VERTEBRATE HISTOLOGY GAGE AND KINGSBURY UNIVERSITY OF CALIFORNIA MEDICAL CENTER LIBRARY SAN FRANCISCO Gift of the Department of Anatomy, U.C \ VERTEBRATE HISTOLOGY A GUIDE FOR COURSE 2, DEPART- MENT OF MICROSCOPY, HISTOLOGY AND EMBRYOLOGY. CORNELL UNI- VERSITY AND THE NEW YORK STATE VETERINARY COLLEGE BY SIMON HENRY GAGE PROFESSOR OF MICROSCOPY, HISTOLOGY AND EMBRYOLOGY AND BENJAMIN FREEMAN KINGSBURY AvSSISTANT PROFESSOR OF MICROSCOPY HISTOLOGY AND EMBRYOLOGY COMSTOCK PUBLISHING COMPANY ITHACA, NEW YORK 1899-1900 Copyright, 1899. BY GAGE AND KINGSBURY. CONTENTS. PAGE. Literature, Equipment and Reagents 7-10 I. INTRODUCTORY u II. WEEKLY EXERCISES 13 Introductory and Technic 15 The Epithelial Tissues 18 The Connective and Supporting Tissues 22 The Blood and Lymph 30 The Muscular Tissues and the Muscular System 37 The Nervous Tissues and The Peripheral Nervous System 41 The Blood and Lymph Vascular System 45 The Digestive System 49 The Respiratory System 57 The Urinary Organs 60 The Genital Organs 63 The Skin and its Appendages 67 The Central Nervous System 74 The Organs of Special Sense 78 III. TECHNIC. Isolation 82 Fixation 87 Sectioning, Free-hand and by 94 (a) The Paraffin Method 96 (b) The Collodion Method 99 (c) The Freezing Method 105 Staining and Mounting Io6 Staining IIJ Mounting J 2o Sealing, Labeling, Cleaning Slides and Covers 123 Special Methods I2 7 Blood "7 Fine Injection I2 9 Bone and Tooth, (a) and (b) 130 Nervous System J 3 2 Silver Nitrate Impregnations 1 2& Formulas (additional) J 37 PREFATORY NOTE. This guide is the outgrowth of the printed and mimeographed sheets fur- nished the students during the last few years in the course of Vertebrate His- tology in Cornell University. The course is meant to be fairly comprehensive in order to meet the needs of the three groups of students who pursue it, viz. : students in the academic department, both graduate and undergraduate, whose aim is general culture and attainment of a sound basis for investigation in morphology and physiology; (2) Veterinary students; (3) Medical stu- dents. For the two latter, the course forms an integral part of their profes- sional training, and is designed to furnish aid in the comprehension of gross anatomy, of physiology, and also as a foundation for the appreciation of the changes revealed by pathological histology. It will be seen in looking through the guide that definite information is given or will be supplied during the course for each specimen studied, and that each student has opportunity and is required to carry on from the beginning all the processes necessary for preparing tissues and organs for study according to the great groups of methods, like the paraffin and the collodion methods, isolation, and the methods of study of living and fresh material. It is believed that this training in independent work is as important as the formal instruction in histology ; for every investigator must, and every practitioner of either human or veterinary medicine, ought to be able to work out by himself some of the problems arising in his study or practice. An effort has been made in this course to combine the excellencies of the three great methods of learning, viz. : ( i ) That of text-books and works of reference with recitations or quizzes 5(2) that of the living teacher in lectures, and in personal instruction and supervision in the laboratory ; (3) finally, and most important, that of personal contact with the truths of nature in actual work where knowledge is gained at first hand. If this guide is looked over or used by teachers in other institutions, we shall deem it a favor if any errors of statement are pointed out to us, and if sug- gestions for improvement are made by those who have had much experience in conducting laboratory courses with definite objects in view. SIMON HENRY GAGE, BENJAMIN FREEMAN KINGSBURY. SEPTEMBER, 1899. LITERATURE, EQUIPMENT AND REAGENTS. TEXT BOOK. Text-Book of Normal Histology : including an account of the Develop- ment of the Tissues and of the Organs. By George A. Piersol, M. D. Fifth Ed. Ivippincott Co. , Phila. REFERENCE BOOKS. The following books and journals will be placed on the reference shelves in the laboratory, or will be referred to : A. Anatomy and Histology. Quain's Elements of Anatomy. Ed. by Schafer & Thane. 1893. Text-Book of Histology. By Dr. Philipp Stohr. Trans, by Dr. A. Schaper. Essentials of Histology. By A. Schafer. Text-Book of Histology. By Clarkson. Manual of Histology. By S. Strieker. Translation. 1872. Handbuch der Gewebelehre des Menschen. By A. Kolliker. Lehrbuch der Histologie und Mikroskopische Technik. By Bohm and and Davidoff. 1898. . Traite Technique d' Histologie. By L. Ranvier. Anatomic du Systeme Nerveux de 1'Homme. By A. van Gehuchten. 1897. B. Physiology. Text-Book of Physiology. By M. Foster. An American Text-Book of Physiology. Edited by W. H. Howell. Essentials of Physiological Chemistry. By Hamersten. Text-Book of Physiology. By E. A. Schafer. 1898. C. Embryology. Human Embryology. By Minot. Text-Book of Embryology : Man and Mammals. By O. Hertwig. Trans. by Mark. Vertebrate Embryology. By Marshall. Quain's Anatomy : Part I, Vol. i; Embryology. D. Journals and Periodicals. Volumes of the following may be referred to and in that case will be placed on the shelves : Archiv fur Mikroskopische Anatomic. Quarterly Journal of Microscopical Science. Journal of Anatomy and Physiology. Journal of Physiology. American Journal of Physiology. Journal of the Royal Microscopical Society. Anatomischer Anzeiger. Proceedings of the American Microscopical Society. Journal of Morphology. Journal of Applied Microscopy. E. Technic. In addition to Stohr, Ranvier, and Bohm & Davidoff, consult: The Microtomist's Vade-mecum. By A. B. L,ee. Methods of Pathological Histology. By A. C. von Kahlden. Practical Histology. By E. A. Schafer. PERSONAL OUTFIT. The student must supply himself with the following : 1. Text-books. 2. 25 quarto portfolios ( 16^x10-^ in. ). 3. 100 sheets of paper, quarto size (8xio^ in.), for laboratory drawings. 4. i oo sheets of paper, ruled if preferred, quarto size (8x10^2), for descrip- tions, etc. 5. One simple microscope (tripod magnifier). 6. Two needle holders and No. 6 needles. 7. Fine forceps, straight or curved. 8. Dividers. 9. One-half gross of slides; more will be needed later. 10. Three slide boxes. 11. Cover glasses ; y z oz., No. i, ^ in. circles; >^ oz., No. i, % in. circles; y z oz. oblongs, No. i, 23x30 mm. 12. One section razor (W. B. & E., Phila. ). 13. One or more scalpels. EQUIPMENT OF THE PERSONAL LOCKER. (Supplied by the Department. ) The personal locker contains the following, for which the student is responsible. Xylene balsam, bottle. %% collodion, bottle. Albumin fixative (Mayer's), homeopathic vial. Eosin, yi% aqueous solution; bottle with pipette. Hematoxylin, large shell vial. Hematoxylin, bottle with pipette. Clearer, carbol-xylene, large shell vial. Clearer, carbol-xylene, bottle with pipette. Picric alcohol, bottle. Castor oil, homeopathic vial with brush. Two Stender dishes for benzin and alcohol. Carbon ink, bottle. Pen and penholder. Glass jar for slides. Glass box for cleaning cover glasses. Two small glass boxes for clean covers. Five medium shell vials. Three small shell vials. Two watch glasses. Box of lens paper. Box of vaseline. Centering card. Thirty centimeter metric rule. Two slide trays. Two towels. Three pipettes. Five bottles for 95% alcohol, 67% alcohol, picrofuchsin, normal salt solu- tion and distilled water. EQUIPMENT OF THE MICROSCOPE LOCKER. Be careful not to put any of these in your personal locker, thereby caus- ing inconvenience to others using the same locker. Do not leave them out on the table. A compound microscope with % in. (No. 3), l /& in. (No. 7), and 1-12 in. oil immersion objective; nose piece; i in. (No. 3 or 4), and 2 in. (No. i) ocular; iris and stop diaphragms; eye-shade. Bottle of benzin for cleaning the immersion objective. Bottle of homogeneous immersion oil. Steel scale, marked in 1-5 mm. Glass micrometer scale, ruled in i-io and i-ioo mm. Slide of Pleurosigma angulatum (a diatom ). Slide showing letters in stairs. 1C GENERAL LABORATORY REAGENTS. Distilled water. Normal salt solution. 67% alcohol. 82% alcohol. 95% alcohol. Cedar-wood oil (thickened). Paraffine for infiltration. Paraffine for imbedding. Ether-alcohol. 1/4% collodion (thin, for infiltrating). 6% collodion (thick, for infiltrating). 8% collodion (thick, for imbedding). %% collodion. Chloroform. Clarifier (castor-xylene). Benzin. Clearer (carbol-xylene). SPECIAL LABORATORY REAGENTS. These will be placed on the supply shelves when needed. There is in- cluded a wide range of stains, fixers and solutions. INTRODUCTORY. The animal body is made up of different "organs" having spe- cial functions. These may be either more or less independent an- atomically, or they may be grouped together into systems. The organs of the body and the parts of the systems are composed of different tissues, which in turn consist of cells and special permanent products of the cells intercellular substance. This analysis may be set forth diagrammatically as follows : f organs or \ Body. 1 systems (of > Tissues. < I "organs), j | intercellular substance. The fundamental tissues of the body are of four kinds : A. Epithelial. Cells only slightly modified structurally and ar- ranged in layers or masses; intercellular substance (cell- cement) small in amount and undifferentiated. B. Connective and Supporting. Cells insignificant in number, slightly modified structurally; intercellular substance pre- dominant and variously modified. C. Muscular. Cells specialized in structure for the purpose of producing movement (i. e., for contracting in one direc- tion). D. Nervous. Cells specialized in structure for the purpose of or- iginating, transmitting, and transferring "nervous im- pulse." In pursuing the study of Histology the following principles are believed to be the most advantageous: 1. The study of the different tissues should precede the study of the organs. 2. Every tissue and organ should be studied fresh as far as possible in order to have correct notions of the structural appear- ances unmodified by reagents. 3. Every tissue and organ should be studied alive as far as possible in order to see the function and thus associate function and structure. 4. Before the microscopic structure of an organ or part is studied t\\z gross anatomy should be first thoroughly understood. 12 Gross preparations for reference should be at hand and constantly referred to while the microscopic study is carried on. 5. In the study of the tissues it should be remembered that, though at first distinct, in the adult body there is more or less mingling of the different tissues; but in this composite structure there is one tissue which from its predominance forms the charac- teristic feature of the structure. The epithelia are the simplest in this as in other respects. The different organs likewise are not isolated and independent units, but are thoroughly permeated by the parts of the vascular and peripheral nervous systems contained within them. In this course the tissues and organs will be studied in the order below: 1. The Kpithelia (including Kndothelia). 2. The Connecting and Supporting Tissues and the Skeletal System. 3. Blood and Lymph; i. e. , the fluids of the body and their corpuscles. 4. The Muscular Tissues and the Muscular System. 5. The Nervous Tissues and the Peripheral Nervous System. WINTER 6. 7- 8. 9- 10. n. 12. The Blood and Lymph Vascular Systems. The Digestive System. The Respiratory System. The Urinary Organs. The Genital Organs. The Skin and Its Appendages. The Central Nervous System. The Organs of Special Sense. During the fall term the principal aim will be to gain a knowledge of the kinds, structure and distribution of the different tissues and a working knowledge . of general histological methods. During the winter term the minute anatomy of the different organs will be studied. The connective and supporting and the muscular tissues are so closely associated with the organs and systems that they principally compose that the tissue and system will be studied together. The peripheral nervous system, likewise, may best be studied with the nervous tissues. METHOD OF LABORATORY WORK. Laboratory work is arranged as weekly exercises, and each week's work is to be completed before the next succeeding is under- taken. Mounted preparations of the various tissues and organs taken up will be studied. These preparations may be divided into four groups: A, those owned by the department and assigned for study; B, those prepared by the department and simply to be mounted by the student; C, those in which the fixing, imbedding and sectioning are done by the department and the staining and mounting by the student; D, preparations made entirely by the student, i. e., fixed, imbedded, sectioned and stained; and E, dem- onstrations of preparations illustrating special features. With all specimens prepared or partially prepared by the department exact data will be posted on the bulletin board as to (a) the mode of fixation, (b) imbedding method, (c) thickness of the section, when known, (d) stains employed. In the case of preparations belonging to class D the student is expected to keep a record of the different steps and times. Since there will be slides owned by the student (B, C, D) and by the de- partment (A), care must be exercised in keeping them separate ; those owned by the student on one slide tray, those of the depart- ment on the other. To gain a working knowledge of histological technic each stu- dent will prepare from the beginning certain organs or tissues, i. e., fix, imbed, section, stain and mount, imbedding them by the para- fine and by the collodion methods. LABORATORY REPORTS. Reports upon each tissue or system are to be submitted when the work on that tissue or system is finished, the reports and the times they are due being posted on the bulletin board. These re- ports are to consist in part of drawings of the preparations studied, in part of descriptions and such discussions as may be specifically called for. In the drawings, name all the parts presented or at least recognized. Large figures are preferable to small ones. Colored 14 pencils, though not required, will be found helpful in making clear interpretations. The descriptions should be made out in accord- ance with the following scheme : Descriptions for Laboratory Reports. 1. Name of the specimen. 2. Names of its principal parts. 3-4. Constituent elements or tissues of each of the main parts and the relation of the parts to one another. 5. Structures present in the specimen, but not recognized. 6. Distribution in the body. 7. Function of the organ and of its principal divisions. RECITATIONS. There will be a weekly recitation, or quiz, covering the ground of the lectures and the laboratory work. Its purpose is not only that of a weekly examination, but it is also intended to unify the work of the week, bring out the important features and clear up difficult points. Weekly references to the text-book covering the matter for which the student is held responsible will be posted, and under Points for Quiz are given the most important features of the work, which the student will be expected to know. In the weekly exer- cises following, these are left blank and should be filled out from the bulletins posted. EXAMINATIONS. There will be a written examination at the end of each term, covering the lectures and the laboratory work. In addition there will be a laboratory examination to test the students' ability to recog- nize the different tissues and organs. In the laboratory examina- tion at the end of the fall term, he will be required to recognize the different tissues in the preparations given him. At the end of the winter term, the identification of organs will be expected of him. In addition, at intervals during the year, especially in the last half of the winter term, unlabeled slides will be assigned for study and report. Also unknown tissues may be given from which prepara- tions are to be made and reported on subsequently as called for. These two methods will help the student in the recognition of the different organs and tissues, and also prepare him for the laboratory examinations. 15 INTRODUCTORY AND TECHNIC. LABORATORY WORK FOR THE WEEK. I. Imbedding tissue. Go to the delivery desk with two small shell vials filled with 82% alcohol from the supply shelf and get two pieces of tissue already fixed and hardened and now in 82% alcohol. One of these is to be imbedded according to the par- affine method and sectioned during the week of the term . The other piece is to be stained in toto in para- carmine imbedded in collodion, and sectioned during the week of the term. By means of a ribbon pin attach to the cork of each bottle a label giving the name of the animal, the tissue or organ, the fixer employed, the method in which it is to be imbedded, and the date: In order that you accomplish this successfully it is necessary for you to study carefully 39-47 ; 50-60. Keep a record of the steps and the times of transfer to each fluid and present it as part of the laboratory report on ( a ) examine the isolated cells unstained, in dissociator ; (6) stain a second preparation with methylgreen and eosin (92, b), and mount in glycerin ; (c) mount a third preparation of the cili- ated cells in alum-carmine and eosin glycerin ( 156). IV. Label the bottles of clearer ; fill and label the bottle for distilled water and those for 95% alcohol and normal salt solution ; prepare and label in their respective bottles no c. c. of picro- fuchsin and 90 c. c. of 67% alcohol. V. Make 4 shellac rings (cells) for ^ in. cover glasses ; pre- pare two for y% in. covers ( 106). VI. Read carefully i ; 18-21 ; 64 ; 71-78. Review what you have so far done on I, II, and III. Study carefully 64-70 ; 79 ; 100-112. THE CELL AND THE TISSUES. x-i_^- LWc*v-tI References : V ***** H, O^- * >W-* % ~~ ^ ** ^%, VI |.l V Points for Quiz : Laboratory Report : -x Z7z^? : i. Ovarian Ova. Starfish (Asterias Forbesii). Assigned for study. Picro-acetic fixation ; paraffine ; hematoxylin and eosin. Study, noting the nucleus (germinal vesicle), cell-body (vitellus), nucleolus, nuclear membrane, and structure of the nucleus and cell-body (spongioplasm and hyaloplasm). This prep- 17 aration affords a demonstration that young ova are single cells with nucleus and cell-body. Sketch one or more that show typically. 2. Ovarian Ova. Amblystoma. Demonstration. Study, recognizing in the young ova nucleus and cell-body. Compare with (i). 3. Early blastula stage. Amblystoma. Assigned for study. At this stage the fertilized ovum has divided up into a few cells, arranged in a single layer around a central cavity (blastoccele), comparable to a simple epithelium. Make a sketch showing out- lines of cells and, if possible, of nuclei. 4. Late blastula stage. Amblystoma. Assigned for study. The cells at this stage have greatly increased in number, are smaller, and the wall bounding the cavity is of several layers of cells, com- parable, therefore, with a stratified epithelium. Sketch, showing the outline of the egg, the outline of the cavity, and for a portion of the wall, the outlines of the cells and nuclei. 