in the bladder being sterile. The manipulation of the instrument is simple, and as it is of service in quantitative chemical urinalysis, no physi- cian can afford to be without it. A number of forms are made and sold by various dealers. That in which the motive power is water is convenient where water-pressure is accessible, but the ordinary hand apparatus answers every purpose. How to Work.—For the examination of urine it is con- venient to use a large glass slip (2 X 3) upon the center of which a quantity of sediment is placed by the aid of a clean pipette, and spread in a thin layer. The low power (% obj.) is first used, the microscope being in the vertical position and the clips removed in order to give greater range of motion to the slip. No cover-glass ts necessary. After examination with the low power the 4 may be used if necessary, but to the experi- enced observer this is generally not the case. Should the front of the objective come in contact with the fluid, it should be immediately removed, wiped dry, and refocused, the layer of urine being spread thinner if necessary. No attention is to be paid to the formation of crystalline substances where the urine has dried upon the slide. They are seldom characteristic and are heterogeneous in character. Light.—Of great importance is the proper quality and quantity of light employed. This can only be learned by experience. Asa rule, one should, by the manipulation of the mirror and diaphragm, cut down the amount of light to a minimum consistent with a clear picture of the field. Urinary sediments both organic and inorganic are small and more or less transparent, and a brilliant light renders URINE. 295 them invisible. Especially when searching for tube-casts is caution in this respect necessary, as hyaline casts are entirely obliterated by any but a very subdued light. Chemical Sediments.—In normal urine these substances are usually held in solution. An overproduction of the chemical body or a decrease in volume of solvent powers of the urine will result in their deposit in amorphous or crystalline form. They may be in solution as the urine is passed and deposit as it cools. Sedimentation, either by the centrifugal machine or by allowing the urine to stand in a conical vessel, is usually necessary unless the amount of amorphous or crystalline matter is very large. Uric Acid.—This deposit occurs in sharply acid urine as a brick-dust sediment which microscopically is seen to consist of crystals of various shapes, chief among them being the rhombic prism, the clusters and rosettes which so often occur being modifications of thisform. They are beautifully colored, being deep yellow or orange red, and are totally unlike any other crystalline bodies found in the urine. There is a tendency on the part of these crystals to form upon the sides of the vessel and upon any foreign body that may be present, so that long strings of them may be found attached to a thread or hair. As pointed out by Purdy, this faculty renders the formation of uric acid calculi all the more likely. Whetstone crystals and dumb-bell forms are not infrequently observed. The presence of uric acid crystals does not by any means signify an increase in the elimination of this substance. As has been pointed out, it may be dependent upon a de- crease in the watery elements of the urine or the cooling of the urine beyond the point where all the uric acid present can be held in solution. Quantitative chemical analysis only can determine the exact proportion present in the case of this and other chemical sediments. All urine de- 296 MEDICAL MICROSCOPY. posits its uric acid if allowed to stand and become am- moniacal. Chemical Significance.—The chief condition of impor- tance is the so-called uric acid diathesis, in which condition there may be an habitual deposit of uric acid in the urine. It occurs also in febrile states with increased tissue meta- Fic. 107.—Uric Acip CRYSTALS.—(Greene.) bolism and a decrease in the watery elements of the urine with a corresponding acidity. Overindulgence in animal food, which is frequently the cause of interstitial nephritis, may be accompanied by the deposit of uric acid. On account of the fact that the urinary pigments tend to hold uric acid in solution, the polyuria accompanying the early stage of interstitial nephritis favors the formation of uric acid crystals. URINE. 297 Acid Urates.—Acid urates of sodium, potassium, ammo- nium, and calcium may be deposited in the urine. ‘The mixed urate is common as amorphous particles which may remain suspended or settle to the bottom of the vessel. They may be easily recognized, as gentle heat quickly dissolves them, a property that is not possessed by any other urinary sediment. Microscopically they appear as agglutinations of yellow- ish or pinkish granules and flakes. Fic. 108.—SomE DEposiITs IN ACID FERMENTATION OF THE URINE. a, Bacteria. b, Amorphous sodic urate. c, Uric acid. d, Calcium oxalate.—(Landois.) Urate of sodium may be amorphous or form in fan- shaped or sheaf-like crystals. Ammonium urate is usually found in alkaline urine that is undergoing fermentation. It may form in the urinary passages and produce serious results on account of the sharp spines setting up an intense irritation of the mucosa. It is a crystalline deposit consisting of spherules with projecting thorns, the so-called ‘‘hedgehog’’ crystals 25—O 298 MEDICAL MICROSCOPY. At times these spines are very numerous and they may be curved and twisted. Clinical Significance of Urates.—Besides the condition brought about by mechanical irritation by the spines of the ammonium urate crystals, the occurrence of urates is of little importance. The amorphous mixed urates are common in febrile diseases and all wasting diseases. They are also deposited during an attack of gout. Fic. 109.—Some DEPOSITS FROM AMMONIACAL URINE (ALKALINE FERMENTATION). a, Acid ammonium urate. 6, Ammonio-magnesium phosphate c, bac- teria.—(Landois.) Calcium Oxalate.—The most common form of calcium oxalate crystal is the envelope crystal. They are small octahedral crystals with highly refractive diagonal planes giving the appearance of crossed diagonal lines. With the low power they appear as bright specks, and it is only when viewed with the 4 objective that their full beauty is revealed. Dumb-bell-shaped crystals some- times occur, but are less frequent than the characteristic envelope form. URINE. 299 Calcium oxalate crystals are readily dissolved by the mineral acids, but are insoluble in water, acetic acid, alka- lies, ether, and alcohol. Acetic acid forms a ready test to differentiate the short forms of triple phosphate from calcium oxalate, the former being quickly dissolved. Usually the microscopic appearance alone is sufficient. Fic 110.—CaLclum OxaLaTE, ‘ ENVELOPE”? CrystTais.—(Greene.) Clinical Significance.—Usually little clinical significance is to be attached to a deposit of calcium oxalate. It occurs in neurasthenic states and in insanity quite fre- quently. Also after the ingestion of food rich in oxalic acid, notably asparagus, tomatoes, oranges, etc., and from excess of alkaline bases in the blood. The amount of calcium oxalate present in the urine can be determined only by quantitative chemical analysis. 300 MEDICAL MICROSCOPY. It is chiefly of interest in that it may form renal and vesical calculi. These crystals are more common in alka- line but may occur in faintly acid urines. Ammonio=-magnesium Phosphate.—This constitutes the so-called triple phosphates, and is the most common of all urinary crystalline deposits. Its most usual form is the coffin-shaped crystals, prismatic figures with beveled ends. These may be so short as to form squares, in which case they may be mistaken for envelope crystals of calcium Fic. 111—Forms of CRYSTALS OF AMMONIO-MAGNESIUM PHOSPHATE, —(Tyson.) . oxalate. More rarely beautiful fern-leaf or feathery crystals occur. These are usually formed when the urine is rendered very alkaline by the addition of ammonia. Various intermediate forms may be observed. Calcium phosphate may occur in amorphous deposits as whitish flakes. It is precipitated by heat, and may therefore be mistaken for albumin. From this it can be distinguished by the addition of a few drops of nitric acid, which rapidly clears up the precipitate but does not URINE. 301 affect the albumin. The crystals of calcium phosphate are usually in the form of rosettes with radiating needle- like or wedge-shaped masses. Basic magnesium phosphate is a rare deposit, occurring in alkaline or faintly acid urine in the form of highly re- fractive plates or rhombic tablets. They are quickly dissolved by acetic acid, and again precipitated on the addition of sodium carbonate. According to Purdy, the following are the chief condi- Fic. 112,—FEATHERY CRYSTALS OF TRIPLE PHOSPHATE.—(Tyson.) tions of the urine which lead to the production of phos- phatic sediments: “(a) If the urine be alkaline from a fixed alkali. ‘‘(6) If the earthy phosphates be in excess (the urine being alkaline or neutral). ““(c) If the urine be alkaline from volatile alkali, the result of decomposition of urea into ammonium carbonate in the urinary passages, the ammonium uniting with the 302 MEDICAL MICROSCOPY. magnesium phosphate to form the triple phosphate of ammonium and magnesium.”’ Clinical Significance.—In cases of phosphaturia from the presence of a fixed alkali in the urine there is an alka- line reaction to the urine when voided, and symptoms of feeble respiration and general debility are often present. Fic, 113. a, Calcium phosphate. 0, Calcium sulphate.—(Jakob.) This may be noted in the later stages of exhausting dis- eases and in flatulent dyspepsia. In the so-called “ phos- phatic diabetes,” in which there is a large deposit of phos- phates, the result of excessive elimination, severe symp- toms of depression, irritability, and dyspepsia may be present. When phosphaturia is due to the presence of a volatile URINE. 303 alkali, the urine undergoes ammoniacal decomposition before being voided, and the precipitation of phosphates takes place as soon as micturition is accomplished. Such a condition is always attended with an inflammatory lesion of some portion of the genito-urinary tract, and is therefore of a serious nature. Pus and mucus are present in addition to the phosphates. This state of affairs is usually dependent upon retention Fic. 114. a, Crystals of cystin. 6, Crystals of oxalate of lime. c, Hour-glass : forms of 6b. of urine on account of mechanical obstruction of the urinary channels. It may also depend upon nervous con- ditions favoring a retention of urine in the bladder. Cystin.—Crystals of cystinoccur in the urine as six-sided crystals or square prisms having a mother-of-pearl luster. If a drop of ammonia be added to the deposit on the slide, the cystin crystals quickly dissolve, to reappear when the 304 MEDICAL MICROSCOPY. ammonia has evaporated. ‘This is a rare deposit, devoid of clinical significance. Tyrosin and Leucin.—These substances are chemically S0Z Be IAE Fic. 115.— Mac- PHATE. —(Gould.) closely related, and result from the decomposition of proteids. They are exceedingly rare, occurring only in the most acute processes, notably in phos- phorus-poisoning and acute yellow atrophy of the liver, though they have been found in severe cases of smallpox, enteric fever, and leukemia. Tyrosin forms sheaf-like bundles of fine needles. Leucin is seen as yellowish globular masses of high refractive power. From fat it is distinguished by its insolubility in ether. Melanin.—Rarely there are deposited in the urine black Fic. 116. u, Leucin. 0b, Tyrosin—(Coplin.) or brown granular masses of pigment. They may occur in wasting diseases, and also in cases of pigmented tumors URINE, 305 of the urinary tract. From the former fact their presence cannot be accepted as indicating melanotic malignant disease, though when other symptoms are present they are a valuable confirmatory sign. LIPURIA. Fat in the urine occurs as globular masses of varying size which are highly refractile. It may be seen after fracture of the bones, in diabetes mellitus, and in acute degenerative renal processes associated with fatty de- generation of the tubular epithelium. HEMOGLOBINURIA. True hemoglobin in the urine is not an infrequent con- dition in those diseases that result in the destruction of the red blood-corpuscles in the vessels. Among these may be mentioned pyemia, poisoning by phosphorus, carbolic acid, scurvy, fat embolism, typhus fever, and malaria. The urine is opaque, dark reddish-brown, and of high specific gravity, and there is a persistent pinkish foam. There may be a quantity of granular matter, and rarely some free blood and casts. If any doubt as to the nature of the coloring-matter exists, the micro-chemical test for blood will give the characteristic rhomboids of hemin in large numbers (see p. 288). Organized Sediments.