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 <V-*- due . (See 
 
 bulletin board). 
 
 II. Fixation of tissue. Fill two medium shell vials with 
 picric-alcohol and go to the delivery desk for two pieces of fresh 
 tissue, to be fixed and hardened, and imbedded according to (a) the 
 paraffine method, and (^) the collodion method. Your name, to- 
 gether with the tissue you are to receive and the imbedding method 
 to be employed is posted on the bulletin board. 
 
 Label the specimen, giving the name of the animal, the organ 
 or part, the fixation method, imbedding method, and the date. 
 
 Keep a record of the steps in the process, and submit it as part 
 of the report for the week of the term. 
 
 In order that this work be successfully pursued, it is necessary 
 to study carefully 18-21 ; 25 ; 39-47 ; 50-60. Be sure that you 
 understand the order of succession of the various steps and the rea- 
 son for them, and follow directions absolutely. Fix in picric-alcohol 
 1-2 days, place in 67% alcohol 1-2 days, in 82% alcohol several days, 
 changing the alcohol to fresh three or four times ( 25). In 
 this work, be as independent of assistance from the instructors as 
 possible. 
 
i6 
 
 III. Isolation. From the stock bottle on the shelves, fill a 
 small shell vial with Miiller's fluid dissociator and go to the delivery 
 desk for a small piece of trachea of cat (sheep or calf). Label it, 
 naming the animal, tissue, and dissociator. Leave the tissue in the 
 dissociator until you come at the second period (1-2 days) when the 
 action of the dissociator will have been sufficient. For this work 
 study carefully 2-7. 
 
 At your second period, in accordance with the directions in 
 5-7 > ( 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 <V/^UX^ ; paraffine ; sections 
 
 i : yw ; stained with hematoxylin ; now in thin balsam. Mount 
 in balsam. 
 
 This is an epithelium in which the scale-like superficial cells 
 form a very thick layer and their nuclei disappear. In the cells of 
 the deeper layers the nuclei are present. This preparation is to 
 illustrate the intercellular bridges in a stratified epithelium and also 
 to show the relation of the deepest cells to the connective tissue 
 upon which the epithelium rests. 
 
 18. Follicular epithelium. Section of the ovary of a young 
 cat. ; paraffine; sections p. 
 
 Stain with hematoxylin 5 minutes, eosin 15-30 seconds.^ The 
 ovary will contain eggs of various ages, covered with a follicular 
 epithelium of a single layer of cells or many layers of cells, depend- 
 ing upon the maturity of the follicle. The follicular epithelium in 
 the older follicles therefore is a stratified epithelium in which the 
 cells are all cuboidal, rounded, or polyhedral. Search the prepara- 
 tion to find ova covered with such an epithelium and note that the 
 cells are approximately alike, and not as in the ordinary stratified 
 or squamous epithelium. 
 
 (10.) Glandular epithelium. Section of the soft palate. As- 
 signed for study. 
 
 In the substance of the soft palate are contained numerous 
 mucous glands, illustrating glandular epithelium. The tubules of 
 the glands are made up of columnar cells with the nuclei in the 
 bases ; the epithelium in these glands is therefore a simple columnar 
 epithelium. 
 
 19. Germinal epithelium. Section of the ovary of a kitten 
 2-3 weeks old. Hermann's fluid ( 23) ; paraffine ( 42) ; iron 
 hematoxylin ( 98) ; sections IJL. 
 
 The germinal epithelium is the cuboidal or low cofumnar 
 epithelium upon the surface of the young ovary, from which the ova 
 are developed. Examine the preparation, observing the surface 
 epithelium, with here and there a much enlarged cell with a large 
 round nucleus a young ovum. 
 
22 
 
 THE CONNECTIVE AND SUPPORTING TISSUES. 
 
 *)^L 
 
 LABORATORY WORK FOR THE-- --WEEK. 
 
 References : 
 Points for Quiz : 
 Laboratory Report : 
 
 Due : 
 
 Go to the delivery desk (; for assigned slides, 20, 22, 27. 
 
 (b) With a slide prepared for a paraffine section ( 67, a), 26. 
 
 (c) With clean slides for 28 and 30, already stained and in 95% 
 alcohol ; clear and mount in balsam. 
 