5. Medullary plate stage. Amblystoma. Assigned for study. At this stage differentiation is well advanced. The three germ layers are seen in the section ; from the entoderm the noto- chord is forming ; from the ectoderm, the central nervous system ; the cavity is the alimentary canal (archenteron). Sketch the dorsal portion, and compare with the figure in the text-book. TOPICS FOR QUIZ 4th Week. INTRODUCTORY AND TECHNIC ; THE CEI,L AND THE TISSUES. 1. Histology, define; what is a cell, nucleus, cell-body; a tissue and its composition, kinds of tissue ; intercellular substance? 2. What is an ovum, and how are the tissues derived from it? 3. What are the ways of examining animal tissues with the microscope ? What is isolation or dissociation ? Give examples. What is fixing and why necessary? What is meant by imbedding, its aim, when necessary? Name three methods. 4. Staining, what is it, why advantageous ; kinds of staining and of stains used in histology. 5. Mounting, kinds of mounting media ? Give exact procedure in mount- ing in (a) glycerine ; (b) in glycerine jelly ; (c) in balsam. i8 THE EPITHELIAL TISSUES. t-t/L LABORATORY WORK FOR THE WEEK. References: Points for Quiz : Laboratory Report : Due: Go to the delivery desk for slides assigned for study, 6, 10, 14, and 19. Prepare a clean slide with albumen fixative ( 67, a} and go to the delivery desk for 18, paraffine section ; carry on according to $ 67, a -70. From the bottle on the delivery desk place a small drop of glycerin jelly on the center of a clean slide and go to the instructor for 9 and 16, already stained. Cover ( 105). Go to the delivery desk with a clean slide for 17, now in thin balsam. Mount in balsam (109). Go to the desk for 7 ; in glycerin ; place a drop on the slide and cover it ( 104). Clean carefully and seal ( no) the preparations of ciliated cells from the trachea, made last week (8). COLUMNAR EPITHELIUM. 6. Small intestine. Cat. Assigned for study. Transec- tion. Flemming's fluid fixation ( 24) ; paraffine ( 42) ; saf- franin stain ( 79) ; sections 10 yu. Study the epithelium covering the villi, noting the two kinds of cells and the structure of each ; the position and shape of the nucleus ; the striated border ; the relation of the epithelium to the underlying tissue. Find a place showing a surface section of the epithelium. 7. Epithelial cells from the small intestine. C-o-fc Isolated in Muller's fluid dissociator ( 4) ; now in alum-carmine and eosin glycerin ( 156). ) _ . 19 Place a small drop of the glycerin on a slide and cover with a cover- glass. Search the preparation to find (a) completely isolated cells, () cells adhering together in clusters and affording a surface view of the epithelium. Note as before the shape and structure of the cells and the shape and position of the nucleus. Try to find goblet cells. CILIATED EPITHELIUM. 8. Ciliated cells from the trachea. Cat. Two preparations made last week. Carefully clean around the cover glass and seal with shellac ( no). Study the preparations, searching for completely isolated cells and cells adhering in clusters. Note the shape of the cells, the structure and position of the nucleus, the cilia, the shape of the basal end. Compare the two slides, noting the identity of structure but different staining. 9. Ciliated cells from the trachea. Horse. Isolated in Miil- ler's fluid dissociator ; stained with V^c~*^, , now in ^U-.i Mount in glycerin jelly ( 105). Study carefully, noting the shape of the cells, the shape and position of the nucleus, the length of the cilia. Compare with 8. 10. Ciliated epithelium. Section of the soft palate. Cat. Assigned for study. The nasal side will show a so-called stratified ciliated epitheli- um. Observe the cilia covering the free ends of the cells ; compare the, apparently, superficial cells bearing the cilia with the deeper cells. Examine 8 again to see if you can find any isolated cells of the deeper layer. n. Living ciliated cells. Go to the desk with a slide for living ciliated cells from the oral epithelium of the frog. Place upon the slide in a drop of saliva or normal salt solution ; cover. Study the activity of the ciliated cells, trying to ascertain the move- ments of the cilia. This may best be done in cells in which the cil- iary movements have considerably slackened. 12. Living ciliated cells. From the trachea. Cat. Demon- stration. Observe the ciliary movements, noting the rate and direc- tion of the vibrations. 13. Ciliated pavement epithelium; From the peritoneal 20 epithelium of an Amphibian (Necturus, Amblystoma. Frog). Demonstration. Note the short cilia projecting up from the flat- tened cells of the peritoneal epithelium. PAVEMENT EPITHELIUM. 14. Peritoneal epithelium (endothelium). Mesentery of cat. Assigned for study. The fresh mesentery was treated with a YZ% aqueous solution of silver nitrate, washed in water, exposed to sunlight until the cell- cement between the cells had blackened, washed in water, stained in hematoxylin to bring out the nuclei, dehydrated, cleared and mounted in balsam. Study the preparation, noting the nuclei and cell-outline as indicated by the stained cell cement. Remember that the cells are flat and a single layer deep. (13). Ciliated pavement (peritonea 1 !) epithelium. Dem- onstration. See 13 above. STRATIFIED EPITHELIUM. (10). Stratified epithelium. Vertical section. Assigned for study. The oral side of the soft palate is covered with a stratified epithelium. Note the shape of the cells and nuclei in the deeper layers and compare with the cells of the superficial layers, observing the grad- ual transition as you pass from the deepest to the superficial cells. Are intercellular bridges to be seen ? 15. Stratified epithelium ; surface cells. With the finger nail gently scrape the inside of your own cheek and mount the scrapings on a slide, adding a drop of normal salt solution if necessary. Examine the preparation, noting the shape and size of the epithelial scales, the shape and size of the nucleus. The sur- face of the cells may be marked by ridges where neighboring cells have overlapped. Can you find groups of two or more cells? Compare with the superficial cells of 10 where you see them in section, or on edge. 1 6. Stratified epithelium ; deeper cells. Isolated cells from the deeper layers of the epithelium of the lip. W-* Miiller's fluid dissociator ; stained with ; now in Mount in glycerin jelly. In studying this preparation find 21 cells in which the intercellular bridges (prickles) are well shown, noting as well the shape and size of the cells and their nuclei. 17. Epidermis. Human finger. Section slightly oblique with the surface. Fixed in o\^S minutes, stain in hematoxylin *, minutes, picrofuchsm f-a minutes ; dehydrate, clear, and mount in balsam. Study carefully, noting the areas of hyalin matrix surrounding the cells and the reticulum of elastic tissue. Compare with sections of hyalin cartilage, 33, 38, and contrast with the ligamentum nuchae (28). Note again the different staining reaction of the elastic fibers of the cartilage and the white fibrous tissue surrounding the cartilage. 35. Fibro-cartilage. From the inter-vertebral disc. Assigned for study. ; ; stained with There are shown the interlacing bundles of white fibrous tissue with the cartilage cells surrounded by hyalin matrix scattered through the tissue. Compare with the elastic cartilage, 34, and in this com- parison recall the staining reaction of the white and the elastic fibers (26, 28). BONE. 36. Dry bone. Homo. Transection of femur. To be ground down to a thin section and mounted in accordance with the special directions, 133-134. 28 Study the preparation very carefully, recognizing (#) the Ha- version systems, () the interstitial lamellae. In the Haversian systems, note the Haversian canal, the concentric lamellae of bone, the lacunae (cavities occupied during life by the bone cells), the canaliculi. Understand from the development of bone, the meaning of the concentric arrangement constituting an Haversian system. In the interstitial bone, observe the lacunae and canaliculi. What is the meaning of interstitial lamellae ? If desired, a tangential section of femur may be prepared to show the lacunae from another aspect, and thus gain a better idea of their real shape. 37. Developing bone. Longisection of the hand (or foot) of a fetal mammaL Fixed in ^iurv*(M collodion; sections 1^ yu. Stain with hematoxylin minutes ; picrofuchsin seconds. This illustrates endochondral ossification (early stage) and the cartilaginous stage of the primary (endochondral) bones in this case the phalanges and carpal (or tarsal) bones. In at least one of the phalanges that is cut longitudinally the early stages of ossification may be seen. Study it carefully, noting the areolation of the cartilage, identifying if possible the osteo- blasts and osteoclasts ; if the section is in the right plane the in- growth of periosteum may be seen. If the process of ossification is far enough advanced, note the formation of the primary marrow cavity and the lamellae of spongy bone. The later processes of os- sification are shown in 28. In addition the preparation illustrates : (#) true or diarthrodial articulations, () capsular ligaments, (<:) muscle and tendon, and (d) developing hair. 38. Developing bone.^^A?V^^<^ongitudmal section of the head of a long bone VXAI\V\&T(^ picric alcohol ; decalcified in nitric acid decalcifier ( 131-132) ; collodion ; sections 50 /*. Stain in hematoxylin 30-45 minutes, picrofuchsin l / 2 -i minute. Mount in balsam. This preparation will illustrate (a) the formation of endochon- dral bone, () the structure of the epiphyses and the longitudinal growth of a long bone. Recognize the following features : (i) the center of ossification of the epiphysis, (2) the center of ossification for the shaft, (3) the marrow, (4) the bone, (5) cartilage, (6) peri- 2 9 osteum. Study carefully the centers of ossification in the epiphysis and shaft, so as to understand the dissolution of the cartilage (de- generation of the cells, absorption of the matrix) and the deposit of bone. How is spongy bone converted into dense bone ? Study the structure of the periosteum, recognizing the two layers ; understand the part it plays in the processes of ossification. In addition, study the preparation for the structure of cartilage, articular cartilage, the general structure and articulation of a long bone^ 39. Transaction of a long bone. VvA?V*^ JUA^/WMJ^-O Picric alcohol; decalcified in nitric acid decalcifier ( 131) ; collodion ;. sections CLi* //. Stain in hematoxylin bO minutes, seconds. Mount in balsam. Study, noting (a) the marrow with its nutrient artery, () the structure of the bone, (c) the structure of the periosteum. Com- pare the structure of the decalcified bone with the dry preparation of bone, 36. Recognize the identity of structure in the two prepar- ations so differently prepared. TOPICS FOR QUIZ 7th Week. THE CONNECTIVE AND SUPPORTING TISSUES CONTINUED. 9. Cartilage, general character ; varieties, and the situation of each? 10. Embryonal cartilage. When found and where situated? ir. Hyalin cartilage, character and distribution? 12. Elastic cartilage, character and distribution? 13. Fibro-cartilage, character and distribution? 14. Bone, the varieties, structure and situation ? 15. Development of bone. Be ready to give the steps as described in Piersol. 1 6. Centers of ossification ? 17. Compare the intercellular or ground substance of bone and cartilage with that of the other connective tissues. 30 BLOOD AND LYMPH. LABORATORY WORK FOR THE WEEK. D f References : Points for Quiz : Laboratory Report : Due: Go to the delivery desk with two clean slides for 45 and 46 now in balsam ; place a small drop of the balsam on the slide, and cover. Obtain from the desk 48, spread on a cover-glass ; stain as directed below and mount in balsam. To economize time, next prepare 42 and 43. Obtain 47 at your second period. HUMAN BLOOD. 40. Fresh blood. Flame a needle and after it is cool, prick the finger near the nail-bed (best) or the side of the finger ; wind a handkerchief around the base of the finger and press the finger with the other hand ; a drop of blood will be obtained. Or, you may dis- infect the needle of a haemospath and with it obtain the blood more quickly and easily ( 120). Touch a -cover-glass to the large drop so obtained and place it upon a slide, blood side down. The drop of blood should be large enough to easily fill the space under a cover-glass. Seal the preparation at once with castor oil. Study the corpuscles carefully, noting the form as determined by observing the face, profile and oblique views ; the corpuscles can be made to turn over by pressing gently on one edge of the cover- glass. Observe that most of the corpuscles are arranged in rolls or rouleaux. Here and there may be seen a leucocyte or white blood corpuscle. 41. Action of reagents. Obtain a large drop of blood as in 40 ; ascertain the effect upon fresh blood of four reagents as given below. Since this will take some time and needs fresh blood it may be necessary to prick the finger more than once. (a) Normal salt solution. Place upon a clean slide a small drop of fresh blood and add to it a small drop of normal salt solu- tion ; apply a cover glass. Is any change observable ? If so, what ? Is the fluid truly normal ? (6) Distilled water . Place upon a clean slide a drop of fresh blood and place beside it a small drop of distilled water ; cover. What is the effect of the water upon the corpuscles ? Where the blood and water meet observe the changes ; find some corpuscles entirely in the water. What is the meaning of the change ? (c) 2 % salt solution. Place a small drop of blood upon a slide, cover, and at the side ot the cover glass place a drop of a 2% solu- tion of common salt. What is the effect of the solution upon the corpuscles with which it comes in contact ? Meaning ? (X) 2 % acetic acid. Make another preparation of fresh blood and at the side of the cover glass place a drop of a 2% solution of acetic acid. Note the effect of the solution upon the red corpuscles and compare with the action of distilled water. What is the effect of the solution upon the white corpuscles? What is the meaning of these experiments in connection with the structure of the red corpuscles ? 42. Dry preparation. With a piece of lens paper wet with 95% alcohol wipe off the finger at the point where it is intended to obtain blood ; prick the finger as in 40, and as directed in 121, ( i ) , prepare six or more blood films upon clean covers ; allow two of them to dry and place the others in a watch glass of ether- alcohol c A^XXVt for 43. Allow the two films saved out to dry thoroughly, then warm gently and mount the best (or both) film side down upon a shellac ring prepared at some previous time ( 103, a). Select a place where the film is thin and even to study the appearance of the dried cor- puscles. Choose several corpuscles that are not distorted and with the ocular micrometer for which you determined the valuation measure their diameter in microns. Compare with the size given in the text -book. 13. Stained preparations. (121-123). Allow the prep- arations that were placed in ether-alcohol to fix for one hour or longer, remove them and allow them to dry for a few minutes, when they may be stained immediately or later (at your second period). Stain the blood films in two ways : (a) with eosin and hematoxylin 32 ( 124) ; rinse in water and dry. Mount in balsam without clear- ing ( IO 9)> (^) with Ehrlich? s triacid mixture ( 125). In the first preparations the red corpuscles will be red or pink, in the second yellow or orange. Compare with 42 as to shape and size. 44. Blood-plates. Demonstration. The tip of the finger was carefully cleaned with 95% alcohol, the finger pricked, a drop of i% osmic acid was placed over the puncture and the blood squeezed out into the drop of osmic acid, which was transferred to a slide, covered, and sealed with castor oil. Note the appearance and size of the blood-plates in comparison with the red corpuscles. AMPHIBIAN BLOOD. 45. Blood of Necturus. A Necturus was pithed, the gills cut, and the drops of blood caught in a vial of i% osmic acid. It was allowed to fix in this 1-2 hours, washed in water, changed sev- eral times, washed in 67% alcohol, stained several hours in paracar- mine ( 86), washed in 82% alcohol, 95% alcohol, absolute alcohol, xylene and xylene balsam. Cover a drop of the balsam containing the corpuscles. Note the shape of the corpuscles, their nuclei, size as compared with human blood and frog's blood (42, 46). 46. Blood of frog. Prepared as was 45. Mount a drop of the balsam containing the corpuscles. Compare with 42, 45. LAMPREY BLOOD. 47. Place a drop of the blood of the lamprey upon the center of a slide, apply a cover-glass, and seal with castor oil. Study carefully the form of the red corpuscles and compare with human (mammalian) blood as to form and size. Do they arrange themselves in rouleaux ? Observe the occurrence of leucocytes, their number and size. Carefully wipe away the castor oil, lift the cover a little, and place under the edge a small drop of 2% acetic acid. What is the effect ? Compare with the effect of acetic acid upon human blood. What differences are there between the red corpuscles of mam- malia, amphibia, and the lamprey ? 33 DEVELOPING RED BI,OOD CORPUSCLES. 48. Smear preparation of red marrow. A drop or red marrow was spread upon a cover-glass as a thin film ( 12 1 ) ; fixed in a saturated solution of mercuric chlorid ( 26) 15 minutes or so, and placed in 67% alcohol. Rinse off the alcohol with distilled water and stain with Bhrlich's triacid mixture ( 125) ifWi for 10-15 minutes, rinse with distilled water, dry and mount in - balsam. In this preparation find () marrow cells, medium sized cells with a large nucleus and scanty protoplasm, resembling leucocytes somewhat ; (^) giant cells, with one or many nuclei ; (e) cells with deeply staining nuclei and more or less hemoglobin in the cell body (stained orange), erythroblasts ; (d) finally, red blood corpuscles, non-nucleated. Find as many forms of erythroblasts as possible, and compare, with the red corpuscles. TOPICS FOR QUIZ 8th Week. THE BlyOOD AND THE IvYMPH. 1. What and where is blood, (a) plasma or intercellular substance; (b) corpuscles ? 2. What and where is lymph, (a) plasma ; (b) corpuscles? 3. Character of the corpuscles in the vertebrates ? 4. Size of the corpuscles in man, horse, ox, dog, necturus? 5. Methods of study ; moist, dry and stained preparations. 6. Compare the blood corpuscles of man, rabbit, camel, lamprey and necturus. 7. Action of reagents on the corpuscles? 8. Development of the corpuscles in the embryo and in the adult? 34 BLOOD AND LYMPH. LABORATORY WORK FOR THE WEEK. References : Points for Quiz : T L , T> z. U l> , U V,U 1 Laboratory Report: Due : Go to the delivery desk for 51; stain as directed below and mount in balsam. Next prepare 53. Make the remaining prepara- tions in order. LEUCOCYTES. 49. Amoeboid movement. Amphibian leucocytes. Place upon the slide a drop of frog's blood ; apply a cover glass at once and seal the preparation with castor oil. Search the preparation until a leucocyte is found undergoing amoeboid movement. Keep it under observation for some time, ob- serving the changes of form and mode of progression. Make sev- eral sketches at intervals of 2-3 minutes showing the changes. 50. Amoeboid movement. Human leucocytes. Place a drop of fresh human blood upon a slide, cover quickly and seal with castor oil. Ten or fifteen minutes after the preparation was made examine it, find a leucocyte in amoeboid movement. Study it care- fully and make several sketches at intervals, as in 49. 