—Red Blood-cells: The red blood- cells may be present in the urine in quantity varying from a few cells, possible of detection only with the microscope, toa large amount. The color of the urine will not be af- fected unless they are present in considerable quantity, but in the latter case it may be bright red or dark brown, opaque, and frothy, the foam being pink. From pus- 26—O 306 MEDICAL MICROSCOPY. corpuscles the red blood-cells are distinguished by their smaller size, pale yellow color, and the absence of granu- lations and nuclei. In urine that is ammoniacal or of low specific gravity the red blood-corpuscles may become distorted and swollen. They then appear as more or less globular bodies of double contour, devoid of coloring-matter and scarcely visible even in a subdued light, or they may be seen as small rings whose outline is hardly distinguishable. GUIDE TO THE ORIGIN OF BLOOD IN HEMATURIA. ORIGIN. QUANTITY. DIFFERENTIAL POINTS. May Occur IN: Kidney. Usually|Clots usually absent. Asso-|Acute and chronic ne- compara-| ciated with blood-casts,) phritis. Malignant tively epithelial and hyaline| growths. Renal small. casts, renal epithelium. |; calculus, tuberculo- Intimately mixed with| sis, embolism, ab- urine. Many swollen! scess, acute febrile (loss of hemoglobin) | processes, hemo- phantom _corpuscles.| philia, and in filari- Sediment slight. asis and distomiasis. Frequently in poi- soning from turpen- tine, carbolic acid, etc. Pelvis of | Variable. | Absence of casts of any | Disease of pelvis, cal- kidney kind or renal epithelium.| culus, etc. and ure- Fibrinous molds of ure- ters. ters may be present. Pus-cells in calculus. : Bladder. Frequent- | Blood-cells well preserved | Stone, cystitis, tu- ly large. —unless urine is am-| mors, varicose veins moniacal. Clots fre-| of vesical neck, etc. quent. Heavy sedi- ment. Pus in cystitis. In papilloma and malig- nant growths may be shreds of such tissue. If from neck of bladder, appears at end of mictu- rition. Urethra. Small, May be expressed. First |Urethritis, trauma, part of micturition. ete. URINE, 307 At times they are shriveled and crenated, and it may be necessary to resort to the micro-chemical test for blood to determine its presence. In this case a few drops of urine are evaporated to dryness on a glass slip and the test proceeded with as already described (see p. 288). The table on page 306 presents in concise form most of the important facts in relation to hematuria. In determining its origin it is of course necessary to take into considera- tion the history and general symptoms presented by the individual case. Even then the origin of the hemorrhage may not be fixed with certainty. PYURIA. Leukocytes.— White blood-corpuscles in small amount are found evenin normalurine. Pus : . . Eé RT a i may be present in the urine in patho- ay a @ ae ¥ 3 ar@ @ logic conditions in large quantity. & ¥ ® It renders the freshly voided urine . a @ & turbid, and white shreds and masses go? ped @ may generally be detected. Micro- '’.@.e~w © scopically these will be seen to consist Fic. 117.—Pus 1n THE of strings and aggregations of pus- yy. a ae i corpuscles. When allowed to stand fragment forms. for some hours, urine’ containing pus AGRE in quantity presents a dense, viscid, whitish precipitate. Pus-corpuscles in the urine are seen under the micro- scope as more or less spherical, coarsely granular bodies, somewhat larger than a red blood-cell and possessing one or more nuclei. If a drop of acetic acid be added to the drop of urine, the granulations of the protoplasm of the pus-corpuscles disappear and the nuclei are brought strongly into view. After a little practice pus is easily distinguishable even without the aid of this reagent. 308 MEDICAL MICROSCOPY. By drying a drop of urine on the slide and fixing by heat, the slide being warmed until slightly uncomfortable to the fingers, the pus-corpuscles may be stained with methylene-blue, which brings out the characteristic nuclei. Pus is the most common of urinary sediments, and may come from any portion of the genito-urinary tract. A large abscess breaking into some part of the genito-urinary tract may produce sudden pyuria with a heavy deposit of pus-corpuscles. It is always important when pus appears in the urine to determine its exact source, but this may be a matter of extreme difficulty. All the facts of the case must be borne in mind, and in doubtful cases several examinations should be made before reaching a conclusion. While the statements in the following table are not absolute, they may serve as an indication of the probable origin of the pyuria. GUIDE TO ORIGIN OF PUS IN PYURIA. DISEASE. REACTION. URINARY SEDIMENTS. Renal Congestion.| Acid. Pus in very small quantity. A few red blood-cells and hyaline casts. Nephritis. Acid. Pus usually in small amount, being greatest in acute disorders. Also present renal epithelium, tube-casts, and perhaps blood. Pyelitis. Acid. Quantity of pus may be large. Epithelium from pelvis of kidney. In uncomplicated cases no tube-casts. May be sudden dis- appearance and return of pus owing to stoppage of ureter upon the affected side. Cystitis. Alkaline. | Pus in small or large amounts. Cells may be disintegrated, a stringy, mucoid sub- stance being voided. Cells from deep layer of mucosa of bladder in severe cases. Absence of renal cells and casts. Urethritis. Acid. Usually gonorrheal. Vaginitis. Acid. Pus and vaginal cells. URINE. 309 The genito-urinary tract is lined with a mucosa the epithelial characteristics of which differ very widely ac- cording to the location and purpose of the part. Certain portions are secretory in function, and from these a vari- ety of glandular epithelium may find its way into the urine. Other parts are merely passageways, or, as in the case of the bladder, temporary receptacles for the urine Fic. 118. a, Epithelium from renal pelvis. 6, Vaginal epithelium.—(Greene.) and other fluids, and here are found coverings of cells the upper layers of which are more or less flattened and sub- ject to constant renewal, the lower strata consisting of cells of a variety of shapes, the whole constituting strati- fied eptihelium. From these facts it will be readily seen that the urine may contain epithelial elements of great diversity of 310 MEDICAL MICROSCOPY. form, and it would seem upon superficial inspection of the subject to be an easy matter to determine their origin. This, however, is not the case, and unless there exist other factors to guide us it may frequently be quite impossible to determine with exactness the origin of epithelium found in urinary sediment. For it must be remembered that detached and single cells from points widely separated may present a similar appearance, and also that such cells Fic. 119.—Various Forms oF RENAL CELLS.—(Greene.) frequently take up watery or other elements from the urine and become swollen and distorted. Their charac- teristics may also be obscured by masses of bacteria which have formed in them after the urine has been passed, or, in the case of cystitis or pyelitis, even while the cells were still within the body. In determining the origin of epithelial cells in the urine a URINE, 311 one should have constantly in mind the histology of the genito-urinary tract, and he must also be in great part guided by the occurrence or absence of other deposits that Fic. 120.—BLADDER EPITHELIUM. frequently may throw light on the subject. a, Surface. b, Deeper layers.— (Greene.) The distin- guishing differences between pus and epithelium are as follows: Pus. EPITHELIUM. 1, Cells globular. 1, Cells globular or otherwise. 2, Protoplasm coarsely granular. 2. Protoplasm finely granular. 3. Nucleus usually indistinct. 3. Nucleus usually indistinct. 4. Nucleus brought out by acetic 4, Nucleus usually single and often acid. large. 5. Nucleus usually multiple (poly- 5. Cells frequently contain fat- nuclear leukocytes). droplets. ; 6. Most frequent cellular deposit. 6. May occur in masses when char- acter of tissue can be seen. 7. Often in large quantity. 7, Usually small in amount. 312 MEDICAL MICROSCOPY. Modern writers classify the epithelium of the genito- urinary tract into: SQUAMOUS EPITHELIUM.* Form. ORIGIN. Flat, irregular outline. Small, dis- Prepuce, vulva, vagina, and blad- tinct, and usually central nu- der. cleus. Slightly granular proto- plasm. COLUMNAR EPITHELIUM. Form. ORIGIN. Polyhedral, spindle, or caudate. Superficial layer of renal pelvis. May show indentations from re- Deep layers of bladder. Ureters cent contact with neighboring or urethra. cells. Often in groups. Nucleus prominent without acetic acid. SPHEROIDAL EPITHELIUM. Form. ORIGIN. Small, round, or irregular, finely Usually convoluted tubules of the granular, often contain fat-drop- kidney. May come from deep lets. Nucleus comparatively layers of mucosa of renal pelvis, large and prominent, usually bladder, and urethra. nucleolus, may occur in groups. Frequent upon tube-casts, form- ing the ‘‘epithelial’’ cast. Epithelial elements from the parenchyma of the kidneys are thrown off only under pathologic conditions of those organs. When they occur in any quantity, they are always associated with other elements that lead to an easy diagnosis of the condition—tube-casts and albumin being present in all serious renal troubles at one or an- other time of the disease. * The table is taken from Purdy’s ‘‘ Urinalysis.” URINE. 313 URINARY CASTS. The multiplicity of terms by which these bodies are known is often confusing to the student, and yet the sub- ject is exceedingly simple. True tube-casts are formed by the collection in the lumina of the uriniferous tubules of a coagulable substance, probably derived from the blood. This collects in fluid form, and upon coagulation shrinks slightly and escapes from the tubule and is cast off with the urine. This then gives us our true hyaline cast. Most other forms are but modifications of this one. Fragments of tubular epithelium or entire desquamated cells, red blood-corpuscles, pus, fat, or bacteria may ad- here to and become embedded in the hyaline cast, thus forming a body which is designated by the name of the constituent predominating. The exact pathologic pro- cess that was present in the individual tubule in which the cast was moulded at the time of its formation is graph- ically set forth by the nature of the cast itself. The following table is intended to portray the mode of the formation of the varieties of true tube-casts and the pathologic condition of the tubule necessarily present in each case. SPECIAL PATHOLOGIC CONDITION OF Tu- BULE WHEN Cast was Founp. RESULT. CENTRAL CORE. Coagulation of al- Integrity of tubular epithelium not | Hyaline casts. buminous mate-| affected. rial from the blood. Hyaline cast plus: |Granules from disintegration of | Granular cast. tubular epithelium. ” ah Epithelial cells, desquamated from | Epithelial cast. inclosing tubule. a on Pus-cells, suppurative process in the | Pus cast. tubule. " wel te Fat, from fatty degeneration of | Fat cast. tubular epithelium. es ce Bacteria, from invasion of the tubule | Bacterial casts. by these organisms. : 314 MEDICAL MICROSCOPY. True tube-casts may be readily differentiated from the pseudo-casts by the addition to the urine of a drop of dilute acetic acid, which quickly dissolves the central core and liberates the bodies embedded in it. Pseudo- and mucous casts are unaffected by acetic acid. Hyaline Casts.—These bodies are of varying diameter, Fic. 121.—SHows THE EFFECT OF Low ILLUMINATION, THE HYALINE CasTs BEING PLAINLY VISIBLE. conforming to the caliber of the tube in which they were formed. ‘They usually are slightly curved and one or both ends are rounded. They may be almost colorless or from a pale yellow to a reddish-brown. Granular Casts.—Purely hyaline casts are seldom seen. They usually contain fine or coarse granules, the products of cellular necrosis, in greater or less abundance. URINE. 315 Epithelial’Casts.—In addition to the above granules, renal epithelium, either entire or necrotic and fatty, may be embedded in the hyaline matrix. The cells may be few in number or in sufficient quantity to obscure the central core of hyaline substance. Pus Casts.—The hyaline cylinder may be covered with Fic, 122.—SHows THE Errsecr oF HicH ILLUMINATION, THE HYALINE Casts BEING Lost IN THE FLOop OF LIGHT, AND ONLY THE RENAL CELLS APPEAR.—(Greene.) leukocytes. More frequently there are granules, pus, and epithelial cells present in the same specimen. Fatty Casts.