 (d?) With clean slides for 24 and 25, now in glycerin ; mount 
 in glycerin jelly ( 105). 
 
 (e) With a clean slide for 31 ; stain and mount as directed 
 below. 
 
 (/") With a clean slide for 29 ; tease as directed below ; mount 
 in glycerin jelly. 
 
 Of) With a clean slide for 21, a fresh preparation ; treat as 
 directed below. 
 
 MUCOUS TISSUE. 
 
 20. Mucous tissue. From 'the umbilical cord of a fetal 
 mammal. (Jelly of Wharton). Spread preparation. Assigned for 
 study. Stained with hematoxylin and eosin. 
 
 In studying this preparation, note the shape of the cells and 
 the anastomosis of their processes ; the general homogeneity of the 
 intercellular substance. Compare with 22. 
 
 AREOLAR TISSUE. 
 
 r 
 
 21. Areolar tissue (subcutaneous). Fresh preparation. 
 The instructor will make for you an artificial edema by injecting 
 normal salt solution into the subcutaneous connective tissue of a kit- 
 ten. This separates the constituents somewhat and enables one to 
 get so thin a layer that the different elements can be easily made out. 
 
23 
 
 Cut out a small piece with the scissors and spread it out in a 
 thin layer on the slide, using a dry slide and new needles. Draw 
 the edges away from the middle, and if they are dry enough they 
 will stick to the slide and hold the film outspread. It will probably 
 be necessary to absorb some of the liquid by means of lens paper be- 
 fore you can make the edges stick. Do not allow the middle to dry. 
 Place on the middle a drop of normal salt solution and a cover-glass. 
 
 Examine the preparation carefully, recognizing (#) the white 
 fibers, in wavy bundles ; () the yellow elastic fibers, straight and 
 single ; if possible find a place where they anastomose (see, how- 
 ever, below) ; ( the connective tissue cells ; can you find more 
 than one kind? (See 22). 
 
 Action of Reagents. Remove the cover glass and place upon 
 the preparation a drop of 2% acetic acid. Note that it makes some 
 of the fibers disappear, the white fibers, while others, the yellow 
 elastic, are sharply brought out. It is now possible to observe the 
 course, anastomosing and branching of the yellow fibres. Note 
 also that the nuclei of the connective tissue cells are more sharply 
 defined. 
 
 22. Subcutaneous areolar tissue. Stained preparation. 
 Assigned for study. Tissue spread on the slide by means of 
 needles, as in 21 ; stained with hematoxylin and erythrosin (or 
 eosin) ( 79). 
 
 Study the preparation carefully, recognizing again the white 
 and yellow fibers and the cells, of which find if possible three 
 kinds (text-book). 
 
 23. Adipose tissue. Demonstration. Spread preparation. 
 Stained with hematoxylin and eosin ( 79). 
 
 In this preparation of subcutaneous adipose tissue note (a) the 
 lobular grouping of the fat cells, (^) the blood vessels and the rela- 
 tion of the fat cells to the capillary network, (<:) the structure of 
 the individual fat cells, the fat globule, the nucleus with the proto- 
 plasm surrounding it. 
 
 WHITE FIBROUS TISSUE. 
 
 24. Tendon. From the tail of a mouse. Tendons pulled 
 out, spread out flat by means of a needle and stained 15-20 minutes 
 
24 
 
 with Khrlich's acid hematoxylin ( 81), now in glycerin. Mount in 
 glycerin jelly ( 105). 
 
 To show the tendon cells (connective tissue cells). Note their 
 arrangement in parallel rows between the tendon fibers, their shape 
 in relation to each other, the position of the nucleus in the cell in 
 relation to the nucleus of the contiguous cell. Consult also 26 for 
 the true shape of the cells. 
 
 25. Tendon. From the tail of a mouse. Silvered to show neg- 
 ative images of the tendon cells ( 147). The tendons were pulled 
 out and treated for a few minutes with a solution of silver nitrate, 
 washed in water, exposed to the light, washed in water ; now in 
 glycerin. Mount in glycerin-jelly. The places occupied by the 
 cells are white, while the cement substance was colored brown by 
 the silver nitrate on exposure to the light. Note the outlines of the 
 cells and compare with the positive images of 24. 
 