51. Ingestion of foreign particles. Leucocytes of rat. A solution containing particles of lampblack ( 157) was injected into the abdominal cavity of a rat ; the following day the rat was killed and smears were made of the abdominal lymph. Stain for 10 minutes with hematoxylin ( 124). Mount in balsam. Study the preparation, noting that the particles of lampblack have been taken up or ingested by the leucocytes of the lymph. Consider, in the light of 49 and 50, how this ingestion took place. (43) Forms of leucocytes. Study the preparations 43, (a) and (), searching carefully for different kinds of leucocytes. The following types will probably be found : (a) small lymphocytes, 35 small leucocytes with a large rouud nucleus and scanty protoplasm, () large lymphocytes, large leucocytes with a large, clear nucleus and rather scanty protoplasm, (V) polymorphonudear neutrophiles, medium-sized leucocytes with irregular, horse-shoe shaped nucleus or several nuclei, the protoplasm granular, and (d) leucocytes with the nucleus irregular or in two or more parts, protoplasm containing large granules that stain intensely with eosin (or other acid staining principle), hence they are called "eosinoph ties" . (V) will be most abundant, (a) and (b) less abundant, (d) least abundant. It is generally believed that (#), (), (V), and (d) are stages in the growth of a leucocyte from youth to old age. Consult FIBRIN. 52. Upon a clean cover glass place a large drop of fresh human blood and cover it with another cover glass, taking care that the two covers are a little eccentric to each other. Place the two covers so prepared in a moist chamber (on the table) for 15 minutes or longer. Transfer the two covers to a slide, flood them with water and carefully separate them. Wash the film side of each very care- fully with water by means of a pipette to remove the red corpuscles. Stain the film side with eosin or erythrosin for 5 minutes, drain off the stain and dry without washing. When dry mount the better of the two preparations upon a shellac ring film side down. Observe the network of the fibrin coagulum adherent to the cover. BLOOD CRYSTALS. 53. Hemoglobin. Place upon a slide a small drop of mam- malian blood (rat, cat, or human) ; place beside it a small drop of a 10% aqueous solution of pyrogallic acid ; cover both drops with a cover-glass and seal carefully with castor oil. Label the slide so prepared with your name and place it upon the window sill. At the second period (i. e., after one or two days), examine it for crystals of hemoglobin. These will be found near the line where the drops of blood and pyrogallic acid met. Observe their color, shape and arrangement. 54. Oxy-hemoglobin. Blood of Nedurus. Demonstration. A plentiful supply of Nedurus blood was placed upon a slide, mixed 36 with a drop of 2% aq. sol. of chloral hydrate, and covered and sealed and allowed to stand for several days, when crystals of oxy- hemoglobin appeared. Note their shape and color. 55. Hemin. Place upon a slide a little powdered blood (from a dried blood clot or stain), and a few granules of common salt (so- dium chlorid), grinding well together. Add two or three drops of glacial acetic acid and cover. Heat gently two or three times until the acid just boils, adding a fresh drop of the acid after each boiling. Allow it to cool, remove the cover and permit the matter on the slide and cover to dry. Examine both, dry and mount in balsam the one showing best the crystals of hemin. If there are rough lumps of blood in the preparation, they may, without injuring the preparation, be removed by scraping before mounting. A large number of hemin crystals will probably be found. Their shape and color are characteristic and afford one of the best tests of blood. THE SPECTRA OF BLOOD. 56. A demonstration of the spectra of hemoglobin and oxy- hemoglobin will be given. Note the characteristic absorption bands of each and their points of occurrence in the spectrum and the dif- ferences between the spectra of hemoglobin and oxy-hemoglobin. It is advisable to read in some work on physics about the spectro- scope and spectrum analysis; also microscopical methods, 179- TOPICS FOR QUIZ gth Week. THE BI,OOD AND LYMPH CONTINUED. 9. L/eucocytes, forms and amoeboid movement ? 10. Ingestion of foreign particles ? 11. Fibrin, (a) of blood ; (b) of lymph ? 12. Blood crystals, (a) hemoglobin ; (b) hemin? 14. Medico-legal importance of the size of the red corpuscles? 15. The spectrum of blood ? 37 THE MUSCULAR TISSUES : THE MUSCULAR SYSTEM. LABORATORY WORK FOR THE-- --WEEK. References : \ Points for Quiz : Laboratory Report : Due: Go the delivery desk for (a) assigned [slide, 69 ; (b) a paraffin section, 61 ; (r) a collodion section, 67 ; (d) 58; 68, now in glycerin ; mount as directed. ( paraffin ; sections //. Stain with hematoxylin 15 minutes, picrofuchsin ^2 minute. Mount in balsam. Study this preparation for (a) the structure of a nerve trunk, noting its component funiculi or bundles, surrounded by connective tissue sheaths, the perineurium, and bound together by the epi- neurium, while within the funiculi, the individual nerve fibers with the endoneurium ; () the structure of myelinic nerve fibers in tran- section, showing the central axis-cylinder, surrounded by the mye- linic sheath (the myelin dissolved out). Find a place where a nerve nucleus is cut. Compare with the transection of a muscle, recognizing the analogy in the relations of the connective tissue. 43 75. Ulnar nerve. . Transaction. Hermann's fluid (23) ; paraffin ; sections yw. Stain for 2-3 minutes with picrofuchsin. This will be a supplementary preparation to 74, since here the myelin is not dissolved out and is stained black by the osmic acid. Compare the two preparations, recognizing in this one the features enumerated above. 76. Vagus and sympathic. Transection ; Her- mann's fluid ( 23) ; paraffin. Stain 2-3 minutes with picro- fuchsin. There will also be included a transection of the carotid artery. Note the myelinic nerve fibers, with blackened myelinic sheaths in both nerves and in the vagus, the variation in their caliber. In ad- dition, recognize if possible the transected amyelinic fibers by the absence of a myelinic sheath. GANGLIA. 77. Sympathetic ganglion. Cat. Section of the semilunar ganglion. Zenker's fluid ; paraffin ; sections /u. Stain i hour with hematoxylin, picrofuchsin 30 seconds. Observe well the characteristic appearance of ganglion cells in section ; their finer structure, indications of their processes ; recall the structure of the nerve cells in 70 and 71, realizing the extent of the dendrites. 78. Spinal ganglion. Section of the ganglion upon the dorsal root of a spinal nerve. Flemming's fluid ; paraffin ; sections yu. Stain with hematoxylin i hour, picrofuchsin 30 seconds. Observe the characteristic appearance of a ganglion, the (apparently) round ganglion cells with their capsules ; the nerve fibers ; the connective tissue within the ganglion and forming a covering sheath. Compare with 77. What is the real form of the nerve cells in the two kinds of ganglia and their relation to the fibers ? PERIPHERAL NERVE ENDINGS. 79. Motor end-plate. From the muscle. Demonstration. The preparation will show the nerve fiber termin- ating in the end-plate with its nuclei and the branching axis- cylinder. 44 80. Sensory. Free nerve endings in Demonstration. Stained by Golgi's rapid method. Illustrates the branching and free termination of the axis-cylin- der in an epithelium. 81. Pacinian corpuscles. Cat. From the mesentery. Demonstration. Note the core (inner bulb) in which the axis-cyl- inder of thenerve fiber terminates and the concentric connective tissue lamellae of the capsules. 82. Meissner's corpuscles. From the human finger. Dem- onstration. There is shown a corpuscle in one of the papillae of the skin. Observe the form and structural appearance. Examine 17 for transected corpuscles in the papillae of the skin. Personal preparations showing 81 and 82 will probably be obtained later. (149, 153). TOPICS FOR QUIZ nth Week. THE NERVOUS TISSUES ; THE PERIPHERAL NERVOUS SYSTEM. 1. Distinction (a) central, (b) peripheral nervous systems. 2. What are neurones, nerve cells, nerve fibers, neuroglia? 3. What is the relation of nerve fiber and nerve cell ? 4. Structure of a nerve cell considering : (a) Nucleus, (b) cell-body, (c) processes, neurites and dendrites. 5. What is the structure of the nerve cells in (a) a spinal ganglion, (b) a sympathetic ganglion? 6. Give two general types of nerve cells. 7. Nerve fibers, varieties. 8. Structure of a myelinic nerve fiber, axis cylinder, myelinic sheath, neurilemma, nerve nuclei or corpuscles? 9. What are nodes of Ranvier ? Internodes ? 10. Amyelinic nerve fibers ; structure compared with myelinic nerve fibers. n. Where found, at least two places? 12. How is the nerve impulse transmitted from one nerve cell to another? 45 THE BLOOD AND LYMPH VASCULAR SYSTEM. LABORATORY WORK FOR THE -1^1 -WEEK. %^ References :_ . . . V(l( ^ - _ vv< Points for Quiz : Laboratory Report : Due: v^ Obtain from the delivery desk (a) assigned slide, 85 ; () paraffin sections, 83, 84, 86, 92, 94 ; (c) collodion sections, 91, 95 ; (*0 93> already stained. ARTERIES. 83. Muscular artery. Cow. Transection of the uterine artery. Zenker's fluid ( 28) ; paraffin ; sections ' /*. Stain with hematoxylin 30 minutes, picrofuchsin 30 seconds. In this preparation identify the three coats forming the walls of arteries and veins, the intima, media and adventitia ; noting the relative thickness of each. In the intima, recognize the vascular epithelium, the internal elastic membrane, and the subepithelial connective tissue. In the media should be found, plain muscle cells, elastic bands and white connective tissue ; recall the staining reac- tions of picrofuchsin (used for this preparation) with muscle, white and elastic connective tissue. Are there longitudinal muscular bundles present ? Note the relative amounts of white and elastic tissue. In the adventitia, observe the relative amounts and arrange- ment of the white and yellow elastic fibers. Is an external elastic membrane present ? 84. Elastic artery. Transection of the carotid artery. C V J , ; paraffin ; sections \ p. Stain with hematoxylin minutes, picrofuchsin seconds. Compare this preparation with 83, recognizing the coats, their relative thickness and structure. Especially compare the media in the two preparations, noting the relative amounts of muscle and elastic tissue in each. 4 6 85. Aorta. Homo. Transection or longisection of the aorta. Assigned for study. Picric alcohol ; paraffin ; stained with hematoxylin and picrofuchsin. Study carefully in comparison with 83 and 84. Are internal or external elastic membranes present ? What is the relative thickness of the coats and the relative amounts of muscle and elastic tissue in the media ? Are vasa vasorum to be seen in the adventitia ? VEINS. 86. Femoral vein (and artery). Transection of the femoral artery and vein. ; paraffin ; sections fj. . Stain with hematoxylin minutes, picrofuchsin seconds. Identify the artery from your knowledge of its structure as gained from a study of 83, and compare with it the vein, observing the differences and resemblances in the following particulars : (a) the thickness of the wall, () its flaccidity, (c) the presence of blood in the lumen, (d} relative thickness of the coats, (e) relative amounts of white connective tissue, elastic tissue and muscle pres- ent. From a study of their structure, compare arteries and veins as to their elasticity and contractility. CAPILLARIES. 87. Vascular epithelium. Demonstration of the vascular epithelium as seen in capillaries and arterioles. Silvered ( 146). Note the shape of the cells as outlined by the blackened cell- cement ; their relative size in the capillaries. Remember that the vascular epithelium is the structure common to all parts of the vas- cular system. 88. Capillaries in striated muscle. Demonstration. Free- hand longitudinal section of muscle that was injected with carmine gelatin mass ( 127). Study carefully the capillary network and its relation to the muscle fibers. 89. Capillaries of plain muscle. Rabbit. Demonstration. The blood vessels of the small intestine were injected with carmine gelatin mass ( 127), the muscular coats stripped off, pieces of the longitudinal coat laid out flat. Balsam mount. 47 Note the distribution of the vessels in the muscular tissue, the shape of the capillary network, and compare it with 88. 90. Capillaries of an organ. Demonstration. Blood vessels injected with carmine gelatin mass ; hardened in alcohol ; collodion. Note the capillary network and the great vascularity. The vascularity of the different organs will be considered subsequently with the organs themselves. LYMPHATIC TISSUES. 91. Peyer's patch. Cat. Transection of the ileum. vom Rath's fluid ; collodion ; sections 10 JJL. Stain with hematoxylin 15 minutes, picrofuchsin 15 seconds. The section passes through a Peyer's patch. In the villi and mucosa will be found diffuse adenoid (lymphatic) tissue. Peyer's patch is an aggregation of lymphatic nodules, "dense" lymphatic tissue. Note carefully the general structural appearance of adenoid tissue, the character of the lymph cells and their nuclei. Can you recognize the lymphoid reticulum ? Does the preparation show the relation of the lymphatic tissue to the epithelium ? 92. Tonsil. Dog. Section of the tonsil. Zenker's fluid ; paraffin ; sections JJL. Stain the section with hematoxylin 30 minutes, picrofuchsin 30 seconds. Study the preparation, recognizing that it is composed of ade- noid tissue, consisting of nodules surrounded by diffuse adenoid tissue. Identify its structure with that of Peyer's patch. Note the relation of the lymphatic tissue to the overlying stratified epithelium of the oral cavity ; is the epithelium destroyed or filled with lymph cells ; are there any lymph cells upon the surface of the epithelium ? 93. Lymphatic gland. Cat. Transection. vom Rath's fluid ; stained in toto in paracarmine ; collodion ; sections yu. Mount directly. Study the general structural appearance of the gland. Identify the following regions or parts : (a) the cortex, medulla and the hilum ; () the lymphatic nodules (follicles) in the cortex ; (c) the capsule and the trabeculae ; can you find plain muscle in the cor- tex ? (d) blood vessels. Compare the lymphatic gland with 91 and 92. Can you recognize the lymph sinuses bordering the trabe- 4 8 culae ? Understand the relation of the afferent and efferent lymph vessels to the gland and the course of the lymph through it. 94. Thymus. Transection. ; paraffin ; sec- tions IJL. Stain with hematoxylin 15 minutes, picrofuchsin 30 seconds. Compare this preparation carefully with 93, identifying as be- fore, capsule, trabeculae, cortex and medulla, lymph follicles (nod- ules), and in addition, the lobular structure. Can you find corpus- cles of Hassall ; what is their significance? Study the structural appearance of the organ so that you may distinguish it from 93 and 95. Study the microscopic appearance of the thymus. 95. Spleen. Dog. Transection. vom Rath's ; collodion ; sections yw. Stain with hematoxylin 15 minutes, picrofuchsin 30 seconds. Examine the gross preparations of the spleen, observing care- fully the structural appearance of natural and cut surfaces. Study the microscopic preparation, recognizing (a) the capsule and the trabeculae ; is plain muscle present in these ? () the splenic pulp containing the Malpighian corpuscles (lymphatic follicles or nod- ules), and blood vessels. Examine the Malpighian corpuscles, not- ing their structure and the eccentric artery ; compare them with the lymphatic nodules in 93 and 94. ' For the supporting framework of the spleen, the capsule and trabeculae, examine the gross, mace- rated preparation. Study carefully the general structural appear- ance of the section of spleen, so that you may distinguish it from lymph gland or thymus. Again consult the gross section of spleen, identifying capsule and trabeculae, Malpighian corpuscles and splenic pulp. Understand the relation of the blood vessels to the Malpighian corpuscles and to the pulp. 49 THE DIGESTIVE SYSTEM. LABORATORY WORK FOR THE-^J? WEEK. ''"* ' ^ d References: L^ * -^ - Points for Quiz : Laboratory Report : \IVx>.-\ Go to the delivery desk for (a) assigned slide 100, () paraffin sections 103, 108, (V) collodion sections 96, 97, 101, 104, (d) 98, 99, 102, 105, 1 06, already stained and in clearer. THE ORAL CAVITY. (10). Epithelium, glands. Section of the soft palate, cat. Assigned for study (10). The oral side of the soft palate affords a demonstration of the stratified epithelium of the oral cavity ; study its appearance. In the substance of the soft palate are numerous mucous glands, which open upon the mucosa of the oral cavity. Serous glands are not present. Study their appearance and struct- ure ; find if possible a duct leading to the surface. These would belong to the group of palatine glands ; others would be lingual, buccal or labial, according to their location. 96. Developing tooth. Early stage. Transection through the cephalic part of the head of an embryo long. ; collodion ; sections yw. Stain with hematoxylin and eosin. Within the section will appear the two nasal cavities and below them the oral cavity, with the tongue projecting up from the floor of the mouth. On each side, opposite the developing lower and upper jaws, will be seen the dental ridge projecting from the epi- thelium of the oral cavity. One of the four sections of dental ridge will probably pass through a developing tooth, showing the cap- like enamel organ covering the papilla which forms the dentine and pulp of the adult tooth. 50 97. Developing tooth ; later stage. Dog. Transection through the lower jaw of a new born puppy. Picric alcohol ; decal- cified ( 131) ; collodion; sections v /*. Stain with hematoxy- lin and picrofuchsin. Be sure your section passes through a developing tooth. In this more mature tooth there should be recognized (a) the enamel organ with its three layers, () the enamel, (c} the dentine lined by the odontoblasts, and finally (d), the dental pulp, containing blood vessels. 98. Tongue. Rabbit. Transection. Miiller's fluid ; stained in toto ( 74 c) ; collodion ; sections . u J*- Study the preparation, noting the surface epithelium with low papillae, in the body of the tongue, the mesal septum and the bun- dles of muscle in general running vertically, longitudinally and transversely ; also nerves and blood vessels. ESOPHAGUS. 99. Esophagus. Dog. Transection. c sections jj.. Stained in toto. ^P &r< In this preparation, as well as in the succeeding regions of the digestive tract, identify the four coats mucosa, submucosa, (inner and outer) muscular and serous coats. In the mucosa and sub- mucosa observe the character of the lining epithelium, the mucous glands ; find if possible a duct opening upon the lining epithelium ; the muscularis mucosa ; the character and direction of its fibers. In the muscular coats determine the character and direction of the muscle fibers. What differences are there in different animals in the muscular coats of the esophagus ? 