—While casts presenting a few fat-droplets in conjunction with other elements are frequently seen, there sometimes occur hyaline cylinders that are com- pletely covered by fat-globules of varying size. They are highly refractile bodies and indicate an exquisite degree of 316 MEDICAL MICROSCOPY. fatty degeneration of the renal epithelium. The addition of ether quickly dissolves the fat-droplets and furnishes a ready means of diagnosis. Bacterial Casts—When the hyaline core is thickly cov- ered with micro-organisms, it may readily be mistaken for a true granular cast. From the latter it may be distinguished by the more even distribution and size of the granulations and by examination with the high power, which will usually reveal their true nature. They are also quite resistant to the strong mineral acids and caustic alka- a, Cast madeup almost purely .. : ; of leukocytes. b,Castcom- lies. Their presence is of great ao ae import, signifying a suppura- tive nephritis or pyelitis. Pseudo-casts differ from the true casts in their mode of formation and physical properties. They are of two kinds, the first consisting of a cylinder of epithelial cells that have desquamated en masse from a uriniferous tubule. This form is hollow and is of rare occurrence, being found 29 695 chiefly in severe cases of desqua- © mative nephritis. The second “e KAS as Fic. 123. . . a ¥ . @ variety is composed of blood that co ZOOS ®@ has escaped into a tubule and fie 124-Broop-ceus coagulated there. It consists, AND BLOOD-CAsT.— (Landois.) therefore, of fibrin, and is thickly studded with red blood-corpuscles, with perhaps here and there a leukocyte. The latter variety is indicative of renal hemorrhage. Cylindroids are hyaline casts of extreme length and URINE. 317 irregular conformation. They vary in thickness in differ- ent portions of their extent, and may be indented, wavy, and ribbon-like. They frequently are twisted or folded upon themselves, and may divide into tail-like processes. Sometimes they contain crystals, and rarely epithelial cells. Their exact significance is not established. They are Fic. 125.—GRANULAR Casts.—(Greene.) dissolved by acetic acid, and as they occur in renal con- gestion and in various forms of nephritis in connection with true casts, their presence in the urine is sufficient to cause apprehension. On the other hand, it is claimed that they have been found in cystitis and in apparently normal urine. Waxy Casts.—This name has been given certain casts of 31 8 MEDICAL MICROSCOPY. uncertain origin and peculiar characteristics. They may be long and slender, but are usually short, broad, and in- dented. They are highly refractile and resist the action of acetic acid. They have been thought to be of an amyloid nature, but may occur in conditions other than amyloid kidney and may be absent in this affection. The Fic. 126. a, Blood-cast and hyaline cast carrying blood-cells. 6, Pus cast. c, Hyaline cast carrying epithelial cells. d, Epithelial cast— (Greene.) theory that they are hyaline casts which have remained a long time an situ and undergone chemical change has been advanced, and is plausible. Crystalline Casts.—Rarely there occur casts of urates or hematoidin crystals. They are of little consequence from a pathologic point of view, and occur chiefly in the urine URINE. 319 of infants, and sometimes in cases of gout or renal con- gestion. Mucus-cylinders.—These may be seen in any specimen of urine containing mucus. They are long, flat, twisted, hyaline bodies of varying diameter. Frequently they divide into tapering processes. They are unaffected by Fic. 127. u, Waxy casts. 0b, Fatty casts.—(Greene.) acetic acid. No practical significance is to be attached to them, Attention is again called to the fact that in searching for tube-casts a very subdued and preferably an oblique light must be used. Strong light shines through hyaline and granular casts and renders them invisible. The special significance of the various forms of casts 320 MEDICAL MICROSCOPY. will be considered under the discussion of nephritis. True casts are never present in normal urine except in small amount, and then are of the hyaline variety and are tran- sient. They may occur, as above, after severe muscular exertion or subjection to cold, and indicate a temporary congestion of the kidney. Their persistence and the presence of albumin are to be taken as indications of renal disease. The importance of tube-casts as an aid in the diagnosis of kidney lesions cannot be overestimated, and their pres- ence in the urine should under all circumstances lead toa further investigation, even though they may be tem- porarily accounted for by the patient having recently undergone severe muscular effort or exposure. Spermatozoa.—The description of these bodies will be found in the section on semen. Spermatozoa may occur in normal urine of the male after coitus or masturbation and nocturnal emissions or those due to muscular spasm in epilepsy. The semen may also be voided in the act of defecation, being pressed from the seminal vesicles by the passage of hard feces. In spermatorrhea they appear in the urine in consider- able quantities, frequently rendering it turbid and forming a whitish sediment. Sediments consisting of phosphates are frequently mistaken by the laity for spermatic de- posits. A microscopic examination readily determines the presence of spermatozoa, for they can be mistaken for nothing else by even the unpractised eye. Spermatozoa may occur in the urine of women after coitus. Fragments of Neoplasms.—In the villous form of papil- loma of the bladder shreds of the tumor are frequently voided in the urine and are easily recognized under the microscope. URINE. 321 If doubt as to their nature exists, they should be em- bedded in celloidin, cut, and stained after the usual method. Single cells from neoplasms of the genito-urin- ary tract afford little positive information, and usually the presence of renal neoplasms cannot be diagnosticated by microscopic examination of the urine. Particularly in the female, where the urethra is short and wide, polypi of considerable size may pass entire. The presence of necrotic masses of tissue containing pigment points to melanotic carcinoma of some portion of the urinary tract, particularly if associated with hema- turia, pain, and cachexia. Bacteriuria.—Normal urine is sterile until it leaves the bladder. If, therefore, micro-organisms in quantity are found in urine immediately after it is voided, the fact of infection of some portion of the urinary tract is estab- lished. To make doubly sure that the organisms do not come from the external genitals, the urine may be drawn with a sterile catheter, the external parts having been previously cleansed, and in cases of a known infection of the urethra that part thoroughly irrigated with a weak permanganate of potash solution. All urine after standing for even a short time contains bacteria which have accumulated after its exit from the bladder, for urine is a very good culture-medium, per- mitting of rapid multiplication of fungi. This is particu- larly true of diabetic urine, in which all of thé lower vege- table forms flourish. Two classes of micro-organisms are found in urine— pathogenic and non-pathogenic. Non-pathogenic Fungi.—These may be either moulds, yeasts, or bacteria. Moulds are seldom seen, and are of little importance. Yeasts occur chiefly in urine containing sugar. They 27—O 322 MEDICAL MICROSCOPY. are to be distinguished from epithelium by their smaller size and the fact that they present budding processes and frequently form bead-like masses. Many of the more adult forms possess a nucleus. They are usually oval in form. Care must be exercised in order to distinguish the non-nucleated cells from red blood-corpuscles. Bacterium termo and other putrefactive organisms are frequently met with in cases of cystitis with ammoniacal urine and in the urine of old and enfeebled persons. Even though the organisms may be non-pathogenic, this is a serious condition on account of the decomposition of the urine 7m situ which is occasioned by their presence. Putrefactive organisms include all forms of bacteria— bacilli, cocci, and spirilla. They are frequently actively mobile, and beautiful examples of spiral forms in active motion may be seen in almost any specimen of urine that has been allowed to stand for a few days until it has under- gone ammoniacal fermentation. Pathogenic Fungi.—It has been well demonstrated that in the effort to eliminate bacteria which have gained en- trance to the circulation the kidneys or bladder may be- come affected. The pyogenic organisms are the ones most frequently found in renal lesions, and, consequent upon this, in the urine. Chief among these are the staphylococci and the streptococci. In enteric fever typhoid bacilli are found in the urine in a large percentage of cases, even in the early stages of the disease. Tuberculosis of the kidneys is quite common as a secon- dary affection following pulmonary tuberculosis. The bacilli may occur in the urine in large numbers. If such is the case, they are often found in S-shaped clumps. For the method of detecting the tubercle bacillus in urine and its differentiation from the smegma bacillus the reader is URINE. 323 referred to the section on tuberculosis. In doubtful cases a guinea-pig should be inoculated with a quantity of the sediment obtained under precautions as to cleanliness. Cultural methods recommended by some text-books are extremely difficult, and therefore of little value. Actinomyces have been found in the urine in cases where the genito-urinary tract was infected by this fungus. The Gonococcus of Neisser is frequently found in the pus-corpuscles in urine from cases of gonorrhea. This subject is treated at length in the consideration of the gonococcus. VERMES IN THE URINE. Distoma hzmatobium.—As mentioned in the section on blood, the eggs of this parasite are frequently found in the urine of persons whose blood is infested with the worm. Their sharp spines cause- an intense irritation of the mucous membranes of the bladder and urethra, and mic- turition is accompanied with pain and hemorrhage. The disease is limited to the tropics, but is occasionally seen in sailors and others just returning from these parts. The eggs should be sought for in urine of suspected cases, and are readily detected and identified by their peculiar form and spines. ‘The urine also frequently con- tains pus and blood. Filaria sanguinis hominis.—This worm, whose normal habitat is the blood, is at times found in the urine of persons infested with it. ' Echinococci.—If the contents of an echinococcus cyst burst into some part of the urinary tract, the character- istic hooklets and scolices may appear in the urine. Pri- mary involvement of the urinary passages is rare. Infusoria.—The novice may be puzzled by the detection in the urine of actively motile cellular organisms, the like ‘sjeysA19 ayeyd -soyd ajdiry,, “peseariout sayeydsoyg ‘snd jo Junowe [ems “erm eu ‘AyQuenb Ay[ensy ‘sjseo relnuess a81e[ ATTensq | ur aq Ae *paseoiouy SLOT 0} OLO'L ‘pry [°° ' ‘Aeupry osha “junoure ‘sjseo Axem aq Avy aBIel *900°T 24 ‘sjseo auyedy May WY ‘Ajueos AJaA | Ur yuasatg ‘peseoiouy | Aww { pasveisaq ‘ply | * ‘Aeupry propAwy ‘speysAi9 ayeleXo UINTD[eo pue poe ‘peystuturp a suQ ‘juasaid Ajarer uimntayyida sa8e}s Id}e, UT Jeuey ‘sported yu9z}1uI1I19] UT 78 s}se9 ‘Ajasrey =—- SOUT}. ‘spuydau auryedy Moy y “Aquos Aroa Ayyensy | “jua}}IWHIIezU] | -au0s = ‘pasvarouy ‘mor Ayyens¢, ‘pry | rennsieqnr sony “Juasaid Ajarer si[90-pooyq : i ee Pex ‘SMWIep JenueiIg ‘peoiq pue aie ore pue a}eutmopaid sjsevo Ienueig ‘juNoure osiey ut uINT[ay} -tda [euer Aye} pue sng ‘padres -qo ale sjseo A}}e] SaMITeUIOG =e] ‘satyy ‘pasearour -oyyide pue ‘rejnuei3 ‘autpeAy oq -uenb a81ey |se8eystoweyT “pa fo ttt ‘styydou ‘Ayyuenb ursjseg ‘JUepunge Ayjensy | ur Ayyensy | -ysturmip AyTensp, “mor ATTenscQ, ‘pry | asnyip ~ “ gtu0IYD ‘sjsvo [eyaypide pue : ; ‘ ‘snd ‘auredy ‘sjsvo-pooig ‘AyWuenb ‘passed ur oumyeyyide [eusy ‘“(uatjoms ‘sotyty ‘uorssaiddns aq |autm yoyunome fo frttttt: ‘srqra pue paj}eueld wszjo) si[ed-poojq pay] -uenb asiey | Av ‘paystarmiq |y}yim satire, ‘poy | -ydau asnyrp ainoy ‘s][20 [erjayyids jeuar sdey : , -iad pue ‘s}seo ourpeAy maj eB ‘sTe} Ofo'l -sAID ple OLIN pue sa}ein snoydioury | junowre[eus “peysturuiq | 0} SZO' pesearouy ‘pry | ‘ermeradAy aatsseg ‘umntyayyida “yunoure : ; ; Ie[nqn} pue sjsvo Iejnue13 10 sur qreurs -eAy May eB aq ABIL “S[[P9-poojq pay| ur juasarg| ‘paseaiourysigyyy| ‘paonpas AYsyS ‘ploy |* ‘ermeredAy aynoy “SLNAWICAS “NIWOETY “SNIYQ¢) dO LNNOWY “ALIAVWUS) OISIDAdS =| ‘NOILOVEY “ASVASIG SINHWIGHS AUVNIYN dO SHONVUVAdIY ‘“HOVA NI VIGOOSOUOIN ANV SHSVHSICG IVNAY AO NOILVINGAVL + Q ro) ‘ayeydsoyd ardr3y ‘s[[20 etpurds pue ayepneo aq Ar ‘OTstIeyVIeyD jou pues junowe [jews urumyayyidy = ‘syonp Azeyided ut s8njd ur urr0y Aew sng sdeyiag ‘s]s@o [eLIojONq pus ‘sjseo Ajvensq. = ‘snyap eliapeg ‘yunoure 321e’T [euar ‘poojq ‘sng ‘wisejdoau ay} jo sassem sdeyiag ‘snd aq Lew ‘Ayuenb ut = sapsndioo-poojq pay ‘payerjsuourap aq Ajyensn Aeur ypeq eypreqny, ‘snyiijep yeuar pUe sasseul snoased ‘snoony, “Blin -eulay payreut aq Av = ‘s}unoure Sursvasour pue ZutArea ut snd 19}e’] ‘s][a0-poojq pel may eB sa8ejs AlIey ‘AyQuenb reurs uy ‘Ayuenb ur 8{qerred “‘Ppoolq st asaya Ft ‘sAeMyY “Ud -said Ayjensy) junouwre [jews ‘pasearouy ‘poonpery “‘jsIg ye posvelouy ‘peseotouy *paseaioaq °900°T 9} 9TO'T pelaMmo’y *parlamo’y “pre Ayjurey ‘Ao “PPT 0} PoyUny x ppy "proe wig W ‘auyey -[e@ I9ye’] “proe qiy 1V + ‘srpyadd otuo1y9 ++ Kaupry peorsing ene ‘TUL [BUI ‘sIsopnoiaqn} [euay 325 3 26 MEDICAL MICROSCOPY. of which perhaps he has never seen. They will probably prove to be infusoria that have gained access to the urine through the vessel in which it was collected having been washed in water inhabited by them. The author has seen such cases a number of times. In one of these vorticelle in abundance and of great activity were found, while in several other instances rotifers were observed. Diatomes and alge are frequently met with. The microscopist should familiarize himself with these various forms by the study of pond- and ditch-water, in which they abound. That interesting little volume ‘‘ Ponds and Ditches,’ by M. C. Cook, may serve as a guide to any so inclined. OTHER FOREIGN BODIES. Besides the above, a great variety of foreign bodies may gain access to the urine, either through the use of unclean vessels or from the dust floating in the air of the room. Among the most common are the fibers of cotton, wool, silk, and hemp, feathers, vegetable matter, hairs,and small seeds. Fecal matter may also be present, either from the pa- tient having defecated in the receptacle in which the urine was collected or having voluntarily introduced it with the purpose to deceive or through a vesico-rectal fistula. Hysterical patients may introduce foreign bodies into the urethra or vagina, or they may be shown the physician as having been voided with the urine. CYSTITIS. Pus and mucus in varying quantity, with red blood- cells in some instances, and bacteria in enormous numbers in the freshly voided urine are the chief microscopic char- acteristics of cystitis. Vesical epithelium is also present, sometimes in large quantity. URINE. 