 26. Tendon. Transection of tendon, biceps of a new-born 
 puppy. Miiller's fluid ( 29) ; paraffin ; sections yw. 
 
 Stain 30 minutes with hematoxylin, 15 seconds with picro- 
 fuchsin. Mount in balsam. Note the relation of tendon-cells and 
 fibers as shown in transection and compare with 24 and 25. Note 
 also the staining reactions of the white fibrous tissue with picro- 
 fuchsin in comparison with the transected muscle fibers (central), 
 and the elastic tissue in 28. 
 
 27. Cornea. Cat. Assigned for study. Free-hand sections. 
 The cornea silvered ( 147), free-hand sections cut from the ental 
 surface and mounted in glycerin jelly. v < 
 
 To show the negative images of the cells of dense connective 
 tissue (i. e., the cornea). Note the shape of the cell spaces, and the 
 anastomosing of the processes ; compare with the similar preparation 
 showing the cell spaces in tendon, 25. 
 
 YELLOW ELASTIC TISSUE. 
 
 28. Ligamentum nuchae. Horse. Transection. ; 
 paraffin ; sections yw. Stained with hematoxylin and picro- 
 fuchsin, now in 95% alcohol. Clear and mount in balsam. 
 
 Note the yellow elastic fibers, their shape and size in transec- 
 tion, the white connective tissue in between them, and the nucleus 
 
25 
 
 of an occasional connective tissue cell. What is the staining reac- 
 tion with picrofuchsin ? Compare with 26. 
 
 29. Ligamentum nuchae. Horse. Teased preparation. 
 With your needles tease out very carefully a small piece of liga- 
 mentum nuchae obtained from the instructor. Remember that the 
 course of the elastic fibers is longitudinal, and therefore tease by 
 pulling the fibers to the side, trying to keep them parallel. Tease 
 carefully and thoroughly. Mount in glycerin jelly. 
 
 Note the anastomosing and branching of the fibers ; are trans- 
 verse markings to be detected ? 
 
 PIGMENT CELLS. 
 
 30. Pigmented pia. Sheep. From the frontal region of the 
 brain. Now in 95% alcohol ; no staining required. Clear and 
 mount in balsam. 
 
 Note the shape and anastomosing of the pigment cells, the pig- 
 ment granules, and the clear space occupied by the nucleus of the 
 cell. 
 
 31. Pigmented peritoneum. Necturus. Picric alcohol ; 
 peritoneum removed and now in 82% alcohol. Stain with hema- 
 toxylin 10 minutes, eosin 15 seconds ( 94). Mount in balsam. 
 Since this preparation is not fixed to the slide, care must be taken 
 in changing fluids ( 65, a). 
 
 Note the shape, the nucleus, the pigment granules, and the 
 state of expansion or contraction of the pigmented connective tissue 
 cells. Does the preparation show cells well expanded and cells 
 quite contracted? 
 
 TOPICS FOR QUIZ 6th Week. 
 
 THE CONNECTIVE AND SUPPORTING TISSUES. 
 
 1. General character of all the connective tissues ? Give the kinds. 
 
 2. Mucous tissues, character and distribution ? 
 
 3. Aroelar tissue, character and distribution ? 
 
 4. Fibrous tissue, kinds and distribution of each variety ? 
 
 5. Special cells found in connective tissue. Where are they found in man 
 and in some domestic animal ? 
 
 6. Compare the cellular and the intertellular or ground substance. 
 
 7. What is the effect of reagents on the cells and on the ground substance ? 
 
 8. From what germ layer is connective tissue developed, and in what form 
 does it first appear ? 
 
26 
 
 CONNECTIVE AND SUPPORTING TISSUE. 
 
 LABORATORY WORK FOR THE ______ WEEK. 
 
 References : 
 Points for Quiz : 
 
 Laboratory Report: 
 
 -. 
 Due : WWu . 
 
 Go to the delivery desk () for 35, assigned for study ; (b) with 
 a slide prepared for a paraffin section, 32 ; (c) with a clean slide for 
 collodion sections, 37, 38, 39 ; fasten to the slide according to 66 ; 
 place in benzin. 
 