100. Esophagus. Homo or sheep. Assigned for study. Transection. Compare with 99, recognizing the coats, the epithelium, mus- cularis mucosa, character and relation of the muscular fibers in the muscular coat. Are glands present ? STOMACH. 101. Stomach. Dog. Vertical section of the wall, cardiac end near the esophagus. X o>rv NfL^vv > collodion; section '.\ /*. Stain the sections with hematoxylin and eosin. Identify as before the four coats, noting the character of the muscular tissue in the muscular coat, the number of layers recog- nizable in it and in the muscularis mucosae. In the submucosa observe the presence of blood vessels, both arteries and veins. In the mucosa are the tubular gastric glands opening, two or three to- gether, into depressions of the surface epithelium. In these gland tubules recognize the two kinds of cells, chief and border cells ; re- call the function of each. Compare the surface epithelium of the stomach with that of the esophagus. If your preparation does not show well the gland tubules and their cells ask the instructor to show you one that does. 102. Stomach. Dog. Vertical section through the wall, pyloric end. ^U^J^SL^ ', stained in toto in paracarmine ; collodion ; sections . f j*. Compare this preparation with 101, recognizing the coats and their structure and noting differences in the muscular coats, espe- cially the increase in the thickness of the inner circular coat, and in the glands of the mucosa. Observe that the pyloric glands differ from the peptic (oxyntic) glands shown in 101 in (a) the absence of parietal cells, () the longer duct into which several tubules open. Recall the relative extent of the regions occupied by the two kinds of glands. 103. Zymogen granules. Calf. Vertical section through the mucosa of the cephalic end of the stomach (fourth stomach or abomasum). i% osmic acid; paraffin; sections 'If ft. No staining or Hcl. carmine ( 87). In the deeper part of the mucosa will be seen the gastric tubules, cut across in various directions and lengthwise. The ends of the cells forming the tubules the chief cells toward the lumen of the tubule will be seen crowded with granules of a yellowish brown color. These granules are the precursors of the ferment, or pepsinogen granules. Find a border cell and note that no such granules are contained in it. SMALL INTESTINE. 104. Duodenum. Cat. Transection. Zenker's fluid ; col- lodion ; sections ' , ^ yu. Stain with hematoxylin and picro- fuchsin. 52 In studying this preparation, observe that the coats recogniz- able in the esophagus and stomach, also form the intestine. Deter- mine the character and direction of the fibers in the two layers of the muscular coat. Is a muscularis mucosa recognizable ? In the mu- cosa, note the tubular crypts of Lieberkiihn extending down into the mucosa, and the villi, tongue-like elevations of the mucosa. Study the character of the epithelium of the intestine, noting the striated border of the columnar cells and the goblet cells. Compare the epithe- lium covering the villi with that of the crypts of Lieberkiihn. In the submucosa are the glands of Brunner with here and there a duct leading through the mucosa. What relation do they have to the pyloric glands ? Study carefully the structure of a villus ; (a) its covering epithelium, () the central core of diffuse adenoid tissue, (r) a strand of plain muscle ; and in comparison, study 105, 106, and 6. 105. Duodenum. Rabbit. Injected with carmine gelatin mass ( 127) ; now in clearer. Mount a piece of the mucosa, villi up. The villi are rather short and leaf-like, flattened. Observe how the arteriole passes up to near the summit of the villus, there to break up into a cascade of capillaries that unite on the opposite side to form one or two venules. 1 06. Ileum. Rabbit. Transection. Injected with carmine in mass ( 127); alcohols; collodion; now in clearer; sec- tions \vr\> yu. In the ileum the villi are longer and more filiform. Note the relations of the blood vessels in the villi and compare with 105. 107. Ileum. Rabbit. Demonstration. Blood vessels in- jected with carmine gelatin mass (red) ; the central lacteal is filled with Berlin blue gelatin mass (blue). Observe the position of the - lacteal (lymph vessel) in the villus, its extent and size in compari- son with the blood capillaries. By means of 6, 104, 106 and 107 a fairly complete idea may be formed of the structure of a villus ; from your physiology recall the part played by the epithelium in absorption and what food stuffs pass into the lacteal and what are taken up by the blood vessels. 1 08. Fat absorption. Intestine of the frog. Flemming's >j fluid ( 24) ; paraffin. The frog was fed with bacon 24 hours be- 5.3 fore it was killed, and the fat is being absorbed by the epithelial cells ; the globules in the epithelium are blackened by the osmic acid of the Flemming's fluid. If desired the preparation may be stained with safranin ( 89, 99). The intestine of the frog has no villi, the elevations of the mucosa shown in the transection are folds cut across. The epi- thelial cells covering the villi in mammals, however, have the same function in the absorption of fat (6). (91). Ileum. Cat. Re-examine this preparation in the light of the knowledge gained of the structure of the intestine, noting the lymph follicles and their relation to the intestinal epithelium ; the occurrence of lymph cells in and upon the epithelium. Identify also the coats and structures mentioned under 104, save, of course, Brunner's glands, which are limited to the cephalic part of the duodenum. TOPICS FOR QUIZ isth Week. THE DIGESTIVE SYSTEM. 1. Constituents of the digestive system? 2. Digestive organs in or communicating with the oral cavity ? 3. Structure of the wall of the oral cavity ? 4. Structure of the tongue ? 5. Structure of a tooth ? Development of a tooth. 6. Structure of the .esophagus (a) different regions in man, (b) in the domestic animals. 7. Cardiac stomach. Pyloric stomach. Stomach of man, horse, rat, pig, ox and sheep. 8. Small intestine. Glands there found? How distinguish trans-and longi-sections ? 9. Injected small intestine ; lymphatics and blood vessels. 10. Fat absorption and zymogen. 54 THE DIGESTIVE SYSTEM. LABORATORY WORK FOR THE--'-^ WEEK. 1 1. - '<-.*) References : C\\ . * ^- i v<* - i o ^ Points for Quiz : Laboratory Report : Due: 'JOb. 6^ Go to the delivery desk for (a) assigned slides, no, 114, 118 ; (b) paraffin sections, 112, 113, 115 ; (c) collodion sections, 109, 116, 117 ; (d) sections already stained, in. LARGE INTESTINE. 109. Colon. (L^A- Transection. Picric alcohol ; collodion ; sections . > yw. Stain with hematoxylin and picrofuchsin. Compare with the sections of small intestine, noting identity of structure of the coats and their general structure and relations, dif- ferences in the absence of villi, greater number of c crypts of Lieber- kiihn, increase in the relative number of goblet cells. no. Rectum. Kitten. Transection. Assigned for study. ; collodion ; sections * ( .ju. Stained with Compare this preparation with 109, observing especially the increased thickness of the muscular coats and the muscularis mu- cosae, and the looser connection of the mucosa with the muscular coat. in. Caecum. Rabbit. Blood vessels injected with carmine gelatin mass ; now in clearer ; mount in balsam, rough side (mu- cosa) up. Note the capillary net work and its denser arrangement in small areas, rudimentary villi. SALIVARY GLANDS. 112. Mucous type. Submaxillary gland. Cat. Mercuric chlorid -f- 5% glacial acetic acid ( 26); paraffin ; section li /u. Stain with hematoxylin and picrofuchsin. Study the section carefully, noting (a) the general structural appearance of the gland, its lobulation, () the component acini and 55 ducts, (V) the blood vessels and nerves (and ganglion), together with the connective tissue. Study carefully the character of the cells composing the secret- ing acini, noting (a) the structure and staining reaction of the cell- body, (b) the position and structure of the nucleus. Can you find a place where duct and secreting portion are continuous ? Recog- nize the presence of demilunes and note the character of the cells. 113. Serous type. Section of the parotid gland. Cat. Mer- curic chlorid + 5% glacial acetic acid ( 26) ; paraffin ; sections 1^ //. Stain with hematoxylin and picrofuchsin. Study the section for the general structural appearance and lobulation, the secreting acini and ducts, blood vessels, nerves and connective tissue. Study the acini carefully, comparing them with the secreting acini in 1 1 2 as to the shape and size of the cells, the position and structure of the nucleus, and the structure and appear- ance of the cell bodies. What are the differences ? Can you dis- tinguish mucous and serous glands ? Compare the glands you found in the esophagus and soft palate. PANCREAS. 114. Pancreas. Horse. Assigned for study. Picro-forma- lin ; paraffin ; hematoxylin and eosin. Examine the gross preparations for general appearance, lobula- tion, compactness, color, etc. In studying the section analyze its structure in the same man- ner as 112 and 113, observing carefully its general structural ap- pearance, the ducts and secreting acini (tubules), the structure and shape of the cells forming these, the position of the nucleus. Are areas of L,angerhans present in the section ? Compare the section with 113 as to (a) compactness, ($) shape of the secreting acini, (<:) the lumen of the acini. Can you distinguish them ? 115. Zymogen granules. Pancreas. Calf. i% osmic acid (22); paraffin; sections I. /^. No staining. In the ends of the cells forming the secreting acini (tubules) observe the presence of light brown granules trypsinogen gran- ules, precursor of the ferment secreted by the gland. Compare 103. 116. Injected pancreas. Rabbit. Blood vessels injected with carmine gelatin mass ; alcohols ; collodion ; stain lightly with hematoxylin. This preparation illustrates the vascularity of the 56 gland and the relation of the capillary network to the tubules of the acini of the gland. LIVER. 117. Liver. Pig. Picric alcohol ; collodion; sections yu. Stain with hematoxylin and picrofuchsin. Study thoroughly the preparations illustrating the gross anat- omy of the liver ; () position, () aspects, (V) lobes, (d) the longi- tudinal and transverse fissures, (>) the appearance of the liver sub- stance as seen in section and the natural surface (covered by the con- nective tissue capsule), (/) the indistinct lobulation. In studying the section of liver note the lobules separated by connective tissue septa. Within the lobules recognize the intra- lobular vein and the arrangement of the liver cells. In the inter- lobular connective tissue will be found inter-lobular veins, inter- lobular arteries, small bile ducts. Can you find intra-lobular blood capillaries passing off from the inter-lobular veins or entering the intra-lobular veins ? Be sure you understand the course of the blood and the origin and relations of the different vessels. What is the relation of the liver cells to the blood capillaries and the bile capillaries ? Note well the structural appearance of the liver so that you can distinguish it from other glands. The liver of the pig has a large amount of connective tissue between the lobules, sep- arating them completely ; hence their distinctness. 118. Liver. Horse or Homo. Assigned for study. Potas- sium dichromate or Krlicki's fluid ; paraffin ; stained with hema- toxylin and picrofuchsin. Study carefully, identifying the parts and structures already recognized in 117; compare with that preparation, noting the in- complete isolation of the lobules, less inter-lobular connective tissue, etc. Again consult the gross preparations, examining them in the light of your present knowledge of the structure of the liver. 119. Bile capillaries. Liver of ox. Demonstration. The bile capillaries are gorged with bile, caused by physiological stasis of bile in Texas fever. Note the fineness of the capillary network and the relation of the capillaries to the cells. 120. Glycogen. Demonstration. Liver hardened in abso- lute alcohol ; paraffin ; sections stained with a solution of iodin ; mounted in iodin-gum arabic. The glycogen is stained a yellowish brown ; note its accumulation in one end of the cells (generally 57 THE RESPIRATORY SYSTEM. LABORATORY WORK FOR References : Points for Quiz : Laboratory Report .-OJUT> cj-v^Ju*. Q,^ t \ Dn _ Go to the delivery desk for (a) assigned slide, 121 ; (b) paraf- fin section, 128 ; (c) colpdion sections, 122, 123 ; (d) sections ready to mount, 124, 126 ; (e) prepare 125. (10) Soft palate. Cat. Assigned for study. The nasal side of the soft palate will afford a demonstration of the ciliated epi- thelium of the nasal fossae. This will be spoken of in your text book as a stratified ciliated epithelium. Compare it with the epithe- lium in the trachea. LARYNX. 121. Larynx. ~. Assigned for study. L/ongisec- tion. ; collodion ; sections /*. Hematoxylin and picrofuchsin. Understand what structures are shown in the section. Of the cartilages shown in the section, the first is the hyoid, the sec- ond the thyroid, followed by the cricoid and the rings of the trachea. Identify the true and false vocal cords and the ventricle of the larynx. Note the character of the epithelium in the different regions, i. e., in the ventricle and upon the false and true vocal cords. Are glands present ? Where are they located ? Note also the structure of the mucous membrane and its variation in the different regions. TRACHEA. 122. Trachea. Cat (young). Transection. Picric alcohol ; collodion ; sections "i ij p. Stain with hematoxylin and picro- fuchsin. Identify the three coats of the trachea, (/) the mucous coat, (2) the submucous coat, (j) the fibrous coat with its incomplete 58 ring of cartilage, studying carefully the structure of each coat. Compare the lining epithelium with that in the ventricle of the larynx and upon the soft palate. Note especially the overlapping of the ends of the cartilaginous rings and the character of the epi- thelium in the fold of the mucosa caused thereby, and the relation of the muscle to the cartilage. In the last respect, compare with the relations in man and sheep, as demonstrated to you. 123. Trachea. Cat (old). Transection. Picric alcohol; collodion ; sections r . //. Stain with hematoxylin and picro- fuchsin. Recognize in this preparation exactly the same structures ident- ified in 122 and compare the two preparations, especially as to the epithelium lining the fold where the cartilages overlap. This will illustrate how the original character of an epithelium is altered by changed conditions. LUNG. 124. Lung. Transection (or longisection) of a lobe (in part). Picric alcohol ; collodion ; sections yu. Stained m to to. y u ^ Examine the gross preparations of lung, showing the lobes, and the appearance of lung tissue seen in surface view and in sec- tion. Is a further division of lobes into lobules recognizable ? The section will illustrate the structure of normal lung. The following features are to be noted : () the general struc- tural appearance of lung tissue, () the structure of a bronchus or bronchiole, (e) the infundibula with air sacs opening into them, and (oT), if possible, the transition of terminal bronchus or alveolar duct to infundibulum. 125. Dried lung. Cat. The fresh lung was inflated and dried. Prepare a slide with a rubber cell ( 103, b), and mount within the cell two sections of lung cut free-hand, a surface section and a deep section, mounting the surface section pleural side up. Cover and seal ( 103, ). This preparation will illustrate well the infundibula and the compartments in their walls, the air sacs. It is possible that there may be a good natural injection of the blood capillaries (filled with blood, yellow) ; if so, note the size of the capillaries and the dense- ness of the net-work. 59 126. Lung. . Injected with Berlin blue gelatin mass ( 128) ; alcohols ; collodion ; sections /^. No staining. Search the preparation to find places showing well the capillary net- work in the walls of the air sacs, and compare it with the natural injection, if present, in 125. 127. Respiratory epithelium. Cat. Demonstration. Free- hand section of silvered lung. The blackened cell cement outlines the cells lining the air sacs, of which recognize the two kinds, large flat cells with irregular outlines and small granular cells. Understand the significance of the two kinds. THYROID. 128. Thyroid. 2><^ ^ cr^ Chrome-oxalic ; paraffin ; sections S J ^^^^.This preparation is to be compared with 135 for (a) the epithelium, its differences in a distended and contracted condition, and () the lay- ers of the muscular coat. URETHRA. 137. Urethra female (and vagina). Cj^V Transection. < V.^_ AJ1 jj^ A/> ^ collodion ; sections 1 u /^- Stain with hematoxylin and picrofuchsin. Study the preparation carefully, noting the coats, the character of the lining epithelium, the layers composing the muscular coat, and the direction of the fibers ; compare with the condition in the ureter and bladder. Are glands present ? Note the blood .vessels of the mucosa. 138. Urethra male. %-v><^ Transection. "h**As>JW* collodion ; sections 'L^ j^. Stain with hematoxylin and picro- fuchsin. Compare this preparation with 137 and note differences. What is the character of the epithelium and therefore what part of the urethra is it ? Are the muscular layers well denned ? TOPICS FOR QUIZ i6th Week. THE URINARY ORGANS. r. What are the urinary organs? 2. General structure of a kidney as seen in longisection ? 3. Medullary rays. 4. Urinary tubule, entire extent with names of parts. 5. Vascular system of the kidney. 6. Malpighian bodies or corpuscles. Bowman's capsule, glomerulus. 7. Epithelium of the urinary tubules, pelvis of the kidney, ureter, bladder and urethra. 8. Structure of the urocyst or urinary bladder (a) empty and (b) distended. 9. Structure of the ureter. 63 GENITAL ORGANS : MALE. LABORATORY WORK FOR THE WEEK. References : Points for Quiz : Laboratory Report : Due : Obtain from the delivery desk, (a) paraffin sections, 141, 143 ; () collodion sections, 139,1 40, 144, (c) 142, cover-glass preparation. TESTICLE. 139. Testis, . Longisection ; collodion; sections ju. Stain with hematoxylin and picrofuchsin. The following parts should be recognized, (a) the testis, and () the epididymis ; in the testis, the three coats, the lobules of the testis, and, composing the lobules, the seminiferous tubules, straight tubules and rete testis ; in the epididymis, note its relation (as a mass) to the testis, the structure of the tubules, the character of the epithelium. Does the section include the globus major or minor or both ? Is the vas epididymis sectioned ? Can you determine the vas deferens ? In addition, note the blood vessels. 140. Testis, . Transection of the testis in the tunica vaginalis. ; collodion ; sections //. Stain with hematoxylin and eosin. Identify, as far as possible, the parts recognized in 139, and in addition, the tunica vaginalis with its two layers, and the mediasti- num. Note the position occupied by the blood vessels. What is the cavity between the two layers of the tunica vaginalis and its lining epithelium ? Identify the vas defefens. 