327 This disease in its various forms is always due to a bacteriologic infection of the bladder by pyogenic organ- isms. In conjunction with the history and clinical symp- toms, this condition of the urine renders diagnosis a matter of no difficulty. Owing to the ammoniacal de- composition of the urine 7m the bladder, triple phosphate and ammonium urate in the form of hedgehog crystals may beseen. ‘The author has observed one case in which the latter crystals were present in such quantity as to form a heavy deposit. VESICAL CALCULUS. In the early stages the frequent presence of crystals of uric acid or oxalate of calcium may, in conjunction with the clinical features, aid in the diagnosis of stone. As the disease progresses infection of the injured vesicular mucosa always results, and to the previous condition cystitis is added. Hematuria of intermittent character is the rule. VESICAL TUBERCULOSIS. From the first there is pus in the urine in gradually in- creasing amount. The cystitis that is set up must be differentiated from other varieties by the clinical symp- toms and by examination of the sediment for the tubercle bacillus (see p. 177). THE SEMEN. The semen as ejaculated consists of the combined secre- tions of the testicles, vasa deferentia, vesicule seminales, 328 MEDICAL MICROSCOPY. the prostate and Cowper’s glands, and the mucus-glands of the urethra. The element of chief importance in this rather complex fluid is the spermatozoon. Spermatozoa are the vitalizing principle of the semen. They are derived from the spermatogenetic cells of the testicles and are elongated bodies (about 50 ) consisting of a thread-like tail, a middle piece, and a head. The latter is about 3 » to 5 # in length, oval, and flattened, the sharper end presenting anteriorly. In normal semen and in the secretions of the vagina these bodies have the power of active motion, being pro- pelled by undulating and rapid movements of the tail. In the vagina they may live for days and weeks. In the seminal fluid they may retain their vitality for a consider- able length of time provided they be kept at body-tem- perature and evaporation guarded against. Besides spermatozoa, the semen contains epithelial cells of various kinds, and if it is allowed to stand for some time these form crystals that chemically and in their shape do not differ from the Charcot-Leyden crystals found in the sputum. They are known as spermatic crystals. Ac- cording to von Jaksch, they are deposited in large num- bers when a 1 per cent. solution of ammonium phosphate is added to pure prostatic fluid, and their appearance in quantity in the semen is an indication of prostatorrhea. Not infrequently the physician is called upon to deter- mine the cause of sterility in married couples. Kehver found that in a series of forty cases of sterile marriages the spermatozoa were absent from the semen of the hus- band in fourteen. The spermatozoa may be present and the cause of sterility be due to their lack of motility. In such instances it will be found that in a short time after ejaculation they become entirely motionless. The semen should be examined as soon after copula- URINE. 329 tion as possible, a drop being placed upon the slide to- gether with a drop of normal salt solution (0.6 per cent.) and covered with a cover-glass to prevent evapora- tion. The 4 objective should be used. MEDICOLEGAL EXAMINATION FOR SPERMA- TOZOA. In Rape Cases.—Interesting and important facts may be gleaned from microscopic examination of stains upon the clothing, deposits or crusts about the external genitals, and scrapings from the vagina. As has been mentioned, the spermatozoa may retain their vitality in the vagina for many days. Scrapings should be made and the product diluted with normal salt solution and examined with the high power It has also been frequently shown that spermatozoa resist drying and putrefaction, and Roussin reported a case in which they were detected in the stains upon a piece of cloth polluted eighteen years previously. The suspected seminal stains should be soaked in nor- mal salt solution for some hours and then squeezed out upon a glass slip. Many spermatozoa, whole or in part will be found if the stain is due to semen. The detection may be facilitated by the addition to the normal salt solution, in which the stain is moistened, of enough aqueous solution of eosin to impart to it a pink color. By this means the spermatozoa will be stained, the head becoming quite red and the tail a delicate pink. Usually this is unnecessary, as the spermatozoa are easily recognized in the uncolored condition. Their absence from the stain does not necessarily prove the same to have been made by some substance other than semen, as the man may have been impotent. In a case 28—O 330 MEDICAL MICROSCOPY. where spermatazoa cannot be found, the stain should be subjected to the chemical test for semen devised by Florence. According to this author, dark brown crystals of iodo- spermin are formed when semen or a dilution of seminal stains is treated with a solution of iodo-potassic iodid after the following formula: Todin crystals) ica cocste rein oererciiads 1.65 Potassium iodid, ................ cece cece 2.54 Water: s:ctesebackecaudsese es kee ees 26.00 Fic. 128.—SEMINAL ELEMENTS, SOME OF WHICH May BE FOUND IN THE URINE. a,a, Spermatozoa. 6, Seminal cells. c, Epithelium. d, d, Seminal granules.—(Coplin.) in pairs, or in the form of rosettes or in long rhombic plates. They are readily recognized under the micro- scope. Examination should immediately follow the ap- plication of the reagent, as the crystals are unstable and may redissolve. CEREBROSPINAL FLUID. 331 It goes without saying that in rape cases a careful note should be made of all other substances detected micro- scopically, such as hairs, fibers of various kinds, epithelial cells, and blood-corpuscles, as these may materially aid in the true solution of the case. VAGINAL SECRETIONS. The normal vaginal secretion is a thin fluid of acid reaction containing mucus and many large pavement epithelium cells and mucus-corpuscles. Bacteria of various kinds are also present, among them a diplococcus that decolorizes by Gram’s method and re- sembles the gonococcus morphologically. For this reason Simon considers it necessary that cultures should be made before reaching a conclusion that gonorrhea is present in the case of married females and children. This precau- tion is, of course, necessary only in selected cases. CEREBROSPINAL FLUID. There are a number of conditions in which an exami- nation of the cerebrospinal fluid is of great importance. In order to obtain this fluid lumbar puncture must be resorted to. Briefly, this procedure is carried out as follows. Strict surgical cleanliness is necessary for the sake of the 332 MEDICAL MICROSCOPY. patient, and also that, if it is deemed necessary, cultures may be made. In the latter instance large quantities of the fluid must be used for inoculation, as otherwise no growth may result owing to the organisms being present in small numbers. The skin of the patient having been prepared for sur- gical procedure, he is placed upon the right side, with the knees flexed and the left shoulder depressed. A long, thin aspirating needle is then introduced 1 cm. to the right of the median line between the spines of the third and fourth lumbar vertebre, and passed upward and inward to the depth of 3 to 4 cm. in children, or 7 to 8 cm. in adults. As soon as the point of the needle enters the sub- arachnoid space cerebrospinal fluid flows from it in drops. The fluid may be allowed to run directly into the culture- tubes and a small quantity be collected in a sterile test- tube for microscopic examination. It is scarcely necessary to add that the needle with which the puncture is made and the hands of the operator must be sterilized as if for a laparotomy. Pathogenic organisms may be detected in the exudate microscopically. It is advisable to centrifuge it and examine the bottom strata in the tube; or, if a centrifuge is not available, to place the tube of exudate in the ice- chest for a few hours so that bacteria and pus-cells may settle. The meningococcus, tubercle bacillus, pneumo- coccus, and many other organisms have been found in the cerebrospinal fluid. Methylene-blue is usually a good stain for routine ex- amination, but, of course, if the presence of the tubercle bacillus is suspected, it is necessary to follow the method laid down for the staining of this bacterium. Descriptions of the meningococcus and other bacteria NASAL SECRETIONS—SALIVA. 333 that may be found in cerebrospinal fluid will be found in the section on bacteriology. NASAL SECRETIONS. The nasal fossee are lined with a mucous membrane covered with stratified columnar epithelium and contain- ing numerous mucous and serous glands. These glands furnish an excretion which is transparent and tenacious, and alkaline in reaction. It consists largely of columnar epithelium, together with goblet cells and leukocytes. Large numbers of bacteria are always present in nasal secretions. Tubercle bacilli, Bacillus mallei, diphtheria bacilli, and the thrush fungus may be found accompanying the lesions in the nasal cavity due to their presence. Diplococcus intracellularis meningitidis has been found in the nasal secretions, and this may account for those cases of meningitis following traumatism affecting the integrity of the brain-cavity. In catarrhal conditions of the nasal membranes the desquamated cells may be found in large numbers. Pustular discharges may contain any of the pyogenic organisms. SALIVA. The normal secretion of the salivary glands of the mouth is a clear, somewhat viscid fluid of alkaline reac- tion. It contains, in various amounts: 1. Salivary corpuscles, which are bodies resembling the 334 MEDICAL MICROSCOPY. leukocytes of the blood, except that they are larger and have a very granular protoplasm. 2. A few red blood-corpuscles may be found. 3. Epithelial cells from the mucous membrane of the mouth. When from the surface, these cells are large, flat plates with irregular borders and a small nucleus. If they are derived from the deeper layers of the mucous membrane, they are polyhedral in form with large nuclei. Bacteria.—Over fifty distinct bacteria have been culti- vated from the normal saliva. Many of these are capable of becoming pathogenic under favorable circumstances. Among them are the pneumococcus, streptococcus, staphylococci, and diphtheria bacillus. The tubercle bacillus may be demonstrated in tuber- cular lesions of the buccal cavity or respiratory passages, and it is claimed that it may sometimes be present even under normal conditions. The causal agents of actino- mycosis, thrush, and gonorrheal stomatitis are treated elsewhere. Leptothrix buccalis.—This is a large organism occurring in threads in which it is difficult to demonstrate segments. It is devoid of pathologic significance, but is frequently found in the sputum of various pulmonary diseases. SPUTUM. By the term sputum is meant those substances that are discharged from the lungs and air-passages through the act of coughing or that rise spontaneously and are ex- pectorated. Dependent upon whether the air-passages are normal, or upon the form and character of the patho- SPUTUM. 