 While the above preparations are being carried on ( 68-70) go 
 to the desk for 33 and 34, from which free-hand sections of the car- 
 tilage are to be cut, stained and mounted as directed below. Return 
 the tissue to the desk when you have finished with it. 
 
 During the week you will make preparation 36, in accordance 
 with the directions, 133-134. Read them carefully in advance. 
 
 CARTILAGE. 
 
 32. Embryonal cartilage. ^-Js-^o ~*~- Transection of 
 
 . Vr<A^iXo. . stained in toto ( 74, c, 86) in 
 paraffin; sections "1 JA. Mount in balsam (benzin, 
 clearer, balsam). 
 
 The skull should be studied. It is at this stage cartilaginous, 
 and the cartilage in an embryonic stage ; note the cells with their 
 nuclei and cell -bodies ; the small amount of matrix deposited be- 
 tween them, and compare with adult cartilage in 33 and 38. What 
 would be the structure of the cartilage at an earlier stage of develop- 
 ment? 
 
 33. Hyalin cartilage. Necturus (Amphibian). You will 
 receive one of the following from which to cut free-hand sections of 
 cartilage, (#) head of the humerus ; (b) head of the femur ; (f) a 
 branchial arch ; (d) part of the hyoid arch ; (e) the pectoral arch ; 
 (/") the pelvic arch. 
 
27 
 
 Cut the sections very carefully and as thin as possible. Place 
 6-10 good sections in an alum solution (^4%) in a watch-glass for 
 5-10 minutes ; place 3-4 sections on a slide in a drop of normal salt 
 solution ; cover and examine, studying the character of the cells 
 arid their nuclei, their grouping, the character of the matrix. 
 
 After the 5-10 minutes, drain off the alum solution from the sec- 
 tions in the watch-glass. Mount one-half of them directly in gly- 
 cerin jelly; the remainder, stain in hematoxylin for 5-10 minutes, 
 wash in water, and mount in glycerin jelly. These two prepara- 
 tions are to be studied in connection with the sections in normal salt 
 solution, for the structure of hyalin cartilage. 
 
 (38). Hyalin cartilage. Mammal. For this, study 38, 
 noting the structure of the cartilage, the grouping of the cells, their 
 shape and arrangement, in the deeper portion and at the articular 
 surface, and the corresponding difference in the density of the matrix. 
 Compare with 33. 
 
 34. Elastic cartilage. From the ear of the ox (or horse). 
 Picric alcohol fixation. Make free-hand sections, cutting them as 
 
 thin as possible. Place three or four of the thinnest in water for ,5 
 
 2.O . . . 1-o rxct>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. (<?) 57 is in thin balsam ; mount a 
 drop of the balsam. (/) Obtain 63, 64 and 65 and prepare them as 
 directed, (g} 60, 62 and 66 take in order. 
 
 NON-STRIATED, PLAIN OR SMOOTH MUSCLE. 
 
 57. Muscle cells from the intestine. Cat. Isolated. 
 The muscular coats of the intestine were dissociated in nitric acid 
 dissociator ( 1 1 ) , washed in water, the cells separated and stained 
 with eosin, now in balsam. Place a drop upon a slide and apply a 
 cover glass. Note the length of the muscle cells, the form, and size 
 of the nucleus in the center. Contraction nodes may also be found 
 in the fibers (cells) ; study them. For the nucleus and striation 
 study 59. 
 
 58. Muscle cells from an artery. Cow. The uterine 
 artery was placed in nitric acid dissociator ( n), the muscular 
 coats teased out to separate the cells. Stained in erythrosin, now 
 in glycerin ; mount in glycerin jelly. Find good examples of iso- 
 lated cells and study, comparing them with the cells in 57 as to rela- 
 tive length and width, and the form and size of the nucleus. 
 
 59. Plain muscle cells. Demonstration. This preparation 
 is intended to show especially (a) the shape, size and position of the 
 nucleus in the cells of plain muscle, and () the longitudinal 
 striation of the muscle fiber. Compare 57 and 58, identifying in 
 them the nucleus and, if possible, the longitudinal striation. 
 