141. Spermatogenesis. Rat. Section of a part of the testis. ; paraffin ; sections //. Stain with hema- toxylin and acid fuchsin ( l /i% solution). Examine different tubules and find if possible five different stages in the formation of the spermatozoa, remembering that the process 6 4 occurs from the outside toward the lumen of the seminiferous tubules, so that in the innermost layer would be found maturing (or mature) spermatozoa, in the outermost layer spermatogonia (parietal cells), and between spermatocytes (mother cells) and spermatids (daughter cells), which metamorphose into the sperma- tozoa. Find also the supporting cells (Sertoli cells) and observe their relation to the ripening spermatozoa. 142. Spermatozoa. Cat. Dried on the cover glass. No staining. Mount on a shellac ring ( 103, a). Recognize the three parts head, middle-piece, and tail, noting the shape and relative length of each ; compare with the sperma- tozoa found in the tubules and ducts (vasa) in 139, 141, noting the staining reactions of the parts. VAS DKFKRENS. 143. Vas deferens. . ; paraffin ; sec- tions yu. Stain with hematoxylin and Determine the number oi the coats and their structure ; the course of the fibers in the muscular layers and the character of the epithelium, and compare it with the epithelium found in the tubules of the epididymis. PROSTATE GLAND. 144. Prostate. . Transection of the gland and ure- thra. ; collodion ; sections yu. Stain with hema- toxylin and eosin. Understand the relation of the gland, to the bladder and the ure- thra. In the section, observe the relation of the gland mass to the urethra. Is a division into lobes indicated ? Note the capsule and its structure, the acini and ducts forming the gland mass, together with connective tissue and plain muscle. Does the section include the prostatic sinus or the ejaculatory ducts (vasa deferentia) ? 65 THE GENITAL ORGANS : FEMALE. LABORATORY WORK FOR THE WEEK. References : Points for Quiz : Laboratory Report : Due : Obtain from the delivery desk, (a) paraffin section, 147 ; (b) collodion sections, 145, 146, 148. OVARY. 145. Ovary. . ; collodion ; sections /*. Stain with hematoxylin and eosin. Examine the gross preparations for the shape, superficial ap- pearance of the ovary and its relations to the ligaments and to the Fallopian tube. In the section, note the regions, roughly defined, the cortex and medulla and the structural appearance of each. As composing the ovary, note (a) the covering tunica albuginea, (b) the ovarian stroma containing (V) Graafian follicles at various stages of maturity, together with (d) the blood vessels and nerves. Points to which attention is called are (z) the peculiar structure of the stroma of the ovary, (2} the large size of the veins, and (j) the structure of the mature Graafian follicle. In studying the Graafian follicle, recognize ( T. , \ vu , \ ,b Due : Obtain from the delivery desk (a) assigned slides 152 and 154, () paraffin sections 150, 152, 157, 158 and 159, (c} collodion sec- tions 149, 151, 156, (d} 153 will be sectioned in your presence dur- ing the week ; read 61-63. THE SKIN. 149. Skin of (palm or heel.) Homo. Vertical section . collodion ; sections o ju. Stain with hematoxylin and picrofuchsin. t>'-oo^c_ Identify epidermis and corium ; in the former recognize the layers and study their structure ; are the layers of the corium dis- tinguishable ? Search the preparation carefully for sweat glands ; corpuscles of Meissner may be present in the papillae of the corium. Are inter-cellular bridges shown in the stratum Malpighii ? (17). Skin of palm or heel. Homo. Surface section (slightly oblique). Preparation already made (17). In this prep- aration note (0) the inter-cellular bridges of the cells of the Mal- pighian layer and the relation of the deepest cells next the corium to the connective tissue, () the transected papillae of the corium. 150. Skin, general body surface. Homo. Vertical section. ; paraffin ; sections r~\ //. Stain with hematoxylin and picrofuchsin. Study this preparation carefully in comparison with 149, identifying the epidermis and corium, the layers compos- ing them, and the relative development of each. Does the section show the presence of hairs ? Sweat glands ? 151. Lip. Homo. Transection. SH^AOAA!/* ; collodion; sec- tions XO JA. Stain with hematoxylin and picrofuchsin. 68 This preparation affords a demonstration of the transition of the epidermis of the skin to the epithelium of the oral mucosa. Note well the character of the epithelium upon the two sides of the lip, and the presence of hairs on the dermal side. Study the section also for the structure of the skin, the layers, glands, etc. As illus- trating the structure of the lip, note the striated muscle, connective tissue, nerves and blood vessels and their arrangement. 152. Lip. Horse or calf. Assigned for study. Transection. ; collodion ; sections //. Stained with Compare this preparation with 151, recognizing the features enume- rated above. Note the greater development of the hair of the skin. NAII,. 153. Finger. Child. Transection through the tip. frozen section ( 61). Now in water. Stain with hydrochloric acid carmine minutes ( 87), and counter-stain slightly with picric alcohol. This preparation illustrates : (#) the structure of the nail, () the skin (of the finger). Note well the structure of the nail and the nail-bed with its longitudinal corrugations cut across ; the relation of nail and nail-bed to the skin of the finger. In addition there are illustrated the structure of Pacinian corpuscles and sweat glands. HAIR. 154. Hair. Homo. Assigned for study. Section of scalp, cutting the hairs at right angles. ; paraffin ; stained with hematoxylin, picrofuchsin, and methyl green. Since the section cuts the hairs at right angles and the hairs are oblique to the surface, there appear transections of hairs at different levels. Study carefully the sections of hair, identifying the layers composing the hair and follicle at different levels, especially compar- ing sections through the mouth of the follicle, through the middle of the root, and through the hair bulb. Study the structure of the sebaceous glands, their relation to the hair follicle and the point at which they open. 155. Hair. Homo. Demonstration. Section of scalp, cut longitudinally with the hair showing the entire length. Note care- fully the root of the hair, the follicle and its relation to the epider- mis, the sebaceous glands and the hair muscle. 6 9 156. Hair muscle. (Arrector pili). Cat. Section of the skin from the dorsal side of the tail, cutting the hairs longitudinally. Picric alcohol ; collodion ; sections x o j*. Stain with hematoxy- lin and picrofuchsin. This preparation will illustrate especially the hair muscles and their relation to the hair follicles. Study, noting their relation to the follicles and sebaceous glands ; their large size in this prepara- tion. Understand their action in raising the hair. Compare 154, identifying in it the hair muscle. MAMMARY GLAND. 157. Mammary gland. CQW. In lactation. paraffin ; sections / O /^. Stain with hematoxylin and eosin. Observe carefully the general structural appearance of the gland, noting the lobulation and the secreting acini. Study care- fully the epithelium forming the acini, noting the character of the cells and their appearance and indications of functional activity. Note also the secretion (if present) in the lumen of the acinus. Com- pare with 158 and 159. 158. Mammary gland. Cow. In lactation. i%osmicacid (22); paraffin; sections \o /* No staining. This preparation supplements 157. The osmic acid blackens the fat globules which in 157 were dissolved out. Observe the num- ber and size of the globules in the epithelial cells of the acini and in the secretion in the lumen of the acinus. 159. Mammary gland. Cow. Not in lactation vi ^ . T_^L, ^V^*^ -t^) ^ b ou Points for Quiz: Laboratory Report : Go to the delivery desk for (#) paraffin section 160, (b) prepar- ations already stained 161, 162, 164-168, (V) section 163 will be given you some time during the first period. MYEL (SPINAL CORD). 1 60. Cervical my el. Transection. votn Rath's fluid ; paraffin ; sections JA.. Stain with hematoxylin 2-3 hours, picric alcohol 30 seconds or longer. Gross anatomy : Examine the preparations of myel, noting the three regions cervical, thoracic and lumbar ; the end of the seg- ment of myel, observing the halves separated by the dorsal septum and the ventral fissure, the central gray matter, cinerea, and the superficial, ectal alba (white matter) ; the spinal nerves formed by the union of a dorsal and a ventral root, the former with an enlarge- ment the ganglion. In the section recognize (a*) the lateral halves, () the ventral fissure, (c) the dorsal septum, (d} the central cavity myelocoele with its lining epithelium endyma. In the cinerea note (/) the rough division into lateral masses connected by a commissure surrounding the myelocoele, (2) the dorsal and ventral cornua. In studying the structure of the cinerea note (a) the general structural appearance of the substantia spongiosa and substantia gelatinosa, () the size and character of the nerve cells in the ven- tral cornua, in the dorsal cornua, and intermediate region. Con- sider the significance of the large cells of the ventral cornu. In the alba, distinguish the dorsal columns and the roughly defined lateral and ventral columns ; remember the physiological tracts, indistin- guishable in this preparation, that compose the alba. Are the points of exit of the dorsal and ventral roots of the spinal nerve shown in the section ? if not, compare with 161 and ,162. 161. Thoracic myel. . Transection. ; stained in toto in ; collodion ; sections JJL. Compare this preparation with the above, 160, noting (a) the general shape of the myel in the thoracic region, () the size and shape of the cornua of the cinerea, (c) the number and size of the nerve cells in the parts of the cinerea, especially the cells in Clarke's column. Is a lateral horn well defined ? Are issuing fibers of the dorsal or ventral roots shown in the section ? 162. Lumbar myel, Transection. ; col- lodion ; stained in toto in ; collodion ; sections JJL. Compare with 160 and 161, noting differences in (#) the gen- eral shape of the myel, (b) the relative extent of the cinerea and the shape of the cornua, (^ the relative number and size of the nerve cells in the regions of the cinerea, (d) the obliquely coursing root bundles of the lumbar nerves, constituting the cauda equina. 163. Myel. Transection. Golgi's rapid method ( 141) ; collodion ; sections ju. No further treatment ; now in clearer. Mount in balsam- without a cover-glass. In this preparation there will be outlined by a black precipitate, one or more large nerve cells and their dendrites, so that their ex- tent and branching may be seen better than in 160, 161 or 162, with which comparison is to be made. In addition, there will probably be stained, neuroglia cells, and many neurites running in the cinerea. THE (MEDUIXA) OBLONGATA. Gross anatomy. In connection with the study of the follow- ing sections, make a careful examination of the preparations of the brain. Ascertain the location of the oblongata in relation to the myel and the remainder of the brain. Upon the ventral aspect recognize (/) the pyramids with an indication of their decussation, ( the olives (olivary bodies), (j) the pons, and O) the cranial nerves springing from this portion, the Vth to the Xllth. Upon the dorsal aspect with the membranous roof (metatela) removed, there should be noted, (a), the nuclei (enlargements) of the gracile and cuneate funiculi, () the restiforra body. Note the relation of the restiform body and the pons to the peduncles of the cerebellum. In examining the following sections, whenever one of these struct- ures is mentioned, examine the gross preparations and ascertain the plane of the section. 164. Decussation of the pyramids. Cat. Transection of the oblongata at the level of the decussation of the pyramids. 3% and 5,% potassium dichromate ; collodion ; Weigert's hema- toxylin stain ( 137) ; sections u. Now in 95% alcohol. Clear and mount in balsam (alkaline). Recall the positions in the myel occupied by the following tracts : (/) the crossed and direct pyramidal tracts, (2) the direct cerebellar, (j) the funiculi gracilis and cuneatus composing the dorsal column. In this section will be seen the decussation of the fibers that will constitute the crossed pyramidal tracts in the myel from their position on the ventral side (pyramids) to the lateral column of the myel. Recognize the dorsal and ventral columns, the substantia gelatinosa Rolandi, the nucleus gracilis. Laterad of the cap of gelatinosa Rolandi is the ascending (sensory) root of the Vth cranial nerve ; ventrad of this, the fibers of the direct cerebellar tract. 165. Decussation of the lemniscus. Transection of the oblongata, through the nuclei gracilis and cuneatus and the pyramids (caudal part). Weigert's hematoxylin method (137); now in 95% alcohol ; sections yw. Clear and mount in alkaline balsam. v At this level, slightly cephalad of 153, the decussation of the pyramidal tracts has not yet begun, the gracile nucleus has en- larged and the cuneate nucleus has appeared. Recognize these features and, in addition, the decussation of fibers from these nuclei to form the lemniscus. Note the pyramids, the diminished funi- culus gracilis (what has become of the fibers?); the dorsal cornu and substantia gelatinosa Rolandi ; can you identify the ventral cornu ? Note increased distinctness of the ascending root of the Vth and the direct cerebellar tract. The nuclei of the Xllth and Xth nerves and the fibers of the Xllth passing to their exit may also be seen. The section may be slightly cephalad or caudad of the level desired for this preparation ; which is it ? 73 166. Olives. Transection of the oblongata through the olives. Weigert's hematoxylin method. Sections yw. Now in alcohol. Clear and mount in alkaline balsam. Identify in this section the tracts already recognized : O) the pyramids, () the lemniscus, (i/i x,^ ^^y^.U Points for Quiz : Laboratory Report : Due : Go to the delivery desk for (a) assigned slide 177 ; (b) par- affin sections 172, 176; (c) sections already stained, 169, 170, 174, I 75 > (*O I 7 I an< i J 73 will De given you during the first period. CEREBELLUM. Gross Preparations. Examine the gross preparations, noting (a) the parts of the cerebellum the vermis, lateral hemispheres, and the flocculus, comparing man and the different animals shown ; () the lodes of the cerebellum and the folia, their size and direction ; () the three peduncles of the cerebellum by which it is connected with the rest of the brain and myel. 169. Cerebellum. Cat. Sagittal section of the vermis ; vom Rath's fluid ; stained in toto in' paracarmine ; collodion ; sec- tions JJL. Note the deep division into lobes and these into folia. As con- stituting the cerebellum, recognize (a) the central core of alba (fibers) extending into the lobes and folia, (b) the covering cinerea cortex. Observe the layers of the cortex, the grouping of the cells of the granular layer, the size of the Purkinje cells, the rela- tively small number of cells in the molecular layer. Remember the real extent of the dendrites of the Purkinje cells, the destination of the neurites of the Purkinje cells and granules, and compare with 171. 170. Cerebellum. . Transection (in part) ; Wei- gert's hematoxylin method ; collodion ; sections /^ ; now in 95% alcohol. Clear and mount in alkaline balsam. This preparation will illustrate the ento-cinerea of the cerebel- lum the nucleus dentatus and the nuclei of the roof, as well as the 75 commissural fibers of the cerebellum. Note the shape of the dentate nucleus and compare with the nucleus of the olive (166). Can you identify the fibers of the pre- and post-peduncles of the cerebellum ? Compare with 169 for the general structure of the cerebellum. 171. Cerebellum. Cat (young). Golgi's rapid method (141) ; collodion ; sections //, cutting the folia transversely. This preparation will afford a demonstration of the extent of the dendrites of the Purkinje cells, the basket cells of the molecular lay^er, and possibly the dendrites and neurites of the granules of the granular layer, neuroglia cells, and nerve fibers. CEREBRUM. Gross anatomy. Consult the preparations, studying them carefully so that you may have clearly in mind (a) the relative size and position of the cerebrum, () that it consists of two halves con- nected with each other (principally) by a large commissure, the cal- losum, (c) the incision of its surface by deep depressions, fissures, dividing it into gyres, {d} the aspects of the cerebrum- dorsal, frontal, occipital, mesal, temporal. The cinerea of the cerebrum comprises (/) the superficial cortex and the hippocamp, (2) masses deeply situated, of which the most important are the caudatum, and lenticula, with which may be considered the closely associated thala- mus and subthalamus. Examine the series of sections through the cerebrum, noting the cortex and the location of these masses of cine- rea, their relation to each other and to the cavities of the diencephal and cerebrum. 172. Cerebral cortex. Cat. Transection of a portion at the dorso-mesal angle, including the callosum ; 95% alcohol ; paraffin ; sections /*. Stain with hematoxylin (2-3 hours) and picro- fuchsin. The points to be noted are (a) the transected gyres with the septums of pia in the fissures, (b) the structure of the cortex upon the summit of a gyre and at the bottom of a fissure. Can you recog- nize the (four) ill-defined layers ? Note the number, shape and size of the nerve cells, and compare with 71 and 173. 173. Cerebral cortex. Cat (young). Golgi's rapid method ( 141) ; collodion ; sections ^ ; in clearer. Mount in balsam without a cover-glass. 7 6 This preparation will demonstrate the real form of the cells already seen in 172. Find good examples of the small pyramidal and large pyramidal cells, and note the extent of the apical process and the number and length of the dendrites arising from it and from the cell body. Trace the neurite as far as possible. Can you find collaterals arising from it ? Find also good examples of well impreg- nated cells from the layer of polymorphous cells. Doubtless glia (spider) cells will also be stained. Compare preparation 172. 174. Hippocamp. Cat. Transection of the hippocamp and adjacent parts. Weigert's hematoxylin method ; collodion ; sec- tions }A. Now in 95% alcohol. Clear and mount in balsam. The parts to be recognized in this preparation are (z) the hip- pocampal gyre, (2) hippocampus, (j) dentate gyre, (^) fimbria, (5) the rima with the plexus, (6) the caudatum. Note the con- tinuation of the cortex of the hippocampal gyre as the grey matter of the hippocamp and dentate gyre ; observe the massing of large pyramidal cells in the hippocamp and the dense layer of small pyra- midal cells in the dentate gyre, the continuation of the fimbria with the central (sub-cortical) alba. Recognize that the hippocamp is an infolding of the cortex. 175. Caudatum, Lenticula, and Thalamus. Transection through a hemicerebrum (or part) including these masses of cinerea (ento-cinerea). Weigert's hematoxylin method ; sections yu. Now in alcohol. Clear and mount in alkaline balsam. i Locate in this preparation the cortex, the caudatum, lenticula, claustrum and thalamus ; between the claustrum and the lenticula the external capsule ; between the lenticula and thalamus (and cau- datum) the internal capsule. The internal capsule is the continua- tion of the crusta (168) ; the subthalmic region is continuous with the tegmentum ; the thalamus and subthalamus constitute the larger portion of the diencephal, intermediate between the mesencephal (168) and the cerebrum (prosencephal). Consult the transections of brain and the special references given. 