335 logic process, the quantity and composition of the sputum may vary greatly. The sputum may be clear, thin, and viscid, or thick and colored by blood, pus, or bacteriologic pigments. Bacil- lus pyocyaneus frequently imparts a greenish hue to sputum in which it occurs. In abscess of the lung, empyema, or gangrene the odor of the sputum may be very offensive. Microscopic Examination of Sputum.—For the general inspection of sputum it is advisable to place a quantity of the collected material in a large Petri dish. It may then be viewed first by transmitted light; then, the dish being placed on a black surface, or a black piece of paper, objects such as cheesy masses, elastic tissue, and fragments of lung-substance and mucous casts of the bronchi are readily detected. Lastly, the dish may be placed upon the stage of the microscope and its contents examined with the low power (# obj.). White Blood-corpuscles. These are present in considerable amount in all sputa. In cases of penumonia and other pyogenic processes of the respiratory tract they may constitute the bulk of the expectorated material. They are frequently granular, fatty, or vacuolated, and may contain foreign bodies due to their phagocytic activity. Red Blood-corpuscles. In pulmonary hemorrhage or hemorrhage from the pharynx or post-nasal space the sputum may contain red blood-cells in greater or less quantity. Under these cir- cumstances these cells are well preserved, while in the red sputum of pneumonia they are usually swollen and pale or crenated, and there is an admixture of leukocytes. 336 MEDICAL MICROSCOPY. The color of sputum containing blood in quantity is not always red. Degenerative changes in the red blood-cells may free the hemoglobin, and chemical action may cause the sputum to assume a greenish or yellowish hue. The micro-chemical test for blood may be useful in certain cases in which the red blood-cells have been broken down and a discoloration by hemoglobin is suspected. Crystals of hematoidin are sometimes observed. The slight discoloration of sputum by blood is usually due to its escape from the pharynx or the bronchial mucosa, and may be of little pathologic significance. Epithelium. Epithelial cells are always present in the sputum in large quantity. They may come from any portion of the respiratory tract, including the nasal fosse and the mouth. Their condition is so frequently changed from the normal, by degenerations of diverse kinds, that it is not possible to identify them positively. The mononucleated, elliptic alveolar epithelium of the lungs may be present in enor- mous numbers in desquamative pneumonia, and also in chronic passive congestion of the lungs due to valvular or other heart lesions. In the latter case these cells contain yellow or black pigment-granules (Hertzfehlerzellen). Upon the whole, the epithelial elements of sputum afford us little information of value. Elastic Tissue.—Elastic fibers occurring either singly or in groups, perhaps with an alveolar arrangement, are of great diagnostic value, indicating a destructive process in the pulmonary parenchyma. Traube has shown that in gangrene of the lung with extensive necrosis of the pulmonary substance these fibers may be absent from the sputum, and accounts for this fact by presuming that they are destroyed in situ. In SPUTUM. 337 examining for elastic fibers von Jaksch recommends that the sputum be spread on a slide and a little caustic potash added. This clears up the sputum and the elastic fibers become visible. If they are present in small quantity and cannot be detected by the above method, that proposed by Fenwick may be followed. He boils a quantity of sputum in an 8 or 10 per cent. solution of caustic potash, and allows the mixture to stand for twenty-four hours in a conical glass. The sediment is then removed with a pipette and exam- ined under the microscope for elastic fibers. On account of the fact that, despite precautionary wash- ing of the patient’s mouth prior to expectoration, elastic fibers that have been taken in as food may get into the sputum, von Jaksch contends that only when the elastic fibers show the alveolar arrangement characteristic of lung structure are they of any sure diagnostic signific- ance, Spirals. Curschmann’s spirals are found in the sputum of a number of conditions, though formerly they were consid- ered to be pathognomonic of asthma. They consist usually of a central thread, around which is wound a mass of delicate fibers in a loose spiral formation. Entangled in these threads are large numbers of leukocytes, often many of them being eosinophiles, epithelial cells, and perhaps.Charcot-Leyden crystals. Czermak believes that they are formed by mucous threads that have become twisted upon their axis and thus formed them artificially. They may be present in tuber- culosis, bronchitis, pneumonia, and bronchial asthma. In connection with the latter affection they are a valuable diagnostic sign, as their presence is proof that the disease 338 MEDICAL MICROSCOPY. is true bronchial asthma and not of the reflex type. The spirals are usually about 1 mm. to 1.5 mm. in length, and may be seen with the unaided eye. Fibrinous casts of the terminal bronchi may occur in the sputa of plastic bronchitis and pneumonia. In the latter condition their presence in quantity indicates a severe type of the disease. According to von Jaksch, they are sometimes several centimeters in length and finely branched. Being fibrinous in character, they resist the action of acetic acid. Organized Tissue.—Pieces of neoplasms of the lung, sarcoma and carcinoma, as well as bits of connective tissue may be coughed up, and with the aid of the micro- scope their structure may be determined. Bacteria. The method for examining sputa for the tubercle bacillus has been already described (see p. 177). When examination fails to reveal tubercle bacilli in sputum, we are not warranted in making a negative diagnosis. ‘The bacilli may be present in numbers so small that they have escaped detection, or they may be found in a subsequent examination of sputum from the same patient, and there may arise cases of pulmo- nary phthisis in which it is impossible at any time to demonstrate Bacillus tuberculosis during the life of the patient. Mittal has shown that the tubercle bacillus willanultiply at 37° C. in sputum. In cases in which the organisms have not been found after repeated examinations, a speci- men of the sputum should be incubated for forty-eight hours and thenexamined. It will by this time have lique- fied, and the sediment should be used for making the preparation. SPUTUM. 339 Simon recommends another method, as follows: ‘About 100 c.c. of sputum is boiled with double the amount of water to which from six to eight drops of a 10 per cent. solution of sodium hydrate have been added, until a homogeneous solution has been obtained, water being added from time to time to allow for evaporation. This is then centrifugated or set aside for twenty-four to forty-eight hours and examined for tubercle bacilli and elastic tissue.’ Influenza. In sputum from cases of nasal or pneumonic influenza smears stained in carbol-fuchsin (see p. 110) may show the influenza bacillus in nearly pure culture. In the early stages of the disease they usually lie free, but as the process becomes older may be found in the pus-corpuscles. They have also been demonstrated in the blood of patients suffering from influenza. Pneumonia. In the early stages of fibrinous pneumonia the pneu- mococcus is often present in the sputum in large numbers. Smears may be stained by methylene-blue and Gram’s method, or animal inoculation may be resorted to (see p. 115). It must be remembered that croupous pneumonia may be caused by a variety of bacteria, and that the pneu- mococcus is present in 20 per cent. of healthy throats. Its presence can therefore only be considered as presumptive evidence of pneumonia. The detection of the pneumococcus in the sputum may be of considerable value in cases of central pneumonia where the diagnosis is in doubt on account of marked physical signs. The organism and its capsule may be stained by Welch’s method (see p. 114). 340 MEDICAL MICROSCOPY. Actinomycosis. When the fungus of this disease attacks the lungs, it may occur in the sputum in the characteristic granular, yellow bodies. Examination of the fresh specimen with the 2 objective may be sufficient to identify them. If doubt still exists, they may be stained by Gram’s method. The characteristic clubs and threads will be stained a deep blue, and can be mistaken for nothing else. Charcot=-Leyden Crystals. These crystals were at one time believed to be patho- gnomonic of bronchial asthma. It is now known that they occur in many other conditions, and their presence in the sputum is of little diagnostic value. They are color- less octahedral bodies, and are probably identical with the crystals of normal semen and those found in blood post- mortem, and in the feces in cases of anchylostomiasis. Hematoidin Crystals. These may be found in sputum from cases of hemop- tysis or pulmonary abscess, and indicate a previous ex- travasation of blood into the lung. They are in the form of columnar or needle-like clusters or rhombic crystals of a ruby red color. ‘When the crystals are contained in cells, they point to a previous hemorrhage; but when free hematoidin is present in considerable quantity, the inference is that an abscess has discharged from some neighboring organ into the lung’’ (von Jaksch). Crystals of Little Importance. Cholesterin crystals, fatty crystals, tyrosin, leucin, oxalate of lime, and triple phosphate crystals have been found in the sputum, but are of little consequence from a pathologic standpoint. SPUTUM. 341 Pulmonary Tuberculosis. The variety of pulmonary lesions associated with tuber- culosis of the lungs renders it impossible that the sputum should be in any degree constant in either its character or amount. In the acute miliary form, when the cough is slight or absent and the tubercles have not yet begun to break down, the sputum may be mucoid and tubercle bacilli absent. In the beginning stage of more chronic forms, the sputum may be muco-purulent and a few bacilli be present, while later in the disease it becomes more puru- lent and tubercle bacilli are present in large numbers. The morning sputum should be invariably selected for examination. When large abscess cavities exist, the pyogenic mem- brane lining them may throw off quantities of pus con- taining pyogenic organisms of every description, but devoid of tubercle bacilli, and the examination of pus from such cavities opened post-mortem is usually negative as regards Bacillus tuberculosis. Acute Pneumonic Phthisis. Here the sputum presents the aspects characteristic of ordinary fibrinous pneumonia, and is usually not exam- ined for tubercle bacilli until after the time of the ex- pected crisis, when the continued consolidation and high temperature sound a note of warning that arouses the suspicions of the attendant, and recourse to the micro- scope reveals the true nature of the malady. In such conditions the tubercle bacilli may be present in enormous numbers, though it may not be possible to demonstrate them for several days after the onset of the disease. Lobar Pneumonia. In the second stage—red hepatization—the sputum is 342 MEDICAL MICROSCOPY. quite characteristic and of the rusty, brick color. Micro- scopically the following elements are distinguishable: 1. Red blood-cells—often pale and swollen or crenated 2. Leukocytes in large numbers. I s Fic. 129.—CuRSCHMANN’S SPIRALS. a, X 80 diameters. 6, X 300 diameters.—(Schmaus.) 3. Epithelial cells from the alveoli and bronchial mucous membrane. 4. Bacteria of different kinds, notably the pneumo- coccus, and perhaps the tubercle bacillus, SPUTUM. 343 Bronchial Asthma. The sputum is viscid, white, or pink from admixture of blood, frothy, and contains Curschmann’s spirals, Charcot- Leyden crystals, and usually large numbers of eosino- philic leukocytes and free eosinophile granules. A smear stained with eosin and methylene-blue will reveal the latter. Method: 1. Sol. alcoholic eosin, ten seconds. 2. Wash. 3. Sat. aqueous methylene-blue, fifteen seconds. 4. Wash, dry, mount in balsam. Pulmonary Abscess. The sputum is copious, there being in some cases a pri- mary discharge of a large quantity, and presents the gen- eral appearance of pus. Upon standing, it separates into an upper, fluid, frothy layer and a lower stratum of pus-cells. The odor may be fetid, but is not so offensive as that produced from pulmonary gangrene. Microscopically may be seen: 1. Pus-corpuscles. Epithelial cells. Shreds of lung tissue. Yellow elastic fibers. Hematoidin crystals. oe ce ts ‘ Fic. 130.—CHOLESTERIN. , Bacteria of various kinds. —(Landois.) WAKH YN Pulmonary Gangrene. The sputum is thin and abundant and horribly offen- sive, and of a brown or greenish tint. On standing, it separates into three layers, the top one thick and frothy; 344 MEDICAL MICROSCOPY. the bottom, thick, opaque, greenish-brown; and a central watery, greenish-brown, semi-transparent portion. Microscopically there are found: 1. Portions of lung tissue. 