 60. Anatomy of muscular coats (intestine). Cat. Obtain 
 from the delivery desk a small piece of the intestine, that has been 
 
38 
 
 macerated in nitric acid dissociator. Demonstrate the outer longi- 
 tudinal and inner circular coats, observing the relative thickness of 
 each. Tease out upon a slide the end of a piece of the longitudinal 
 coat, fraying the end with a needle, and spreading it out like a fan ; 
 cover in a drop of water and examine. Observe the course of the 
 fibers (cells) and their relation to each other. Compare with the 
 longisection and transection in 61. 
 
 61. Transection and longisection of plain muscle. Cat. 
 Transection of the intestine. Picric alcohol ; paraffin ; sections 
 
 //. Stain with hematoxylin 15 minutes, eosin y? minute. 
 Mount in balsam. 
 
 The outer muscular coat will afford a transection of plain muscle, 
 the inner coat a longisection. In the outer coat, note the variation 
 in the size of the sections of different fibers ; and the occurrence of 
 nuclei in only a few ; why is this ? Compare the section of the inner 
 coat with the appearance found in 60. Study well the general 
 structural appearance of plain muscle in section ; what are the stain- 
 ing reactions of muscle and connective tissue with picrofuchsin ? 
 
 STRIATED MUSCLE. 
 
 62. Fresh muscle fibers. Frog. Tease out upon a slide 
 in normal salt solution a small shred of the muscle from the leg of a 
 frog. Apply a cover glass and examine. 
 
 Observe the semi-translucent appearance of the fresh muscle. 
 With a y% objective study the individual fibers, noting the trans- 
 verse striations. Here and there broken fibers may be found ; ex- 
 amine the broken ends to detect the torn sarcolemma. Lift the 
 cover and add a drop of 2% acetic acid, and note the effect upon the 
 fibers in bringing out sharply the muscle nuclei. 
 
 63. Stained muscle fibers. Macerated in acetic 
 acid and glycerin and stained with Ehrlich's hematoxylin (Sihler's 
 method). Now in thin balsam. Carefully separate the individual 
 fibers of the muscle, keeping them parallel with each other and 
 tearing them as little as possible. Mount in balsam. 
 
 The transverse striations and the muscle nuclei will show with 
 great clearness. Note also the difference in caliber of different 
 fibers. 
 
39 
 
 64. Fibrillae. (Longitudinal striations). Biceps of rabbit. 
 Treated with ^% platinum bichlorid 6-24 hours ; now in 82% alco- 
 hol. Carefully tease apart with needles the fibers of a shred of 
 muscle and stain 5 minutes with erythrosin ; dehydrate and clear. 
 On the slide in the clearer tease again, trying to split the individual 
 fibers ; you cannot tease too finely. 
 
 If the teasing is fine enough in many places the fibers will be 
 separated into their component fibrillae. Observe this feature and 
 the structure of the individual fibrillae, the alternate light and dark 
 zones, lateral and transverse discs, the dark intermediate disc in the 
 midst of the light disc, and possibly the light middle disc in the 
 midst of the transverse disc. Ask the instructor to show you a 
 longisection of striated muscle stained with iron hematoxylin, in 
 which the discs are more sharply differentiated. 
 
 65. Tendinous ends of muscle fibers. Cat. Intercostal 
 muscle, dissociated in nitric acid ; now in 82% alcohol. With the 
 needles gently separate the fibers of a small fascicle, taking care to 
 have the ends of the fibers well separated. If this is successfully 
 done, the continuity of fiber and tendon will be shown. Stain the 
 preparation with picrofuchsin five minutes, wash with 67% alcohol, 
 dehydrate, clear, and mount in balsam. The muscle fiber will be 
 stained yellow or orange, the tendon fibers red. Note the shape of 
 the end of the muscle fiber and its relation to the fibrous tissue of 
 the tendon. Can you determine the relation of the sarcolemma at 
 the end of the fiber ? 
 
 66. Anatomy of a skeletal muscle. Obtain from the desk 
 an entire muscle which has been macerated in 20% nitric acid. Dis- 
 sect it carefully, determining the course and arrangement of the 
 component muscle fascicles in relation to the tendons (if present) of 
 origin and insertion. Are the fascicles parallel with the axis of the 
 muscle ? Is it a penniform or bipenniform muscle ? 
 