176. Olfactory bulbs. Rabbit. Transection ; vom Rath's fluid ; paraffin ; sections yu. Stain with hematoxylin ( i hour) and picrofuchsin. Recognize the following layers : ( i ) the layer of olfactory nerve fibers, (2) the layer of glomerules, (3) the large nerve cells 77 (mitral cells), (4) the granular layer, (5) the olfactory tract, (6) the entocinerea, (7) the central cavity (rhinocoele) with its lining endyma. Understand the course of the olfactory impulse from the olfactory nerve fibers to the olfactory tract (leading to other parts of the brain). Remember that in the human brain there is no rhino- coele (in the adult). ADRENAL BODY. 177. Adrenal body. Cat. Assigned for study. Chrome- oxalic ; paraffin. Stained with hematoxylin and picrofuchsin. One of the " ductless " glands. In studying this preparation, note (a) the regions, cortex and medulla, () the zones of the cor- tex and their structure, (<:) the structure of the medulla and the number and character of the contained blood vessels. Other duct- less glands studied by you are the thyroid (128), and the thymus (94). Note the diversity in structure and origin of these three glands. It should be recognized that the inclusion of the adrenal body with the organs of the central nervous system is a matter of con- venience rather than because it may logically be grouped with them. On an embryological basis, however, it might be placed in the peri- pheral nervous system. The thyroid (128), likewise, should only be grouped with the respiratory organs, in a classification the basis of which is embryology. 78 THE ORGANS OF SPECIAL SENSE. LABORATORY WORK FOR THE _?-.-- WEEK. 1/**M. 5 -- References : ^K - AVI i >~ Points for Quiz : Laboratory Report : Obtain from the delivery desk, (a) paraffin sections, 178, 179, 180, 183, 185 ; (b) collodion sections, 181, 182, 184. EYE. 178. Eye. Pig. Section through the anterior quarter of the eye-ball. Zenker's fluid ; paraffin ; sections //. Stain with hematoxylin and picrofuchsin . This section will include cornea, iris, ciliary body (muscle and processes), schlera, choroid and retina. Study well the structure of each of these parts, observing the relation of () schlera and cornea, () iris and choroid, (c} ciliary and sensory parts of the retina. Note also the canal of Schlemm, ciliary arteries, sphincter of the pupil. Is there any trace of the suspensory ligament of the lens ? 179. Eye. Cat. Section through the posterior half showing the entrance of the optic nerve. Zenker's fluid ; paraffin ; sections /^. Stain with hematoxylin and picrofuchsin. This preparation is to illustrate the structure of the three coats of the eye, the layers of the retina, and the relation of the optic nerve to the eye-ball, especially to the retina, and should be studied to afford demonstration of these points. 1 80. Retina. Pig. Vertical section. ; paraffin ; sections yw. Stain with In this section identify the layers of the retina already seen in 178 ; be sure you understand the real structure of the retina as re- vealed by other methods, and the meaning of the layers brought out by ordinary methods. Distinguish the rods and the cones ; re- member which side of the retina faces outward, i. e., is toward the other coats of the eye. What course would the sensory impulse pursue in order to reach the optic nerve ? 79 (27). Cornea. Cat. Assigned for study. Silvered prepar- ation. Again examine the preparation of cornea already studied, and in connection with the structure of the cornea (177) note the cell spaces and their relation to each other. 181. Optic nerve. Transection. vom Rath's fluid; collodion ; sections //. Stain with hematoxylin and picrofuchsin. Note the connective tissue sheath and its relation to the schlera (178), the fibers in bundles. Compare the individual fibers with the fibers of the peripheral nerve given (74) ; what is the differ- ence ? Can you ascertain ? Are the retinal vessels included ? 182. Eye-lids. Transection. collo- dion ; sections /*. Stain with hematoxylin and eosin. In this section recognize (a) the dermal and conjunctival sides and the character of the epithelium of each, () the cilia (eye- lashes), (c} the Meibomian gland and compare with the sebaceous glands of the hair follicle (154) ; (W) the orbicularis muscle. Can you find the glands of Moll ? 183. Lachrymal Gland. . ; ; . ; sections /*. Stain with hematoxylin and eosin. Determine the type of gland, noting the secreting acini and the ducts ; and compare with the salivary glands (112, 113). THE NOSE. 184. Nasal fossae, . Transection through the head and nose. ; decalcified ( 131) ; collodion ; sections //. Stain with hematoxylin and picrofuchsin. The preparation illustrates (a) the respiratory and olfactory (sensory) regions in the nasal fossae of this animal and their epi- thelium, () the nasal septum and the turbinal bones. 185. Olfactory mucous membrane. Guineapig. Transec- tion of a part of the more dorsal turbinal bones. Flemming's fluid ; stained in toto ; paraffin ; sections /t. The general structure of the sensory epithelium is shown, the glands of Bowman, and the olfactory fibers in the mucosa. Can you distinguish the sustentacular and sensory cells in the epithelium ? 8o THE ORGANS OF SPECIAL SENSE. LABORATORY WORK FOR THE-?*-' ___ WEEK. Vw-*x / -* ,/ References : -i hour, and harden in 50%, 67%, and 82% alcohols. This appears to be a good general fixer for many organs ; it penetrates well, the tissue cuts well after this method of fixation, and the staining is sharp and strong. % 33. Alcohol. 95%, 67-70%, absolute alcohol. The employment of most of the fixers so far mentioned requires the expenditure of considerable time, rendering them inapplicable or unsuitable in many instances. 95% alco- hol itself answers admirably for most histologic objects, fixing well, hardening and likewise dehydrating (\ 41) preparatory to imbedding in paraffin or col- lodion, affording thus a considerable economy of time. It is also most service- able in pathological tissues where the presence of bacteria is suspected. In some instances 67% alcohol answers as well or better, while in other cases ab- solute (99%) alcohol should be employed. The addition of 5% glacial acetic acid increases the penetrating power and improves the cutting quality of objects containing much connective tissue. Fix in 95% alcohol for 1-3 days, changing two or three times, after 3 or 4 and after 24 hours. The tissue will probably be found to be dehydrated and ready for the next step of the imbedding process ($ 44 or 52). Stain as you wish. \ 34. Formaldehyde. Solutions of this chemical have been found to be good preservatives and hardeners and fairly good fixers. It penetrates rapidly, and preserves -the natural transparency and pigmentation of the tissue, making it valuable for gross anatomy and museum purposes. As a fixer, an aqueous solution of 2-4% strength may be employed, or it may be used in conjunc- tion with other chemicals, as picric acid, inpicro-formalin, or chromic acid and acetic acid. Fix 12-24 hours, remove to 67% alcohol for a day, 82% alcohol one to several days. Stain as you wish. $ 35. Perenyi's fluid. Formula : 10% aq. sol. nitric acid, 4 parts ; 95% alcohol, 3 parts ; >% aq. sol. chromic acid, 3 'parts. An embryological fixer of much value. It is also serviceable for general work. Fix tissue for 4-5 hours, place in 67% alcohol for 24 hours, 82% alcohol several days. | 36. Picro-nitric. Formula: Water, 95 c.c. ; nitric acid, 5 c.c. ; picric 94 acid, to saturation. This is a valuable embryological fixer, especially service- able with eggs that have a good deal of yolk. Fix for 4-12 hours, transfer to 67% alcohol for i day, 82% alcohol, several days. It is necessary to wash the tissue well in several changes of the alcohol, as the picro-nitric is washed out with difficulty. | 37. Picro-sulphuric. Formula : Distilled water, 100 c.c. ; sulphuric acid, 2 c.c. ; picric acid, to saturation. This may be used full strength or diluted with 1-3 volumes of distilled water. It is a embryological fixer, useful for many eggs, chiefly invertebrate, and for many invertebrate animals. In general, fix for 1-6 hours, remove to >7% (7%) alcohol for a day, and place in 82% alcohol, in which it should remain until most or all of the yellow color has been extracted. $ 38. Nitric acid. A 10% solution of nitric acid is serviceable in fixing the blastoderm of the chick. SECTIONING. 39. In addition to the examination of tissue by the separa- tion of the component elements isolation it may be examined microscopically after cutting very thin slices or sections of it. This may be done free-hand or by means of a special machine, a micro- tome, and with or without an imbedding and supporting mass. For the finer work an imbedding mass and a microtome must be used. Free-hand sectioning without an imbedding mass, and even without previous hardening, is, however, necessary or advis- able when economy of time is a desideratum, as in clinical exam- inations of tissue, when one wishes to study the part alive or fresh (i. e., not treated with reagents), or if the reagents necessary for fixing and imbedding destroy or alter the structural features to be investigated. The ability to recognize tissues and organs unaffected by re- agents and without the employment of methods involving the ex- penditure of time and effort is very desirable, especially in patho- logical work, when haste often forbids the employment of the finer methods, were facilities for their use available, as in many cases they are not. Great skill in the use of simple tools may be gained and counts for much. It should be remembered also that the greater one's knowledge of a structure the less the need to resort to special methods of preparation for its recognition. 95 IMBEDDING METHODS. 40. When the consideration of time is not so important and finer results are more to be desired, the sections should be prepared according to some method in which an imbedding mass is used. The interstices of the tissue are completely filled with some sub- stance that will give support and greater consistency and homegen- eity to the tissue, and thereby enable the cutting of much thinner and more perfect sections. There are three methods that are generally employed, (#) the Paraffin method, () the Collodion method, and (c) the Freezing method ; the imbedding masses to fill the spaces being respectively paraffin, collodion and a congelation mass, ice. The last is the simplest ; it requires less expenditure of time, fewer reagents, and its results are the crudest. As in cutting free-hand sections without imbedding, the freezing microtome should be employed when haste is necessary and finer detail unimportant, as in clinical work. The two remaining methods may be employed in most cases and give good results. A choice between them must be determined by the following considerations and the special exigencies of the case : (i) As a general rule paraffin may be employed when very thin sections are desired and the piece or organ is not very large ; collodion, when larger sections are necessary but which need not be so thin. (2) For paraffin, heat is necessary, which is not required in the collodion method. (3) In the case of paraffin, the imbedding mass is removed from the sections before they are stained and mounted ; the collodion is (usually) not dissolved out. With most nuclear stains the collodion is colored more or less, affecting the ap- pearance and excellence of the preparation unless it is bleached, a process not possible in many cases. (4) In paraffin sections there is apt to be more or less crushing together of the parts of the tissue ; by the employment of collodion, the form of the organ may be more exactly preserved, sometimes an important consideration. 41. The following will indicate the steps in the employment of the two methods : 9 6 Living tissue v Fixing Hardening in alcohols (50-67-82-95 %) Dehydration (in 95-99 % alcohol) Paraffin Method Cedar-oil Clearing . Melted paraffin Infiltrating < Collodion Method Ether-alcohol Thin collodion (1-2 % solutions) V Thick collodion (6-8 % solutions) Paraffin mass 4 cooled quickly Imbedding ( . v I Collodion hardened in chloroform \ I Collodion block clarified In Paraffin --------- Sectioning ---------- In Collodion THE PARAFFIN METHOD. 42. As seen by the above scheme, the aim is to fill all the interstices of the tissue with paraffin of the right degree of hardness to have it cut well. Paraffin is not soluble in water or alcohol, but is soluble in a number of fluids which in turn are miscible with alco- hol. Hence the following steps are necessary : (i) the tissue must be first water-rid, thoroughly dehydrated with strong alcohol; (2) freed from the alcohol, cleared by a fluid that mixes with melted paraffin, which (3) takes the place of the clearer in the tissue, infiltrates it, filling the spaces ; (4) finally, the tissue is imbedded in paraffin of the right degree of hardness, the mass cooled, and it is ready (5) to cut, or section. 97 43- Dehydration. After the various steps pertaining to the proper fixing and hardening (21) of the tissue have been prop- erly pursued it may be stored in alcohol of 82^-95% strength de- pending on the tissue and its purpose. The dehydration necessary in imbedding may be accomplished by immersion in alcohol of 95% strength. For more delicate work it is perhaps better to employ stronger (absolute) alcohol. Immerse small pieces 2-3 mm. in diameter for at least 6-8 hours in 95% alcohol changed once or twice. A longer time, even days, does no harm and is preferable to ensure complete dehydra- tion. For larger pieces of tissue or entire organs a correspondingly longer period of dehydration should be employed, a several days' stay, with the alcohol changed daily, being often advisable, In any case, dehydrate thoroughly, changing the alcohol 1-3 times, letting the tissue remain in it for a longer rather than a shorter period. 44. Clearing. The alcohol must next be replaced by some solvent of paraffin which is miscible with alcohol, a step spoken of as clearing . Cedar-wood oil is perhaps most generally serviceable, although for special purposes other media, such as xylol, bergamot oil, or chloroform may be preferred. When the tissue is dehydrat- ed, it is removed to a vial of thickened cedar- wood oil. It will float at first, but as the alcohol used for dehydration is displaced by the oil, it sinks to the bottom, the currents of alcohol rising from it. When the alcohol is entirely removed by the oil, such currents will have ceased to rise from it and the tissue will be clear and translu- cent, except, of course, such as is dark in color. 45. Infiltration. After the tissue is completely cleared by the cedar- wood oil, remove it to a dish of melted infiltration paraffin and place in the paraffin oven for 2 to 24 hours, depending on the size of the piece. Quite large pieces may be left longer. The melted paraffin replaces the cedar-wood oil, filling in the interstices of the tissue. Paraffin melting at about 48 C. is.used, and is pre- pared by mixing equal parts of 43 and 54. paraffin. It is best not to expose to a higher temperature than is necessary, or for a long period of time. The paraffin oven will be maintained at a temperature of 5o-52 C. 46. Imbedding. It is best to use fresh paraffin for imbed- ding and generally with a melting point higher than that of the infil- 9 8 tration paraffin, 50 C. paraffin (42 paraffin i part, 54 paraffin 2 parts), answers well in a room of 19 to 20 C., and will be gener- ally used. If the cutting is to be done in a room of lower tempera- ture, a softer grade of paraffin may be used for imbedding ; if at a higher temperature, a harder paraffin should be chosen ; 54 C. par- affin giving good results when summer work is necessary. Make a small paper box, fill it with the melted imbedding paraffin ; transfer to it the tissue from the paraffin oven, arrange it carefully in the box in the way you wish it for cutting, and cool the mass by floating the box on a dish of cold water. 47. In imbedding in paraffin observe the following rules ; (i) Take no more paraffin (no larger box) than is needed to form a mass of convenient size around the specimen. The aim is to have as homogeneous a mass as possible ; paraffin tends to crystallize if it cools slowly, hence the smaller the mass the more rapidly may it be cooled. (2) Let the imbedding paraffin when poured into the box be several degrees above its melting point, and the tissue like- wise should have an equal temperature. Should the imbedding paraffin (or the tissue) be too cool it will not set well around the specimen, and a film of air may be enclosed. On the other hand, take care that the paraffin is not hot enough to "cook" the tissue, thereby shrinking it and rendering it hard and tough or ruining it altogether. (3) Cool by floating on cold water. Paraffin in cooling must contract greatly if it does not crystallize ; the more homo- geneous it is the more it must contract, and if it is cooled on all sides it will crystallize in the center of the mass, because it cannot shrink. 48. Cutting the sections. The essentials for good paraffin sectioning are (i) well-imbedded tissue, (2) a sharp knife (or sec- tion razor), (3) a room of the proper temperature, and (4) the par- affin block properly trimmed and arranged in the microtome. Fur- thermore, tissue fixed and hardened in different ways cuts very dif- ferently. Tissue fixed in Hermann's, Flemming's, Miiller's, or Zenker's fluid cuts well ; picric acid and mercuric chlorid tissue is more apt to be tough or hard, etc. The different organs and tissues have of course very different adaptabilities to the method. After the imbedding mass is well cooled, remove the paper box and trim the part containing the tissue in a pyramidal form, two of 99 the sides at least being as nearly parallel as possible. Clamp the block of paraffin in the holder of the microtome so that the tissue will be at the proper level for cutting, being careful to have the par- allel sides also parallel to the edge of the knife. If a ribbon micro- tome is used, heat the holder and melt the end of the block upon it. Cool and place the holder in its place in the microtome, again hav- ing the parallel sides and the edge of the knife parallel. Use a very sharp, dry razor for cutting the sections. Clamp it in the (Minot) microtome slightly inclined to the cutting surface of the tissue. If the temperature of the room is right for the paraffin used, the sec- tions will remain flat, and if the directions given above for trimming and arranging the block be observed, they will adhere and thus form a ribbon. If the room is too cold or the paraffin too hard, the sec- tions will roll ; if it is too warm, the sections will crush or be imper- fect. If a microtome in which the knife is not fixed, is used, make the sections with a rapid straight cut as in planing. Do not try to section with a drawing cut as used in collodion sectioning. io// will be found the most convenient thickness for the sections, though in special cases they should be thinner or even thicker. Remember to have the paraffin block trimmed with two sides parallel and the knife edge parallel to these. Also, do not attempt to cut if the temperature of the room is too high, above 21 C. 49. Resume of the method. To obtain as good results as possible with a certain organ fixed and hardened in a certain way, the steps must be carefully and exactly followed. Let the dehydra- tion be complete, clearing thorough, infiltration sufficient ; imbed, carefully observing the three cautions mentioned ; and in cutting, remember to have a sharp knife, a cool room, and the imbedding block properly trimmed. Success also depends largely on the pre- vious treatment in the fixer and on the care with which the fixer is washed out. Properly employed, the paraffin method is widely serviceable, being only useless where the tissue is very large, very hard, hard- ened or injured by heat, or where the exact form of a large organ is important. THE COIvLODION METHOD. 