2. Elastic fibers. 3. Granular detritus. 4. Rarely crystals (hematoidin). 5. Bacteria and moulds of various kinds. Von Jaksch denies the existence of elastic fibers in this disease, but the consensus of opinion of authorities differs with him in this particular. Acute Bronchitis. In the primary stage the sputum is mucoid in char- acter, sticky, and viscid, and may show streaks of blood. Later it becomes muco-purulent and more abundant. Microscopically will be seen: 1. Pus-cells. 2. Mucus. 3. Perhaps a few red blood-cells. 4. Bacteria of a non-specific character. Chronic Bronchitis. Sputum more profuse than in the acute form, and may in the case of bronchorrhea be quite largeinamount. Itis usually thin, greenish-yellow, and consists largely of pus- cells with occasional red blood and epithelial elements. ‘Putrid Bronchitis. The sputum is similar to that from gangrene, but does not contain the broken-down pulmonary tissues. Brown Induration. Brown induration from heart disease producesa sputum which may at times be quite red in color owing to the STOMACH CONTENTS. 345 presence of hemoglobin derived from extravasated blood or hemorrhagic infarcts. Free red blood-cells may also be present. Attention has already been called to the epithelial cells of the sputum of this affection, the so-called ‘ Hertz- fehlerzellen” (heart-failure cells). They consist of des- quamated alveolar epithelium which contains granules of yellowish pigment (hematoidin). Pneumonoconiosis. The sputum of the various mechanical pigmentations of the lungs shows epithelial cells containing granules of the special kind of pigment present—coal, iron, stone, etc STOMACH CONTENTS. The normal gastric juice is a clear or slightly turbid fluid, acid in reaction, and containing a few squamous cells, which have desquamated from the mouth or esoph- agus, and bacteria of a variety of kinds. Some of the latter are known to take part in the physiologic digestion of food through the action they exert upon the carbo- hydrates and proteids. MICROSCOPIC EXAMINATION OF VOMIT. A large drop of the vomit is placed upon a glass slip, covered with a cover-glass, and examined unstained, first with the low and then with the high power. A great variety of substances may be seen, including partly 29—O 346 MEDICAL MICROSCOPY. digested food, muscle fibers, connective-tissue, yellow elastic fibers, fat-globules, vegetable cells and fibers, mucus, mucus-corpuscles, sarcine, and bacteria of other forms, etc. In pathologic conditions there may be blood, in cases of gastric ulcer or carcinoma; or pus, denoting suppurative conditions of the mucosa, or, if it is present in large quan- tity, the rupture into the stomach or esophagus of a neigh- boring abscess. In carcinoma of the stomach there may be found por- tions of the malignant growth. ‘These must be differ- entiated from shreds of the mucosa. The peculiar arrangement of the carcinoma cells within alveoli of connective tissue, and the fact that the char- acter of the cells differs from that of the cells constituting the normal mucosa, are usually sufficient to enable one to come to the correct conclusion. On the other hand, the old idea that the cancer cell possesses features peculiar to itself, so that a diagnosis nay be made from individual detached cells, 1s entirely erroneous. Blood in vomited material, if in small quantity, may be difficult of demonstration, from the fact of the probable destruction of the corpuscles. In such a case the micro- chemical test for blood already mentioned should be tried with some filtrate from the vomit which has been dried upon the glass slip. FECES. The feces consist of the remains of undigested food, secretions of the intestinal tract, desquamated epithelium from the gastro-intestinal mucosa, and bacteria. Microscopic Elements. Upon minute examination the bulk of the feces will be found to be composed of remnants of food, the character of the passage therefore depending much upon the pre- vious diet. Usually there are seen striated muscle, often much altered in form and devoid of striations, connective and elastic tissue, starch granules, vegetable cells, spirals, and fat-globules. Needle-shaped crystals occurring singly orin bundles may be seen. These are derived from the fatty acids. Triple phosphate crystals are not infrequent, as are other crystalline forms, but these are of no diagnostic im- portance. A large amount of granular material that cannot be identified remains; this is called detritus. Formed Elements. These are derived from the intestinal tract. Red Blood-corpuscles. It is seldom possible to find red blood-corpuscles in feces unless from a hemorrhage very low down in the intestine or rectum. Usually even in severe hemorrhage the red cells are destroyed by the intestinal juices, so that while the feces may be intensely colored by the liberated hemo- globin, one may search in vain for red blood-corpuscles. The microchemical test performed with a drop of the feces will show the characteristic hemoglobin crystals if the color is due to free blood. Leukocytes. In normal stools these elements are uncommon, but in catarrhal or inflammatory diseases of the intestines they may occur in large numbers. If a quantity of pus is sud- 348 MEDICAL MICROSCOPY. denly expelled per rectum, it may be due to the discharge of an abscess into the intestinal tract. Epithelium. In health few epithelial elements are found in the feces, though a variety of forms may be present. In desquama- tive processes in the intestinal tract they may occur in large numbers. It is only when they are present in quan- tity that they are of any clinical importance. Bacteria. Enormous numbers of bacteria occur constantly in the feces. They are largely derived from the food ingested and from the saliva. Many of them are saprophytes and harmless, while others may, under conditions favorable to them, produce serious lesions. The more important organisms that have been isolated from the feces are Bacillus typhosus, the colon bacillus, Bacillus lactis aerogenes, Bacillus proteus vulgaris, the spirillum of Asiatic cholera, and the tubercle bacillus; these have already been considered. Trichina spiralis—Trichinosis is contracted by man by eating meat infected with the parasite, which is com- mon in hogs (measly pork). The male worm is about 1.5 mm.long, the female 3 mm. long. After fertilization in the intestinal canal the parasites penetrate the mucosa, and the adult forms eventually become encysted in the skeletal muscles, the psoas and the muscles of the thigh being chiefly affected. In sections of muscles viewed with the low power (2 obj.) the parasites are seen coiled within the oval cysts. A rich capillary plexus is formed about the cysts, as is well shown in Fig. 131. Pieces of suspected meat may be pressed thin between two glass slips and examined without staining. Cats are FECES. 349 frequently affected by this parasite. Rarely it is found in human feces in infected cases, or where infected meat has been ingested. The eosinophilia occurring in trichinosis is mentioned in the section on blood. Ameeba coli.—In the amebic form of dysentery and in liver abscesses Lésch in 1875 discovered bodies possessing the power of ameboid movement, but these cells were not recognized by him as being in any way the cause of the disease. It remained for Kartulis to point out that tropic dysen- tery and liver abscesses follow- ing these cases were due to Amoeba coli, and his observa- tions have been repeatedly con- firmed by other observers. At rest, the ameba is a cell- like circular or oval body of 10 to 20 win diameter. The ameba may present great irregularity of form, and, in addition to a nucleus, contain granulations and vacuoles. In fresh feces from a case of amebic dysentery, on the warm stage they may show active amebic movement, pro- jecting pseudopodia, and streaming of the granular pro- toplasm. ‘They may also be demonstrated in stained sections of colon or liver infected with them, and in the pus of liver abscess, of which they are the causative agent. hy rat U, MTT Fic. 131.—TRICHINA SPIRA- Lis.—(Reeves.) Hydatid Cysts. The microscopic appearances of the fluid from a hydatid cyst are very characteristic. The fluid should be allowed to stand in a conical glass, and the sediment, examined 350 MEDICAL MICROSCOPY. with the low power (3 obj.), should show the hooklets and scolices of the ecchinococcus or portions of membrane which on their inner surface are granular and distinctly striated. The hooklets look like minute cat’s claws, while the scolices are composed of a double circular row of hooklets and four suctorial discs attached by a constricted neck to a short pouch-like body. The adult form of the parasite is found in the intestinal canal of dogs as Tenia echinococcus. The cysts may form in various portions of the body or break into the respiratory or genito-urinary tract, in which case the hooklets or membrane may be found in the sputum or urine. They may also occur in feces. Ovarian Cysts. The contents of ovarian cysts vary greatly according to the nature of the cyst. Usually the formed elements are very numerous. In dermoid cysts there may be found in the aspirated fluid numerous squamous epithelial cells, hairs, and crystals—fatty, hematoidin, or cholesterin. The latter may be recognized as clear plates, often super- imposed, square or rhomboidal, with a square notch in one corner. INDEX. A. Abbe’s condenser, 33 Aberration, chromatic, 22, 28 how corrected, 23 spherical, 21, 29 Absolute alcohol, fixing by, 46 Achorion Schonleinii, 195 Achromatic lenses, 23 Acidophile granules in white blood- corpuscles, 236 Actinomyces bovis, 183 botanic position of, 185, 198 pure culture of, 184 Actinomycosis, 183 bacteriologic diagnosis of, 186 in human beings, 183 in urine, 323 of animals, 183 staining of, 185 Acute lymphatic leukemia, 263 Adenocarcinoma, 223, 226 Adenomata, 221 alveolar, 221 papilliferous, 233 tubular, 222 Adjustments, 37, 38 Aerobic bacteria, 77 stivo-autumnal. autumnal. Agar plates, 99 Agar-agar plates, 99 preparation of, 85 Agglutination phenomenon in ty- phoid fever, 143, 144 Air, hot, disinfection by, 89 Alcohol, absolute, fixing by, 46 commercial, fixing by, 47 synthetic, fixing by, 46 Alveolar sarcoma, 217 Amebz, examination of stools for, 349 : Amebic dysentery, 349 See Estivo- American type of microscope, 35 Anaerobic bacteria, 77 cultivation of, 105 facultative, 77 obligate, 77 Anesthetic leprosy, 181 Angiomata, 203, 204 Angiosarcoma, 216 Angular aperture, 25 Anilin-water-gentian-violet, 110 Animals, autopsy on, 116 inoculation of, 115 Anthrax, 157 bacilli, cultivation of, 159 infection with, through lungs, 157 infection with, through skin, 157 morphology of, 158 sporulation of, 158 bacteriologic diagnosis of, 159 occurrence of, in animals, 157 in human beings, 157 spores, resistance of, 159 Aperture, angular, 25 numerical, 26 of condenser, 33 Aplanatic lens, 23 Apochromatic objectives, 29 Arm of microscope, 35 Asthma, bronchial, 343 bacteria in, 338 Autoclave, 93 Autopsy methods, 103 on animals, 116 Avian tuberculosis, 172, 180 Axis of lens, 19 B. Bacilli, 71, 73 Bacillus aerogenes capsulatus, 154 351 352. Bacillus anthracis, 157 coli communis, 122, 138, 146 compared with B. typhosus, 142 diphtherie, 161 persistence of, 169 cedematis maligni, 149 icteroides, 192 lactis aerogenes, 146 mallei, 147 of anthrax, 157 of bubonic plague, 156 of cholera, 186 of diphtheria, 161 of Eberth, 139 of Friedlander, 133 of glanders, 147 of influenza, 159 of leprosy, 181 of malignant edema, 149 of plague, 156 of smegma, 178 of syphilis, 119, 183 of tetanus, 151 of tubercle, 171 of typhoid, 139 staining of, 141 Widal reaction with, 143 of yellow fever, 192 pestis, 156 prodigiosus, 119 proteus vulgaris, 137 pseudo-diphtheria, 166 pyocyaneus, 122, 129 subtilis, 159 tetani, 151 tuberculosis, 171 typhi abdominalis, 139 Bacteria, aerobic, 77 anaerobic, 77 chromogenic, 77 culture-media for, 75 facultative anaerobic, 77 food of, 75 forms of, 71 Gram’s method of staining, 107 growth of, 75 how to study, 117 in smegma, 120 light, effect upon, 76 motility of, 74 multiplication of, 73 obligate anaerobes, 77 of the normal human body, 120 pathogenic, 121 INDEX. Bacteria, peptonization of milk by, 119 pigment produced by, 77 pyogenic, 121 rod-shaped, 71, 73 spheric, 71, 72 spiral, 71, 73 spore formation of, 73 staining of, 113 staining of, 107 by Gram’s method, 107 temperature of growth of, 76 unstained by Gram’s method, 111 Bacteriologic methods, 78 work, necessities for, 78 Bacterium coli commune, 138 differentiated from B. ty- phosus, 142 termo, 137, 322 Basophile granules, 236 Bismarck-brown, 112 Bladder, epithelium, 312 Blood, the, 231 acidophile granules in, 236 anemia of, 266 pernicious, 266 secondary, 267 bacteria in, 287 basophile granules in, 236 cells in, 239 cells, nature of granules in, 239 chlorosis of, 268 