 67. Transaction of a skeletal muscle. Cat. 
 
 ; collodion ; sections yu. Stain with hematoxy- 
 
 lin 15 minutes, picrofuchsin 30 seconds. Mount in balsam.- 
 
 Observe the connective tissue sheath of the muscle, the epi- 
 mysium, the transected muscle fascicles surrounded by the perimy- 
 sium, the individual fibers held together by the endomysium ; finally, 
 in the transection of the fibers, note the punctate appearance of the 
 
40 
 
 cut ends, due to the transected fibrillae, and the muscle nuclei entad 
 of the sarcolemma. Can you recognize areas of Cohnheim ? The 
 transections of the fibers are of different sizes. This may or may 
 not mean a difference in the caliber of the fibers. Why ? Compare 
 with 61. You may be shown transections stained with iron hema- 
 toxylin in which the fibrillae are better differentiated. 
 
 CARDIAC MUSCLE. 
 
 68. Muscle cells. Isolated in caustic 
 potash dissociator ( 14) ; now in glycerin. Place a small drop of 
 the glycerin containing the cells upon the slide ; cover carefully ; 
 seal with shellac. 
 
 Study the preparation, examining many cells and noting the 
 shape, the position of the nucleus or nuclei, the trans-striations, the 
 relation of the cells to each other, and their processes. 
 
 69. Transection of myocardium. Human auricle or ven- 
 . tricle. Assigned for study. Miiller's fluid ; paraffin ; sections 
 
 10 /*. Stained with hematoxylin and picrofuchsin. 
 
 Observe the shape and relation of the cells, the muscle columns 
 and fibrillae, and the position of the nucleus ; compare with the 
 transections of plain and striated muscle (61, 67). You may be 
 shown transections and longisections of cardiac muscle stained with 
 iron hematoxylin, in which the fibrillae and cross-striations are bet- 
 TOPICS FOR QUIZ loth Week. 
 
 THE MUSCULAR TISSUES ; THE MUSCULAR SYSTEM. 
 
 1. Muscular tissues, variety and characteristics of each ? 
 
 2. Plain muscle cells, structure? <^ 
 
 3. Compare the muscle in an artery and that in the intestine. 
 
 4. Name five regions of distribution of plain muscle. 
 
 5. Give relative length and width of muscle cells in arteries. 
 
 6. Compare plain muscle in transections and in longisections. 
 
 7. How, distinguish plain muscle from connective tissue? 
 
 8. Stilted muscle ; structure of a muscle fiber, considering sarcolemma, 
 muscle nuclei, sarcoplasm and fibrils? 
 
 9. Give the structure of the fibrillae and the various discs. 
 
 10. What is the structure of a skeletal muscle, fasciculi, epi- peri- and endo- 
 mysium. 
 
 11. What is the position of muscle nuclei in mammalian and immammalian 
 muscle ? Best determined by transactions. 
 
THE NERVOUS TISSUES : THE PERIPHERAL NERVOUS 
 
 SYSTEM. 
 
 LABORATORY WORK FOR THE WEEK. 
 
 References : 
 Points for Quiz : 
 Laboratory Report: 
 
 Due : 
 
 Go to the delivery desk for (a) assigned slide, 73 ; () paraffin 
 sections, 74, 75, 76, 77, 78 ; (c} 72, stained ; now in clearer; tease 
 as directed below and mount in balsam ; (a?) 70 and 71 may be pre- 
 pared while the paraffin sections are being carried through. 
 
 NERVE CELLS. 
 
 70. From the myel (Spinal cord). Calf. The myel was 
 split lengthwise and placed in formaldehyde dissociator ( 8 and 9). 
 Place a small piece of the grey matter upon a slide in a drop of ^% 
 eosin ( 94), and proceed as directed in 10. 
 
 Search the preparation carefully until you find a nerve cell with 
 quite long processes (dendrites). Observe (<z) the nucleus with its 
 nucleolus, () the number, extent, and branching of the processes 
 of the cell-body, (V) the structural appearance of the cell-body and 
 its processes. Can you determine the identity of the neurite ? If 
 desired, you may be shown nerve-cells in section stained with, 
 methylene blue, for Nissl's corpuscles. 
 