50. A comparison with the paraffin method has already been given (40); there may be emphasized here three points: (i) 100 with paraffin heat is required, with collodion no heat ; (2) paraffin must be removed from the sections subsequently, collodion need not and usually is not dissolved out ; (3) by the paraffin method may be obtained small sections (2 cm. square or less), and thin, by the collodion, larger sections, but thicker. With paraffin heat (melting and cooling) is necessary, and the mass is sometimes spoken of as a fusion imbedding mass ; collodion is a solution, arid the mass is left in the tissues by evaporation or its equivalent. In the collodion method the imbedding mass with which the spaces of the tissue are to be filled is collodion, a solution of pyroxy- lin (soluble cotton) in ether and alcohol, hence the steps, which are comparable with those of the paraffin method (see 41), are (i) Dehydration, removal of the water ; (2) Saturation with ether-alco- hol, the solvent of the collodion ; (3) Infiltration with collodion solutions, a thin and a thick ; (4) Imbedding in a thick collodion mass, which is hardened and (as employed in this course) clarified and (5) sections cut. .51. Dehydration. Let it be complete, as in the prepara- tion for paraffin imbedding ( 43). Immerse the tissue in 95% alcohol for 12-24 hours or longer, changing 1-3 times. Consult also 43 upon the dehydration of tissue. 52. Saturation with ether-alcohol (equal parts). Re- move the tissue from the strong alcohol and place it in a vial of ether-alcohol for 12-24 hours. In addition to preparing the tissue for the collodion solutions, it completes the dehydration, should it be imperfect. In special cases, or if the dehydration is very thorough and the specimen small, this step may be omitted. A satisfactory infiltration is, however, more certain if ether-alcohol be used. 53. Infiltration: (a) with thin collodion. Pour off the ether-alcohol and add the thin (i^%) solution of collodion ( 154). This, being a solution in ether-alcohol with which the tissue is sat- urated, readily permeates it. It is best to allow at least a day for this to take place, although if there is time a stay of several days is better, there being little or no danger of deterioration while in the solution. With large (i c. c. + ) objects an infiltration of a week or even a month is advisable. 54. Infiltration : (b) with thick collodion. Pour off the 101 thin collodion solution and add thick (6%) solution. In this there is gradual concentration of the solution in the tissue. Allow small specimens to remain a day, or, better, several days ; larger objects should be given a proportionately longer time, a week to a month, or even longer. If the object to be imbedded, such as many embryological specimens, is one with large interior cavities with thin walls the transfer from the thin solution to the thick solution may be attended by a collapse of the walls and a consequent shriveling and distortion of the specimen. Avoid this by allowing the thin solution to thicken very gradually by evaporation until the solution has at- tained the right consistency. To accomplish this it is only neces- sary to have the cork of the vial containing the specimen perforated by a small hole. A small piece of paper may be inserted with the cork, or with porous corks no special effort need be made. Unless the thick solution has itself thickened by evaporation, with large specimens it is advisable to follow the 6% bath with a stay in a thicker solution, as 8%, for a day or so. 55. Imbedding. Pour off the 6% solution and add for a short time at least an 8% solution of collodion. The tissue is now ready for imbedding in 8%, which may be accomplished in either of two ways : (a) on a cork or other holder that may be clamped in the microtome, or (b) in a paper box. Only those specimens need be imbedded in a box that, from their shape, or for purposes of careful orientation or serial sectioning, require a larger imbedding mass around them. (a) On a holder (cork). Choose a cork of a convenient size ; put a drop or two of collodion upon one end and insert a pin verti- cally to the surface near the edge. Transfer the tissue from the vial of thick collodion to the cork and lean it against the pin. The shape of many tissues will obviate the need of a pin. Pour the thick collodion onto the tissue, drop by drop, moving the cork in such a way that the thick viscid mass may be made to surround and envelop the tissue. Continue to add drops of collodion at in- tervals until the tissue is well surrounded, and then as soon as a slight film hardens on the surface invert the cork bearing the tissue in a shell-vial of large diameter containing enough chloroform to float the specimen and cork. If the piece of tissue is of awkward 102 size and shape, oiled paper may be wound around the end of the cork and tightly tied, the projecting hollow cylinder being long enough to receive the object. The tissue may be put into the cyl- inder as before, the collodion slowly poured in drop by drop until the specimen is completely covered. When a film has formed, place in chloroform as before. (b). In a paper box. When a box is required for imbedding proceed as follows : The inside of the paper box should be slightly oily to prevent the collodion from sticking to it. Rub upon the paper that is to be folded to form the box a little vaseline, and then with a cloth or lens paper remove as much as possible. Fold the paper into a box of convenient size and shape. Remove the object from the thick collodion and place it in the box, arranging it in the manner wished with a view to sectioning it later. Pour over it slowly, drop by drop or a little at a time, an 8% solution of collodion until the specimen is well covered and the box sufficicently filled. It is better to have a deep layer over the specimen. The 8% solu- tion does not afford the best mass for cutting, so that, with large objects, it is better to allow the mass in the box to thicken by evap- orating it slowly under a bell-jar (aquarium jar) until it has at- tained the consistency of a very thick syrup. When thick enough allow a film to harden upon the surface and immerse it in a glass box or jar of chloroform. 56. Hardening and clarifying. The chloroform into which the collodion mass is placed takes out the ether- alcohol and hardens the collodion mass, for which a few hours is sufficient. Allow the chloroform to act for 6-24 hours. If it acts long enough the imbedding mass is rendered entirely transparent when no water is present. The hardening action of the chloroform may be quickened and itensified by carefully heating the chloroform until bubbles of ether begin to come from the specimen. When the collodion mass is hard, whether clear or not, pour off the chloroform and add castor-xylene clarifier ( 151), in which the tissue is to remain until the sections are cut. In a few hours the collodion mass will become quite transparent (clarified) and hardly discernable, so that the tissue is readily seen. Sometimes, however, the collodion remains white and opaque, due to the presence of moisture, and considerable time is required for its 103 clarification. In such cases the process may be hastened by placing the tissue in the clarifier in a warm place, and changing the clarifier several times. If the block still remains opaque, remove to 95% alcohol for a day for dehydration, pass through chloroform, and into clarifier. In this way the mass may usually be cleared perfectly. Change the clarifier to fresh after the first and second days. The sectioning may be done after a few hours' immersion, although a several days' clarification is preferable.* If a paper box were used, after the collodion is hardened and clarified, remove the paper box, absorb the castor-xylene on the sur- face, trim the end and put some fresh, thick collodion on the cork or other holder. Press the block firmly against the holder ; within two minutes it will be firmly cemented and one may proceed at once to clamp the holder in the microtome and commence cutting ( 57). 57. Cutting the sections. For collodion sectioning, a long drawing cut is necessary in order to obtain thin, perfect sections. The knife should, therefore, be set at an obliquity of 15-20* or less, so that half or more of the blade is used in cutting the section. Re- call that in the paraffin method the knife is set at right angles to the direction of the cut, and the stroke is a rapid straight one. Trim away the surrounding collodion mass, as in sharpening a lead pencil, so that there is not more than a thickness of about two millimeters all around the tissue, being careful, however, to leave a broad base as a support to the tissue and prevent its bending under the impact of the knife ; if the collodion mass is too tapering, bending will occur and thin sections cannot be cut. Clamp the object in the jaws of the microtome, placing it so that the mass of collodion is opposite the side to which the pressure of the knife is applied in cutting. It is advantageous also to have the ob- ject placed with its long diameter parallel with the edge of the knife. When knife and tissue are properly arranged wet the tissue well with clarifier and flood the knife with the same. Make the sections with a slow, steady motion of the knife. With a small ob- ject (3x5 mm.) and a good sharp knife, sections 5jw to 6yu can be cut without difficulty. In addition to a sharp knife, however, there are *The imbedded object may remain in the castor-xylene clarifier indefi- nitely without harm. The collodion grows somewhat tougher by a prolonged stay in it. After cutting all the sections desired at one time, the imbedded tis- sue is returned to the clarifier for future sectioning. 104 necessary well-infiltrated tissue and a hard, firm collodion mass. 58. Transferring the sections. If the sections are quite thick they may be transferred from the knife to a slide or a dish by means of forceps or a brush ; if they are thin, however, it is better to handle them by means of an absorbent tissue paper, as follows : Flood the sections well with clarifier and then by means of a pipette remove the clarifier from the knife and place over the sections the end of a piece of the tissue paper, pressing it clown upon the sec- tions if necessary. Carefully pull the paper off the edge of the knife ; the sections will adhere to the paper. .Place the paper, sec- tions down, on a slide, taking care that the sections are in the de- sired position. With the finger carefully press the sections (through the transfer paper) to the slide, and then lift the paper, with a roll- ing motion, from the slide ; the sections will adhere to the slide. Should they stick to the paper instead, lower the paper again and again firmly press the section to the slide. For further proceedure see 66, 68. If it is not desired to mount the sections upon a slide immediately, or if they are to be kept in bulk, as for class work, the transfer paper may be shaken gently in a dish of clarifier or 95% alcohol and the section (or sections) will float free and sink to the bottom. 59. If it is desired to mount the sections in a series, proceed as follows : With an artist's brush draw the first section, when cut, up toward the back of the knife and make the next section. Place this section to the right of the first, and so on, arranging them in serial order, section after section, and line below line, until enough are cut to fill the area that the cover-glass will cover. Flood the sections as before by letting the clarifier flow over them, being care- ful, however, not to float them from their places. Absorb the clari- fier from the knife with a pipette, and place over the sections a piece of the transfer paper twice the width of a slide ; press it down if necessary, and slowly draw it off the edge of the knife. Should some of the sections adhere to the knife instead of the paper, it means that the clarifier had been allowed to thicken* on them, * If one is a long time cutting a series of sections, it sometimes occurs that the xylene evaporates leaving the castor oil that is thick and viscid and also a solvent of the collodion, so that the sections are not easily transferable but stick rather firmly to the knife. In such a case, fresh clarifier or even a little xylene to dissolve the castor oil must be used. 105 cementing them to the slide, and the preliminary flooding to insure their being free, was insufficient. In that case it is best to flood the paper with clarifier, carefully lift it, arrange the sections again, flood them with clarifier, place a clean piece of transfer paper over them and try again. One soon becomes accustomed to the behavior of the sections, and accidents are rare. In cutting a series of many small sections, some time is consumed and it is necessary to flood the sec- tions frequently with clarifier while cutting in order to prevent the clarifier thickening and cementing them to the knife. 60. Resume of the method. Success in the employment of the collodion method depends upon the thorough infiltration with the collodion solutions, requiring days or even months, and the em- ployment of a thick imbedding mass giving when hardened a firm unyielding support to the tissue. This may be gained by employ- ing a relatively long period of infiltration, and taking pains in im- bedding to have the collodion mass well thickened. Observing these two cautions, collodion may be used in almost all cases as an imbedding mass, except such as are affected by the conditions of the methods already mentioned ( 40 and 50) . THE FREEZING METHOD. 6 1. This method is expeditious and of use in the rapid ex- amination of tissues, and therefore especially serviceable in the pathological laboratory and in clinical diagnoses. It may also be' used in cutting tissues that are too hard to be cut satisfactorily by means of either the collodion or paraffin methods. Both fresh and fixed tissue may be cut by means of the freezing microtome and with or without any special mass such as is used in paraffin or collo- dion imbedding. When no mass is employed the tissue is simply frozen and cut, or, if it is fixed tissue, soaked well in water first and then frozen. When extreme haste is not so essential it is better to first saturate the tissue with some solution that does not crystallize on freezing, but simply hardens, since the formation of the ice crystals is hurtful to the tissue. Such are solutions of gum arabic or sugar and anise- seed oil, and they are spoken or as Congelation masses, 62. Infiltration. Gum arabic or anise-seed oil may be used, (a) Gum arabic. If the tissue has been fixed and is in alco- io6 hoi remove the alcohol by soaking it for several hours to i day in water. Remove to a thick solution of gum arabic in water, in which it may remain for about 24 hours. It is then ready to freeze and cut. (b) Anise-seed oil. For this method the tissue should be first dehydrated ( 43). When dehydration is complete, transfer the tiSvSue to anise-seed oil, in which it may soak for 12-24 hours ; it is then ready to freeze and cut. 63. Cutting. Place a drop of the solution of gum arabic (or anise- seed oil) upon the object carrier of the freezing microtome and turn on the carbonic acid (or ether) spray. When the mixture begins to harden, place the object upon it in an abundance of the solution and freeze it solid. Covering with an inverted cup hastens the freezing. When the tissue is completely frozen, cut it with a straight movement of the knife, as in the paraffin method, holding it firmly upon the knife rest and making the strokes as rapidly as possible, at the same time rapidly raising the tissue a few microns at a time by means of the microtome screw. For cutting frozen sections, a strong, wedge-shaped knife must be used. The mass of sections is transferred to a dish of water in which the gum arabic is dissolved away and the sections are ready for stain- ing ( 7 r ~79)- If anise-seed oil is used, the sections are to be transferred to 95% alcohol which will dissolve out the oil ; stain ( 69-79). If the tissue has been stained in toto the sections may be transferred to anise-seed oil (or other clearer) and mounted in balsam directly. STAINING AND MOUNTING. 64. The remaining steps in making permanent histological preparations are usually done at one time and are conveniently con- sidered together. In all the processes seemingly complicated, if it is remembered that the succession of media, as in paraffin imbed- ding, depends on their miscibility, and the reason for the various steps is recognized, much of the difficulty in remembering the order in which they come will be avoided. There are here to be considered, (0) Paraffin and Collodion sec- tions, (b} Free-hand and Frozen sections, (r) Isolation preparations. Of these, the paraffin and collodion sections require some prelimi- nary treatment not needed with the others, and to them only, there- fore, 65-70 apply. Collodion Sections Paraffin Sections . V fasten to slide v v not fastened to slide fasten to slide f > b / y v >rbing clarifier (a) albumen fixative > / :r alcohol (^) ^Af % < :ollodion v \ 1 f V Benzin Alcohol (95 Water Aqueous stain (hematoxylin) Aqueous Counter-stain Water Alcoholic Counter-stain v Alcohol Alcoholic stain (Hcl. carmine) v v v Dehydrate (95 % alcohol) V Alcohol v Clear Mount in balsam io8 PARAFFIN AND COLLODION SECTIONS. 65. These may be carried on (a) as loose sections, or (b) fastened to the slide. (a) Sections not fastened to the slide may be carried on in watch glasses, or larger glass vessels if there are many of them, the sections either being transferred from vessel to vessel by means of forceps or a section-lifter, or the fluid decanted, care being taken not to pour off the sections, and the succeeding medium added. Single sections may best be carried on upon the slide, which must be kept horizontal. When the fluid is to be changed place a brush or needle gently on one corner of the specimen and pour off the liquid, if necessary first absorbing most of it by means of a pipette ; in this way the section may be retained on the slide. (b) Fastening the Sections to the slide. This is of great advantage in carrying the sections on through the different steps. With serial sections it becomes an absolute necessity. Different methods are employed for collodion and for paraffin sections. 