corpuscles, differential count of, 248 enumeration of, 249 number of, 248 pathologic increase of, 260 peculiar to pathologic blood, 240 physiologic increase of, 260 transitional forms of, 237 white, normal blood of, 236 counting, 251 cyclostomata in, 231 deficiency of hemoglobin in, 259 development of red cells of, 232 differential counts of, 248 Distoma hematobium in, 290 dust, 235 Ehrlich’s stain for, 236, 245 eosinophiles of, 238 eosinophilic myelocytes of, 240 erythrocytes of, 232, 235 examination of, 241 fat in, 287 INDEX. Blood, filaria in, 289 fixation of, 244 Goldhorn’s stain for, 277 granular degeneration of, 258 granules of cells, nature of, 239 histology of, 231 increase in number of white cor- puscles in, 259 in feces, 347 in pneumonia, 341 in sputum, 335, 341 Jenner’s stain for, 236, 245, 247 leukocytes, large mononuclear of, 237 polynuclear of, 238 loss of staining reaction of, 259 lymphocytes of, 236 malaria in, 269 malarial parasites in, distinguish-. ing features of, 284 mast-cells of, 239 megaloblasts of, 232 megalocytes of, 232 methylene-blue and eosin stain for, 244 microblasts of, 232, 233 micro-chemic test for, 288 microcytes of, 232, 233 myelocytes of, 240 eosinophilic, 240 neutrophile cells of, 239 granules of cells of, 236 normoblasts of, 232, 233 normocytes of, 232, 234 oxyphile cells of, 239 granules in, cells of, 236 plasma, 235 plates, 235 polychromatophilic degeneration of, 258 polynuclear leukocytes of, 238 red cells of, 231 relapsing fever of, 192 staining reaction of white blood- cells of, 237 technique, 241 of counting red cells of, 251 of counting white cells of, 254 of fixation of smears of, 244 of making smears of, 242 test for typhoid fever, 143 triacid stain for, Ehrlich’s, 245 typhoid bacillus in, 140 varieties of red cells in, 232 Blue pus, bacillus of, 122 129 30—O 353 Bouillon, 80 Brownian movement, 74 Bubonic plague, bacillus of, 156 Buchner’s method for cultivating anaerobes, 106 Cc. Calculus, vesical, 327 Capsule, staining of, 114 Carbol-fuchsin, 110 Carbol-thionin, 277 Carbol-xylol, 68 Carcinomata, 223 adeno-, 223 226 classes of, 221 giant-cell, 227 glandular, 225 medullary, 227 melanotic, 228 myxomatoides, 227 scirrhous, 227 simple, 227 Casts, bacteriul 316 blood, 313 cellular, 315 classification of, 313 crystalline 318 epithelial, 315 examination of, 294, 319 fatty, 315 granular, 314 hyaline, 314 pseudo-, 316 pus, 315 urinary, 313 waxy, 317 Cedar oil, 24 Celloidin method, 49, 55 sectioning tissues in, 60, 70 sections, staining of, 65 Chain cocci, 126 Charcot-Leyden crystals, 337 Chlorosis, blood-changes in, 268 Cholera spirillum, 186 Chondromata, 208 Chondromyxomata, 208 Chondrosarcomata, 208, 219 Chromatic aberration, 22, 28 Chronic lymphatic leukemia, 263 Clearing tissue, method of, 67 Clips, 38 Coarse adjustment, 35 Cocci, 71 354 Comma bacillus, 186 Compensating ocular, 29, 33 Compound microscope, 35 Concave lenses, 19 Condenser, Abbe, 33 aperture of, 33 achromatic, 34 focusing of, 34 purpose of, 33 substage, 33 Continental microscope, 35 Convex lenses, 19 Cornet forceps, 108 Corrected lenses, 28 Counting red blood-cells, technique of, 251 white blood-cells, technique of, 254 Culture-media: agar, 85 alkaline blood-serum, 87 blood-serum, human, 88 bouillon, 80 Dunham’s, 87 filtering of, 84 gelatin, 83 glucose, 83 glycerin-agar, 86 litmus milk, 86 L6ffler’s serum, 81 peptone solution, 87 potato, 86 Wertheim’s, 135 Cultures from blood, 101 from feces, 102 from living animals, 101 from sputum, 103 from the throat, 102 hanging-drop, 106 plate, 99 Curschmann’s spirals, 337 ‘Cylindroids, 316 Cyst, dermoid, 230 ovarian, 350 Cystitis, 326 Cystocarcinoma, papillary, 228 Cystomata, epithelial, 228 papillary, 228 D. Deep cultures, 105 Degeneration, granular, of Grawitz, 258 polychromatophilic, 258 INDEX. Dehydration, method of, 48 Dermoid cyst, 230 Diaphragm, 22, 38 Diphtheria, bacillus of, 162 bacteriologic diagnosis of, 166, 170 methods of examination in, 166, 170 mixed infection in, 168 pseudo-membrane in, 169 Diplobacillus of Friedlander, 133 Diplococci, 71 Diplococcus intracellularis menin- gitidis, 122, 130 lanceolatus, 122, 131 pneumonia, Frankel, 131 Distoma hematobium, 290, 323 Double staining, 67 Draw tube, 37 Drum-stick bacteria, 151 Dry objectives, 23 Dysentery, amebic, 349 E. - Earthy phosphates, 293, 301 Eberth’s bacillus, 139 Echinococci, 323, 349 Edema, malignant, bacillus of, 140 Ehrlich’s granules, 246 staining method, 245 triacid mixture, 245 Embedding tissues, 50, 53 Endotheliomata, 216 Enzymes, 75 Eosin, 67 Eosinophile cells, 238 granules, 238 Eosinophilia, 259, 261 Epithelial casts, 315 cystomata, 228 pearls, 224 tumors, 219 Epitheliomata, 224 Epithelium in feces, 348 in nasal secretion, 333 in sputum, 336 in urine, 308 in vaginal secretion, 331 in vomit, 346 renal, 312 vesical, 312 Erysipelas, coccus of, 126 Erythrocytes, 232, 233 INDEX, Estivo-autumnal fever, 282 organism of, 269, 282 Eye lens, 31, 37 compensating, 29, 33 F. False membrane, 169 Farcy, 147 Fatty casts, 315 Favus, 195 Feces, 346 Ameeba coli in, 349 bacteria in, 348 blood in, 347 epithelium in, 348 leukocytes in, 347 microscopic elements of, 347 red blood-corpuscles in, 347 Trichina spiralis in, 348 tubercle bacillus in, 348 typhoid bacillus in, 348 Fermentation, method of obsery- ing, 106 tubes, 106 Fibers, elastic, in feces, 347 in sputum, 337, 343, 344 in vomit, 346 Fibrolipoma, 207 Fibroma, 2))4 Fibromyxoma, 207 Fibrosarcoina, 212 Field, flatness of, 29 Filiria sanguinis hominis, 289, 323 Films, 107 fixing of, 108, staining of, 108 Fine adjustment, 38 Fixation of tissues, 45 Fixing fluids, 46 Flagella, 74 staining of, 114 Flagellated organisms of malaria. 272, 276 Flaming test-tubes, 97 Fluorescein, 129 Focal point, 19 Focus, principal, 19 virtual, 20 Focusing, 41 Forceps, cover-glass, 108 Formalin, fixing in, 47 Formed blood-elements, variations of, 257 355 Fowl tuberculosis, 172, 180 Fractional sterilization, 93 Frankel’s pneumococcus, 131 Friedlander’s pneumobacillus, 133 Fuchsin, carbol-, 110 Fungi, budding, 194 G. Gas production, method for deter- mining, 106 Gelatin, preparation of, 83 Gentian-violet, 110 Giant-cell carcinoma, 227 sarcoma, 218 Glanders, 147 bacillus of, 147 bacterial diagnosis of, 148 Glandular carcinomata, 225 Gliomata, 210 Glycerin agar, 86 Gonococcus, the, 134 culture of, 135 pus in, 122 specific pathologic significance of, 134 Gonorrhea, 134 bacteriologic diagnosis of, 136 urine in, 323 Gram’s method of staining, 111 pathologic organisms stained by, 112 important organisms stained by, 112 iodin solution, 111 Granular casts, 314 Granules in colorless blood-cor- puscles, 246 not H. Hematozoon malarie, 269 Hairs in urine, 326 Hanging-drop cultures, 106 Hayem’s fluid, 251 Hedgehog crystals, 297 Hematoxylin, Bohmer’s, 65 method of staining with, 65 Hematuria, 305 Hemocytometer, Thoma-Zeiss, 249 care of, 255 Hemoglobin, estimation of, 255 rhombic crystals of, 288 356 INDEX. Hemoglobinuria, 305 Lenses, 17 Herpes tonsurans, 197 concave, 19, 20 Hodgkin’s disease, 265 convex, 19 Homogeneous immersion lens, 23 fluid, 24 How to study bacteria, 117 Hyaline casts in the urine, 313, 314 Hydatid cysts, 349 Hydrophobia, 194 Hyperleukocytosis, 259 Hypoleukocytosis, 259 I. Illumination, method of, 40 Image, virtual, 20 Immersion fluid, 24 objectives, 23 Incubators, 98 regulation of, 99 Indol, formation of, 87 Infiltration methods, 49, 52 Influenza bacillus, 159 bacteriologic diagnosis of, 161 Inoculation, methods of: of animals, 115 of test-tubes, 95 Intracanalicular fibroma, 205 Iodin solution, Gram’s, 111 Iris diaphragm, 41 Isolation of bacteria in pure cul- tures, 99 J: Joint for inclination, 39 K Kidney, blood from, 306 casts from, 313 epithelium from, 312 urinary sediments in diseases of, 324 Klebs-Léffler bacillus, 161 Koch’s law, 120 L. Laverania malarie, 269 Laveran’s crescents, 272, 284 Leiomyomata, 199 Leiomyxomata, 200 Lens, achromatic, 23 aplanatic, 23 apochromatic, 29 converging, 21 corrected, 23 correction of, 28 diverging, 20 forms of, 18 meniscus, 18 optical properties of, 18, 21, 22 Leprosy, bacillus of, 181 bacteriologic diagnosis of, 182 Leukemia, 262 changes in blood in, produced by intercurrent disease, 265 diagnosis of, 263 lymphatic, 262 lymphatico-splenic, 262 myelogenic, 264 pseudo, 265 splenic, 262 varieties of, 262 Leukocytes in blood, 236, 240 in feces, 347 in malaria, 273 in sputum, 335 in urine, 307 in vomit, 346 Leukocytosis, 259 inflammatory, 260 Leukopenia, 259 Light, refraction of, 17 modification of, 41 sources of, 40 Lipemia, 287 Lipomata, 207 Lipomyxomata, 207 Lipuria, 305 Litmus milk, 86 Loffler’s bacillus, 161 methylene-blue, 163 serum medium, 88, 165 Lumbar puncture, 332 Lungs, infection through, by B. anthracis, 157 Lustgarten’s bacillus, 183 Lymphangioma, 204 Lymphocytosis, 259, 261 mixed, 259 M. Macroglossia, 204 Magnifying power, 24 increase of, 27 INDEX. Malaria, 264 exciting agent of, 269 morphology of organism of, 269 Malarial fever: changes in the red_blood- corpuscles in, 280, 282, 285, 286 double quartan, 281 double tertian, 279 estivo-autumnal parasite, 269, 282 leukocytes in, 273 pigmentation of, 273 parasites, pigmentation of, 269, 270, 272, 279, 281, 283, 284 parasites of, in peripheral cir- culation, 272 quartan parasite of, 269, 281 quotidian parasite of, 269 tertian parasite of, 269, 278 organism, appearance of, in stained specimens, 278, 279, 281, 283 . appearance of, in unstained specimens, 269 biology of, 269 crescents, 272 examination of fresh blood preparation for, 275 examination of stained blood preparation for, 276 in peripheral circulation, 272 morphology of, 269 ovoids, 272 pigmentation of, 269, 270, 272, 279, 281, 283, 284 presegmenting forms of, 270 pseudopodia of, 269 segmentation of, 270 spores of, 269 stains for, 276 technique of examining blood for, 274 Manipulation of the microscope, 40 Medullary carcinoma, 227 Megaloblasts, 232 Megalocytes, 232 Melanin, 217, 228 Meningitis, 130 bacteriologic diagnosis of, 131 Meningococcus, 122, 130 Meniscus lens, 18 Methylene-blue, 109, 163 Microblasts, 232, 233 Micro-chemic test for blood, 288 357 Micrococcus lanceolatus, 131 of gonorrhea, 134 pneumoniz croupose, 131 pyogenes tenuis, 122 tetragenus, 122, 128 Microcytes, 232, 233 Microcythemia, 258 Micro-organisms, methods of stain- ing, 107 Microscope, the, 17 American type of, 35 care of, 39 classification of, 17 compound, 17 Continental type of, 35 focusing, 41 how to look through, 43 mirror, 33 mirror bar, 38 simple, 17 Microscopic examination of the blood, 231 of cerebrospinal fluid, 332 of feces, 346 of hydatid cysts, 349 of nasal secretions, 333 of ovarian cysts, 350 of saliva, 333 of semen, 328 of sputum, 335 of tumors, 198 of urine, 292 of vaginal secretions, 331 of vomit, 345 Microsporon furfur, 197 Microtomes, 56 care of, 58 forms of, 56 freezing, 64 knife for, 59 sharpening knife for, 59 Milk as culture-medium, 86 tubercle bacilli in, 180 staining for, 180 Mixed leukocytosis, 259 Monococci, 71 Mordant, 114 Mucous cylinders, 319 Miiller’s fluid, 47. Muscle-fibers in feces, 347 Mycoses, 195 Myelenic neuroma, 20 Myelogenic leukemia, 264 Myomata, varieties of, 199 Myxochondromata, 207 358 Myxomata, 206 Myxosarcoma, 207 N. Naming objectives, 24 Nasal secretions, 333 Neisser’s gonococcus, 134 stain, 164 Nephritis, 324 Neuroglioma ganglionare, 221 Neuromata, 202 amylenic, 202 myelenic, 202 Nielsen’s stain for tubercle bacilli, 175 Normal body, bacteria in, 120 Nose-piece, 35 Numerical aperture, 26 Nutrient media, preparation of, 80. See also Culture-med‘a. oO. Object, to illuminate, 0 Objectives, 23, 37 angle of, 25 apochromatic, 29 classification of, 24 dry, 23 immersion, 24 naming of, 24 penetration of, 27 power of, 25 rating of, 24 resolving power of, 26 Oculars, compensating, 33 par-focal, 37 Oil-immersion fluid, 24 objectives, 24 Oligocythemia, 257 Optical axis, 19° Osteoma, 209 Osteosarcoma, 210, 219 Ovarian cysts, 350 Overcorrection, 29 Oxaluria, 298 Oxygen, influence of, on bacteria, 77 Pr Papillary cystocarcinoma, 229 cystoma, 229 INDEX. Papillomata, 220 varieties of, 222 Paraffin, embedding in, 51, 53 method, tabulation of, 54 mounting tissues in, 54 mounting sections cut in, 62 staining sections cut in, 69, 70 Parasites, facultative, 74 obligate, 73 of blood, 192, 284, 287, 289 of feces, 348, 349 of malaria, 284 of sputum, 117, 338, 339, 340, 345 of urine, 316, 321, 323 Pathogenic bacteria, 121 stained by Gram’s method, 112 Penetration of objectives, 27 Pericanalicular fibroma, 205 Pernicious anemia, 266 Petri dishes, 99 sterilization of, 89 Phagocytosis, 276 Phosphates, earthy, 293, 301 Pigment formed by bacteria, 77 malarial, 269, 270, 272, 279, 281, 283, 284 Pillar of microscope, 35 Pityriasis versicolor, 197 Plague, 156 bacilli in culture, 156 morphology of, 156 Plate cultures, 99 Plates of agar-agar, 99 Platinum needles, 96 Pneumobacillus, Friedlander’s, 133 Pneumococcus, Frankel’s, 131 Pneumonia, blood in, 341 sputum in, 339 Poikilocytosis, 257 Polychromatophilic degeneration, 258 Polycythemia, 257 Potatoes, preparation of, 86 Power, magnifying, 25 Preparation of tissues, 45 Principal focus, 19 Prism, the, 17 Protozoa, 260 Protozoon of malariz, 264 Pseudo-diphtheria, bacillus, 166 Pseudo-leukemia, 265 Pseudo-lymphocytosis, 241 Pure culture, how to obtain, 99 Pus in feces, 347 INDEX. 359 Pus in sputum, 335 in urine, 307 in vomit, 346 Pustule, malignant, 157 Pyocyaneus bacillus, 122, 129 Pyocyanin, 129 Pyogenic bacteria, 121, 122 Pyuria, 307 Q. Quartan malaria, 269 R. Rabies, 194 Rack and pinion, 38 Radius of lens, 19 Ray fungus, 183 Red blood-corpuscles, 231 Refraction, 17 Relapsing fever, spirillum of, 191 Renal casts, 313 epithelium, 312 Resolving power, 26 Revolving nose-piece, 35 Rhabdomyoma, 201 Rod-shaped bacteria, 71, 73 Ss. Saliva, 333 bacteria in, 334 salivary corpuscles in, 333 Sapremia, 122 Saprophytes, 73 Sarcina lutea, 119 Sarcine, 71 Sarcomata, 211 alveolar, 217 angio-, 216 fibro-, 212 giant-cell, 218 lympho-, 213 medullary, 211 melanotic, 217 mixed, 213, 216 myeloid, 218 ptimary, of uterus, 201 round-cell, 213, 214 varieties of, 213 Scirrhous carcinoma, 227 Screw, Society, 37 Secondary anemia, 267 spectrum, 28 Sectioning tissue, freezing method of, 64 in celloidin, 60 in paraffin, 62 Sections of tissue, cutting, 56 mounting, 63, 68 staining, 65, 69 staining, tubercle bacilli in, 180 thickness of, 64 Semen, 327 Septicemia, 121 bacteriologic diagnosis of, 281 Simple angioma, 203 carcinoma, 227 microscope, 17 Skin wart, 221 Slide for hanging-drop, 106 Smegma bacillus, 178 Society screw, 37 Spectrum, the, 22 secondary, 28 Spermatozoa, 320, 328 medicolegal examination for, 329 Spherical aberration, 21, 29 Spiral bacteria, 71, 73 Spirilla, 71, 73 Spirillum cholere Asiatica, 186 Spores, 73 staining of, 113, 153 Sputum, actinomyces in, 340 bacillus of influenza in, 339 of tuberculosis in, 177, 338 bacteria in, 338 blood-corpuscles in, 335 Charcot-Leyden crystals in, 340 crystals in, 340 Curschmann’s spirals in, 337 elastic tissue in, 336 epithelium in, 336 fibrinous casts in, 338 hematoidin crystals in, 340 Hertzfehlerzellen in, 345 of abscess, pulmonary, 343 of acute pneumonic phthisis, 34 of asthma, bronchial, 343 of bronchial asthma, 343 of bronchitis, acute, 344 of bronchitis, chronic, 344 of bronchitis, putrid, 344 of brown induration, 344 of gangrene, pulmonary, 343 of lobar pneumonia, 335 of pneumonic phthisis, acute, 341 of pneumonoconiosis, 345 of pulmonary abscess, 343 360 Sputum of pulmonary gangrene, of pulmonary tuberculosis, 341 organized tissue in, 338 pneumococcus in, 339 pneumonia bacillus in, 339 tubercle bacillus in, 117, 338 Stage, 38 revolving, 38 Staining, double, 65 hematoxylin method of, 65 of bacteria, 107 of capsules, 114 of cover-glass preparations, 107 of flagella, 114 of sections of tissue, 65, 69 of spores, 113, 153 tubercle bacilli, 177 in sections, 180 Staphylococci, 71 Staphylococcus cereus albus, 122, 126 flavus, 122, 126 epidermidis aibus, 125 lesions of, 122 pyogenes albus, 122, 125 aureus, 122 characteristics of, 122 citreus, 122, 125 Steam sterilization, 93 sterilizer, 92 Sterilization, 92 by dry heat, 92 by live steam, 93 fractional, 93 of blood-serum, 93, 95 * Stomach-contents, 345 Stop, 22 Streak culture, 97 Strepto-bacilli, 73 Streptococci, 71 lesions of, 122 on false membrane, 168 Streptococcus pyogenes (erysipela- tis), 122, 126 Subcutaneous inoculation, 115 Substage, 38 condenser, 33 Synthetic alcohol, 46 Syphilis, bacillus of, 183 T. Tenia echinococcus, 350 Temperature, maximum, 76 INDEX. Temperature, minimum, 76 optimum, 76 Teratomata, 229 Test-tubes, filling, 90 flaming, 97 inoculating, 95 labeling, 97 preparation of, for cultures, 88, 89 sterilizing, 89 Tetanus, 151 bacillus of, in soil, 151 cultural characteristics of, 153 staining spores of, 153 bacteriologic diagnosis of, 154 Tetrad arrangement, 71 Thrush, 198 Tissue, fixing of, 45 infiltration of, 49, 51 sections, mounting of, 50 staining of, 45, 46, 65, 180 Triacid mixture, 245 Trichophyton tonsurans, 197 Triple nose-piece, 35 Tube length, 35 Tubercle bacillus, 171 culture of, 173 diagnostic demonstration of, 174 from cold abscess, 179 in feces, 180 in milk, 180 in sections, 180 in sputum, 175 in urine, 178 morphology of, 172 pathogenic activity of, 171 pus in, 179 staining of, 175, 177, 178, 180 Tuberculosis, avian, 172 vesical, 327 Tumors, 198 classification of, 199 Typhoid bacilli, 139 cultural characteristics of, 141 differentiated from B. coli commune, 142 ~ U. Urine, the, 291 acid urates in, 297 actinomyces in, 323 alkaline urates in, 293 ammonio-magnesium-phosphate in, 300 ammonium urate in, 297 INDEX. Urine, bacteria in, 321 bacterial casts in, 316 basic magnesium phosphate in 301 blood in, 305 calcium phosphate in, 300 oxalate in, 298 urate in, 297 carbonates in, 303 casts in, 313 bacterial, 316 blood, 313 cellular, 315 classification of, 313 crystalline, 318 epithelial, 315 examination for, 294, 319 fatty, 315 granular, 314 hyaline, 314 pseudo, 316 pus, 315 chemical sediments in, 295 crystalline casts in, 318 cylindroids in, 316 cystin in, 303 Distoma hematobium in, 323 earthy phosphates, carbonates, and alkaline urates in, 293 echinococci in, 323 epithelial casts in, 315 epithelium in, 309 fat in, 305 fatty casts in, 315 Filaria sanguinis hominis in, 323 foreign bodies in, 326 fragments of tumors in, 320 gonococci in, 323 hemoglobin in, 305 hyaline casts in, 314 infusoria in, 323 leucin in, 304 leukocytes in, 307 microscopic examination of, 292 molds in, 321 mucous cylinders in, 319 nonpathogenic fungi in, 321 organized sediments in, 305, 324 oxalate of calcium in, 298 parasites in, 316, a2, 323 pathogenic bacteria in, 322 potassium urate in, 297 pseudo casts in, 316 pus in, 293, 307 pus casts in, 315 31—O 361 Urine, pyuria of, 307 sedimentation of, 293 sediments of, in ‘disease (tabula- tion of), 324 sodium urate in, 297 solids in, 291 spermatozoa in, 320 technique of examination of, 294 triple phosphate in, 300 tuberculosis of, 178 typhoid bacilli in, 322 tyrosin in, 304 urate of ammonium in, 297 of calcium in, 297 of potassium in, 297 of sodium in, 297 urates, clinical significance of, 298 uric acid in, 293, 295 vermes in, 323 waxy casts in, 317 yeasts in, 321 Vv. Vaginal bacteria, 331 secretions, 331 Variations in formed blood-ele- ments, 257 Vascular nevi, 203 Venereal wart, 221 Vesical calculus, 327 tuberculosis, 327 Villous wart, 221 Violet, gentian-, 110 Virtual image, 20, 21 focus, 20 Vomit, microscopic examination of, 345 w. Wart, skin, 221 venereal, 221 villous, 221 Widal reaction in typhoid fever, 143, 144 Wool-sorter’s disease, 157 Working distance of objectives, 30 x. Xerosis bacilli, 166 Xylol, carbol, 68 362 INDEX. Y. Zz. Yeasts, 194 Ziehl-Nielsen’s stain, 175, 178 Yellow fever, bacillus of, 192 Ziehl’s carbol-fuchsin, 110, 175 sarcina, 119 MEDICAL BOOKS There have been sold more than 140,000 copies of Gould’s Dictionaries See Page 12 P, BLAKISTON’S SON & COMPANY PUBLISHERS OF MEDICAL AND SCIENTIFIC BOOKS 1012 WALNUT STREET, PHILADELPHIA Montgomery's Gynecology A PRACTICAL TEXT-BOOK A modern comprehensive Text-Book. By Epwarp E. 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It will be found wanting in none of these respects. OERTEL Medical Microscopy NEARLY READY A GUIDE TO DIAGNOSIS, ELEMEN- TARY LABORATORY METHODS, AND MICROSCOPIC TECHNIC By T. E. Oerre., M.D., Professor of Pathology and Clinical Microscopy, Medical Depart- ment, University of Georgia. WITH 120 ILLUSTRATIONS 27 JACOBSON’S Operations of Surgery The Operations of Surgery. By W. H. A. JACOBSON, F.R.C.S., Surgeon to Guy’s Hospital; Consulting Surgeon Royal Hospital for Children and Women; Member Court of Examiners Royal College of Surgeons; Joint Editor Annals of Sur- gery; and F. J. Srewarp, F.r.c.s., Assistant Surgeon Guy’s Hospital and to the Hospital for Sick Children. Fourth Edition, Revised, En- larged and Improved. 550 Illustrations. Two Volumes, Octavo, 1524 pages. Cloth, $10.00 Sheep, $12.00 PRESS NOTICES OF FORMER EDITIONS “The author proves himself a judicious operator, as shown by his choice of methods and by the emphasis with which he refers to the different dangers and complications which may arise to mar success or jeopardize life.”—Aew York Medical Record. ‘«The important anatomical points are clearly set forth, the conditions indicating or contraindicating operative inter- ference are given, the details of the operations themselves are brought forward prominently, and frequently the after- treatment is considered. Herein is one of the strong points of the book.””—New York Medical Journal. 28 The Pocket Cyclopedia of Medicine and Surgery Full Limp Leather, Round Corners, Gilt Edges, $1.00 With Thumb Index, $1.25 Uniform with ‘ Gould’s Pocket Dictionary’’ A concise practical volume of nearly 600 pages, containing a vast amount of infor- mation on all medical subjects, including Diagnosis and Treatment of Disease, with Formulas and Prescriptions, Emer- gencies, Poisons, Drugs and Their Uses, Nursing, Surgical Procedures, Dose List in both English and Metric Systems, etc. By Drs. Gould and Pyle Based upon their large “ Cyclopedia of Medicine and Surgery.” % 2 *,* This is a new book which will prove of the greatest value to students. It is to the broad field of general medi- cal information what ‘‘Gould’s Pocket Dictionary’’ is to the more special one of definition and pronunciation of words. The articles are concise but thorough, and arranged in shape for quick reference. If no other book can be found so much exact detailed knowledge so conveniently classified, so evenly distributed, so methodically grouped. It is Multum in Parvo. 29 A NEW EDITION CROCKER ON THE SKIN The Diseases of the Skin. Their Description, Pathology, Diagnosis, and Treatment, with Special Reference to the Skin Eruptions of Children. By H. RADCLIFFE CROCKER, M.D., Physician to the Department of Skin Diseases, Uni- versity College Hospital, London. With new Illustrations. Third Edition, Rewritten and Enlarged NEARLY READY; CLOTH, $5.00 *,* This new edition will easily hold the high position given the previous printings. The author is a member of American, English, French, German, and Italian Dermato- logical Societies, and a recognized authority the world over. STURGIS—MANUAL OF VENEREAL DISEASES By F. R. SrurGis, M.D., Sometime Clinical Professor of Venereal Diseases in the Medical Department of the Uni- versity of the City of New York. Seventh Edition, Revised and in Part Rewritten by the Author and FoLLeN Casot, M.D., Instructor in Genito-Urinary and Venereal Diseases in the Cornell University Medical College. 12mo. 216 pages. Cloth, $1.25 *,* This manual was originally written for students’ use, and is as concise and as practical as possible. It pre- sents « careful, condensed description of the commoner forms of venereal diseases which occur in the practice of the general physician, together with the most approved remedies. 30 FOR THE DISSECTING ROOM Holden’s Anatomy—Seventh Edition 320 Illustrations A Manual of the Dissections of the Human Body. By JoHN LANGTON, F.R.C.S. Carefully Revised by A. HEWSON, M.D., Demonstrator of Anatomy, Jefferson Medical College, Phila. delphia, etc. 320 Illustrations. Two small compact vol- umes. I2mo. Vol. I. Scalp, Face, Orbit, Neck, Throat, Thorax, Upper Extremity. 435 pages. 153 Illustrations. Oil Cloth, $1.50 Vol. II. Abdomen, Perineum, Lower Extremity, Brain, Eye, Ear, Mammary Gland, Scrotum, Testes. 445 pages. 167 Illustrations. Oil Cloth, $1.50 Lach volume sold separately. Hughes and Keith — Dissections Illustrated A Manual of Dissections by ALFRED W. HUGHES, M.B., M.R.C.S. (Edin.), late Professor of Anatomy and Dean of Medical Faculty, King’s College, London, etc., and ARTHUR KEITH, M.D., Joint Lecturer on Anatomy, London Hospital Medical College, etc. In three parts. With 527 Colored and other Illustrations. I. Upper and Lower Extremity. 38 Plates, 116 other Illustrations. Cloth, $3.00 II. Abdomen. Thorax. 4 Plates, 149 other Illus- trations. Cloth, $3.00 III. Head, Neck, and Central Nervous System. 16 Plates, 204 other Illustrations. Cloth, $3.00 Lach volume sold separately. *,* The student will find it of great advantage to have a “Dissector”? to supplement his regular text-book on anatomy. These books meet all requirements, and as they can be purchased in parts as wanted, the outlay is small. 31 EDGAR’S OBSTETRICS A NEW TEXT - BOOK Tue ILLustrations in Edgar’s Ob- stetrics surpass in number, in artistic beauty, and in practical worth those in any book of similar character. They are largely from original sources, are made to a scale, and have been drawn by artists of long experience in this class of medical work. Tue Text has been prepared with i‘ great care. The author’s extensive ex- perience in hospital and private prac- ~ tice and as a teacher, his cosmopolitan knowledge of literature and methods, -. and an excellent judgment based upon these particularly fit him to prepare .: what must be a standard work. ogg IN PRESS ‘o POET EN I Sara CRI ela er aOR E ‘s ‘S | aay > . a7 oe PPPS BODE: a tea eit a ate ae Da Sea