66. (i) Collodion sections. If the sections are trans- ferred to the slide from clarifier, absorb the oil thoroughly by placing over the section some absorbent paper and pressing it down gently and firmly, repeating the operation several times with fresh paper. After the oil is well absorbed, with a pipette drop upon the section enough ether-alcohol to moisten it (1-2 drops). This soft- ens the collodion and fastens the section to the slide. Allow the ether-alcohol to evaporate until the collodion has again set and the surface of the section looks dull or glazed, and then place it in ajar of benzin. Take care that the specimen does not dry. If the sections are in series, it is better to put the ether-alcohol on one end of the slide and let it run quickly over the sections and drain from the other end of the slide, repeating the operation two or three times. If it is found that the sections float off of the slide in the process of staining, their adhesion may be insured by using albumenized slides or removing the slides from the alcohol ( 69) and again treating with ether-alcohol. 67. (2) Paraffin sections. If the sections are unwrinkled, or with a few wrinkles that may be easily " ironed out," fasten them to the slide with (a) albumin fixative and y% collodion, or (o) io 9 albumin fixative and heat ; if the sections are wrinkled, they may be extended and the wrinkles removed by the method given be- low ( 95-99 % Alcohol v Clear in Carbol-xylene I v Mount in Balsam 120 99- Staining with Safranin Flemming's or Hermann's fluid fixation (best) v Paraffin sections i v Benzin i v 95 % Alcohol I v Safranin (Babes') 2-12 hours i v Distilled water (rinsed) [ 95 % Alcohol | Acid v$% alcohol ( T V Hcl.) for a few seconds i v 95-99% Alcohol i v Carbol-xylene v Mount in Balsam MOUNTING. loo. Whether stained or unstained, prepared for microscop- ical examination by isolation or sectioning, and especially if it is de- sired to keep the preparation, it is necessary to mount it in some way, i. e., so arrange it upon some suitable support (glass slide) and in some suitable mounting medium that it may be satisfactorily studied with the microscope. Mounting may be I. Temporary, or II. Permanent, as A. Dry, or in air, B. In a medium miscible with water, or C. In a resinous medium, in which case it is neces- 121 sary first to remove all water by either (a) drying Desiccation, or (d) a series of displacements, i.e., i. Removing the water with strong alcohol Dehydration; 2. Removing the alcohol with clearer Clearing ; 3. Replacing the clearer with balsam or other resinous mounting medium. 10 1. Temporary mounting. Used in this course princi- pally in the examination of blood corpuscles and living ciliated cells (2). Temporary examination of tissues is quite simple, though important, and for this it is only necessary to place the teased tissue or section on the slide in a drop of the fluid in which it is at the time, normal salt solution, dissociator, or alcohol, and cover. The examination of preparations intended for permanent mounts during the staining or before mounting will often serve to detect faulty treatment at a time when it may be remedied without great expend- iture of time, or discard the specimen as worthless. 102. Permanent mounting. In this course are employed () mounting dry on a ring or in a cell, () in glycerin or glycerin jelly, media miscible with water, and (c^) in xylene balsam, a resin- ous medium. 103. Mounting dry. The preparation may be either upon the under side of the cover-glass (best if possible) or rest upon the bottom of the cell. In the first case a shallow cell made by a shellac ring will be sufficient ; in the second, a shellac ring may not give a deep enough cell and a paper, hard rubber, or metal ring may be cemented to the slide. (a) When the preparation is on the cover. Prepare a shellac cell ( 1 06) on the slide of a size slightly smaller than the cover to be used, and allow it to dry for a day or so. Warm the cover bear- ing the preparation to remove the last traces of moistuie, and place it film side down upon the ring. Warm the slide until the edge of the cover may be made to adhere to the shellac ring and press the cover down until it adheres all the way round. Seal the cover with shellac and label ( in, 113). (b) Mounting in a paper or rubber cell. With a brush, cover one side of the ring with a layer of shellac and place it on the center of the slide, shellac side down ; place within the cell the prepara- tion, arranging it in the manner desired, and place upon the ring a cover-glass of a suitable size, and seal it with shellac ; label. 104. Mounting in glycerin media, (a) Pure glycerin ; (b) glycerin and acetic acid, i %; (V) glycerin, alum carmine and eosin ( 156). Glycerin and glycerin-jelly are most serviceable in mounting isolation preparations. For both of these mounting media the ob- ject must be mounted from water or an aqueous solution. Arrange the section or teased tissue in the center of the slide, drain off the water or aqueous solution in which the preparation is and add a small drop of glycerin. Take a clean cover in the forceps, breathe on the under side and carefully lower it upon the object ; gently press it down. It is best to use only a small drop of glycerin so as not to get it outside the cover, as it is hard to clean away sat- isfactorily. Clean carefully and seal with shellac in accordance with 111. 105. Mounting in glycerin-jelly. The preparation should be mounted from some aqueous solution. Warm the slide gently and put it upon the centering card ; in the center of the slide place a drop of warmed (melted) glycerin-jelly. Remove the object from the water or aqueous solution and arrange it in the glycerin -jell) 7 . Grasp a cover-glass with the fine forceps, breathe on the lower side, gradually lower it upon the object and gently press it down. Allow the glycerin-jelly to set, keeping the slide horizontal meanwhile. Scrape away the superfluous glycerin : jelly around the cover-glass and seal with shellac ( in). 106. Preparation of shellac mounting cells. Place the slide upon the turn table and center it (i. e., get the center of the slide over the center of the turn table). Select a guide ring on the turn table which is a little smaller than the cover-glass to be used ; take the brush from the shellac, being sure that there is not enough cement adhering to it to drop. Whirl the turn table and hold the brush lightly on the slide just over the guide ring selected. An even ring of the cement should result. If it is uneven, the cement is too thick or too thin or too much, was on the brush. After a ring is thus prepared, remove the slide and allow 7 the cement to dry spontaneously, or heat the slide in some way. Before the .slide is used for mounting, the cement should be so dry when it is cold that it does not dent when the finger nail is applied. A cell of con- siderable depth ma) 7 be made with shellac by adding successive lay- ers as the previous one dries. 123 107. Mounting in balsam. Certain preparations may be mounted in balsam by drying, the method of desiccation ( 100), e. g., cover- glass preparations of bacteria, stained cover-glass prepara- tions of blood, etc. For this it is only necessary that the prepara- tion be absolutely dry, a small drop of balsam placed upon it or upon the under side of the cover- glass, which is carefully placed over the specimen and pressed down. Mounting in balsam by desiccation is serviceable for but few preparations in histology, and in most cases the removal of the water by a series of displacements is resorted to ( 100). For this the following steps are necessary : Dehydration, Clearing, Mount- ing in balsam. Dehydration. The sections are entirely freed from water by the use of 95% or absolute alcohol. The slide or free section may either be placed in a jar of alcohol or alcohol from a pipette be poured over it. Treat the preparation to be mounted for 5-15 min- utes. The thicker the section the longer the time required ; collo- dion sections require a longer time than paraffin sections. In any case, be sure that the dehydration is complete, giving a longer rather than a shorter time, and then clear. 108. Clearing. This is accomplished by putting the slide in a jar of clearer or dropping the clearer upon the section from a pipette. When the section is cleared it will be transparent. Test it by holding it against a dark background ; if it is not cleared it will be* cloudy, white, and opaque. 109. Mounting in balsam. Drain off the clearer and allow the section to stand until there appears the first sign of dullness from evaporation of the clearer from the surface. Then place a small drop of balsam upon the section or upon the cover-glass which is then inverted over the specimen. Remember that in mounting in this way you must always ' ' De- hydrate, Clear and Mount in Balsam" and that the three steps are inseparable. SEALING THE PREPARATIONS. This is only necessary when the preparation is a glycerin or glycerin-jelly mount. It is better not to seal balsam preparations, or only quite late after the balsam has thoroughly dried out. 124 no. Sealing glycerin mounted specimens. Wipe away the superfluous glycerin as carefully as possible with a moist cloth or a piece of lens paper. Place four minute drops of cement care- fully at the edge of the cover at the four quarters and allow them to harden for half an hour or more ; these will anchor the cover-glass and the preparation may then be placed upon the turn-table and a ring of shellac cement put round the edge while revolving the turn- table. ni. Sealing glycerin-jelly mounts. Allow the glycerin- jelly to harden for 12 hours or longer. With a knife scrape away the superfluous jelly and then carefully wipe around the cover-glass with a cloth moistened with water. Place the slide on a turn-table, carefully center the cover-glass, and with a brush seal the edge of the cover by a ring of shellac while revolving the turn-table. A second coating may be given subsequently if needed, after the first coat has dried. 112. Sealing balsam mounts. This is necessary only with special preparations, and should in any case be done only after the preparations have dried out for several weeks. With a knife scrape off all superfluous balsam from around the cover-glass and wipe it carefully with a cloth moistened with alcohol or benzin (or xylene). Seal as with glycerin-jelly mounts. When the oil im- mersion is to be used often, it is advantageous to seal the prepara- tion with shellac (after it has dried) to facilitate cleaning away the immersion fluid. LABELING MICROSCOPIC SLIDES. 113. Every permanent microscopic preparation should be carefully and neatly labeled in ink, the label being placed upon the right hand end of the slide. The label should furnish at least the following information : EXAMPLE. (1) The number of the preparation, the thickness of the cover- glass and of the section. (2) The name, kind, and source of m Transection. the preparation. No. C. 15. S. Ileum of Cat. (3) The date of the specimen. November, 1898. 125 In the case of specimens with which it is advantageous to have more information at hand a second label may be placed upon the other end of the slide, and it may bear the following information : (1) Mode of fixation. (2) Imbedding method. (3) Stains employed. (4) Mounting medium (generally not necessary). (5) Special purpose of the preparation. A catalog giving the full data of the specimen, age, condition of the animal, mode of preparation in detail, special points illus- trated, etc., is not required for the preparations in this course, but is valuable particularly in special investigations and with standard specimens. CLEANING SLIDES AND COVER-GLASSES. 114. Cleaning cover-glasses. Fill the large glass box one-half full of cleaning mixture and put in the new covers, one at a time, being sure that they are entirely immersed and the cleaning mixture reaches all points. The one cover may be pushed under by the next. Let them remain over night (or longer) and then wash them thoroughly in running water, until all trace of the cleaning mixture is removed. Then place the covers in 50 or 6j% alcohol. 115. Wiping the cover-glasses. When ready to wipe the cover-glasses, remove several from the alcohol and put them on a soft, dry cloth, or on some of the lens paper to let them drain. Grasp a cover-glass by its edges, cover the thumb and index finger of the other hand with a soft, clean cloth, or some of the lens paper. Grasp the cover between the thumb and index finger and rub the surfaces. In doing this it is necessary to keep the thumb and index well opposed on directly opposite faces of the cover so that no strain will come on it, otherwise the cover is liable to be broken. When a cover is well wiped, hold it up and look through it to- ward some dark object. The cover will be seen partly by transmit- ted and partly by reflected' light, and any cloudiness will be easily detected. If the cover does not look clear, breathe on the faces and wipe again. If it is not possible to get a cover clear in this way it should be put again into the cleaning mixture. When the covers 126 are wiped, put them in a clean glass box. Handle them always by their edges, or use fine forceps. Do not put the fingers on the faces of the covers for that will surely cloud them. 116. Measuring the thickness of the cover-glasses. With the cover-glass measurer determine the thickness of the cover- glasses and sort them into three groups : (a) those with a thick- ness of . 13-. 17 mm., () those less than .13 mm., and (c) those thicker than .17 mm. Groups (a) and () only should be used ; (V) should be discarded or used only with objects for low magnification. It is advantageous to know the thickness of the cover-glass on an object for the following reasons : (a) That one do not try to use objectives in studying the preparation of a shorter working distance than the thickness of the cover-glass (Microscopical Methods, 57) ; () In using adjustable objectives with the collar graduated for dif- ferent thicknesses of cover, the collar might be set at a favorable point without loss of time ; (c) For unadjustable objectives the thickness of cover may be selected corresponding to that for which the objective was corrected (see Microscopical Methods 27 table). Furthermore if there is a variation from the standard one may rem- edy it in part at least by lengthening the tube if the cover is thinner and shortening it if the cover is thicker than the standard (Micr. Meth. 96). 117. Cleaning slides. Rinse 'new slides thoroughly in clean water and then wipe them with a soft towel. In cleaning the slides handle them by their edges to avoid soiling the face of the slide. After the slides are cleaned, to keep them free from dust store them again in the box in which they came, or in a covered glass dish or jar. 118. Cleaning used slides and covers. If only water, glycerin or glycerin -jelly has been used on them, they may be cleaned with water, preferably warm water, and then, if necessary, wiped out of 50% alcohol. If balsam has been used, heat the slides until the balsam is soft and then remove the cover-glasses. Scrape from the slides all the balsam possible and place them (and coveglasses) in cleaning mixture for several days. If then they cannot be readily cleaned, place them in fresh cleaning mixture for a period of several days. Wash away the cleaning mixture thoroughly with water and wipe them with a clean towel. 127 SPECIAL METHODS. THE BLOOD. 1 19. Special methods in the examination of the blood include (i) Examining fresh ; (2) Technic of staining blood films; (3) Determination of the number of red and white corpuscles per cubic millimeter ; (4) Determination of the relative amount of hemoglo- bin ; (5) Spectroscopic examination of blood (hemoglobin), (i) and (2) are briefly given here ; for (5) see Microscopical Methods, 201-203. 120. Examining fresh. This consists in covering a drop on a slide and immediately sealing the cover-glass to prevent evapo- ration, observing the following cautions: (i) The drop of blood (from the finger or the lobe of the ear) should flow freely and not be obtained by pressure. The drop should be a medium-sized one, which will spread out in an even, thin layer under the cover. (2) The drop should be received upon a cover or slide, covered, and sealed at once with castor oil. Examination of fresh blood may be used in clinical examination for the detection of some abnormal conditions, and it is of value in the rough diagnosis of many others. 121. Stained preparation of blood, (a) Preparing the blood film. This may be best done in one of two ways : (i) The edge of a slide is first drawn through a drop of fresh blood and then moved quickly across the surface of a clean cover-glass, in this way spreading the blood in a thin, even layer upon the cover. Success depends upon getting the right amount of blood upon the edge of the slide and the quick, even movement by which it is spread upon the cover-glass. A second, possibly better, method is the following : (2) Have ready two thin clean cover-glasses and obtain a drop of fresh blood. Take one of the covers in the forceps, touch it to the drop of blood and place it upon the second cover-glass eccentric- ally, with* one edge projecting slightly. Slip the two covers apart in the plane of their surfaces and dry them quickly by waving them in the air or by passing them rapidly over the tip of a flame. The lower cover-glass will have the better film. () Fixing the hemoglobin with (a) ether-alcohol or (b) heat. 128 122. Fixing with ether-alcohol. When the blood films on the covers are dry, place them in ether-alcohol (equal parts) for YZ-I or several hours. Let them fix for a longer rather than a shorter time, as the quality of the stain (with triacid mixture) will be improved. After they have fixed a sufficient time remove and again dry them in the air. They may now be stained, immediately or at convenience. 123. Fixing with heat. Place a gas or alcohol flame under the apex of a triangular copper tabte (or other similar warm table or incubator). When it is well heated determine the region that has a temperature equal to the boiling point of water (100 C) by plac- ing on it drops of water at varying distances from the flame. Just within the point so determined (nearer the flame) place the covers bearing the dried film of blood, film side down upon the copper plate. Leave them for 15-30 minutes or longer. When the covers have cooled they are ready to be stained. (< c.c. ; l /z % aq. sol. eosin, 7^ c.c. $ 157. Lampblack mixture. Lampblack, i gram ; gum arable, i gram ; common salt, T % gram ; water, 20 c.c. $ 158. Neutral (alkaline} balsam. Canada balsam is liable to be slightly acid. This is of advantage for mounting sections stained with carmine or with acid fuchsin (as when picro-fuchsin is used), and for injected preparations where carmine or Berlin blue is used as the coloring matter. For hematoxylin and other stains easily affected by acid media it is often advantageous to use neutral or slightly alkaline balsam as a mounting medium. To obtain this slightly alkaline balsam, add some pure sodium carbonate to the thin xylene balsam and shake thoroughly at intervals for a day or so. Allow the balsam to stand until the soda has settled, then decant and thicken by evaporation till of the desired consistency. ( The Microscope, 7th ed., p. 176, g 300). UNIVERSITY OF CALIFORNIA Medical Center Library THIS BOOK IS DUE ON THE LAST DATE STAMPED BELOW Books not returned on time are subject to a fine of 500 per volume after the third day overdue, increasing to $1.00 per volume after the sixth day. Books not in demand may be renewed if application is made before ex- piration of loan period. 57n-7,'52(A2508s2,