 71. From the cerebral cortex. Cat. The hippocamp was 
 cut up into pieces and placed in formaldehyde dissociator. Care- 
 fully prepare two or three preparations according to 10. The best 
 preparation may be retained as a permanent mount. 
 
 Examine the slide carefully, finding as many cells with long 
 processes as possible. Compare with 70, recognizing the identity of 
 structure of the cells, but observe that these cells are of a pyramidal 
 form, with a long apical process and basal processes. Can you find 
 the neurite springing from the base of the cell ? 
 
42 
 
 NERVE FIBERS. 
 
 72. Myelinic (medullated) nerve fibers. Cat. Isolated 
 fibers from the sciatic nerve. Hardened in Miiller's fluid 8-10 
 days ; hardened in alcohols ; dehydrated ; in chloroform 1-2 days ; 
 95% alcohol ; 82% alcohol ; water ; stained in Delafield's hematoxy- 
 lin 12-24 hours ( 82) ; now in clearer. With needles tease out a 
 small bundle of fibers, keeping them parallel if possible. Mount in 
 balsam. 
 
 In studying this preparation, determine the axis cylinder, the 
 myelinic sheath, the nodes of Ranvier, the internodes, their 
 length, the nerve corpuscles (nuclei). Can you recognize the 
 neurilemma ? Does the preparation show medullary segments or a 
 reticular structure of the myelinic sheath ? 
 
 73. Amyelinic nerve fibers. Ox. Isolated fibers from the 
 splenic nerve. Assigned for study. Dissociated in -$% osmic acid 
 ( 17) ; teased apart with needles. 
 
 Both myelinic and amyelinic fibers are present, and care must 
 be exercised in distinguishing between delicate myelinic fibers and 
 the amyelinic ones. When a good place is found, study the amye- 
 linic fibers, noting, as compared with the myelinic nerve-fiber, the 
 absence of the myelinic sheath and the greater number of nerve 
 nuclei. Compare the myelinic fibers in this preparation with those 
 in 72. The myelin of the sheath is retained and blackened in 73 ; 
 in 72 it has been dissolved out. 
 
 PERIPHERAL NERVES. 
 
 74. Sciatic nerve. Cat. Transection ; chrome-oxalic 
 ( 3 2 ) > 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 
 <o /*. Stain with hematoxylin and eosin. Consult the gross 
 preparations for the position, shape (general), and color of the 
 glands ; their appearance superficially and in section. In the sec- 
 tion there are to be noted, the shape, size, and structure of the 
 acini ; the colloid mass ; the connective tissue dividing the gland 
 into lobes and lobules ; the blood vessels. Study well the epitheli- 
 um in connection with the formation of the colloid mass. Can you 
 recognize two kinds of cells ? This is a ductless gland ; in what 
 way may the secretion be utilized by the body ? 
 
 TOPICS FOR QUIZ isth Week 
 
 THE RESPIRATORY SYSTEM 
 
 1. Constituents of the respiratory system. 
 
 2. Compare the respiratory canal of the nose and the structure of the oral 
 cavity. (Shown in the section of the soft palate.) 
 
 3. Structure of the larynx. 
 
 4. Structure of the trachea. What does the trachea merge into ? 
 
 5. Structure of the lung ? 
 
 6. Ivobules or infundibula, and air cells or sacs. 
 
 7. Character of the epithelium in different parts of the respiratory tract. 
 
 8. Vascular supply of the lungs. 
 
 9. How distinguish a transection and a longisection of trachea ? 
 10. Thyroid. Where is it, and what is its structure and fuftcbkm ? 
 
6o 
 THE URINARY ORGANS. 
 
 LABORATORY WORK FOR THE--^-- WEEK, "i^- S -I 
 
 References: \\sj<rJ&- Ok. 
 Points for Quiz : 
 
 Laboratory Report : ^^.w^JU^. \ x v-i % "fctcxoo t -x. n_ t \-^^ i . if 
 
 rr- w- 
 
 Due: *** ^ 
 
 Obtain from the delivery desk (a) paraffin sections 129, 132, 
 i33- W collodion sections 130, 134, 135, 136, 137, 138. 
 
 KIDNEY. 
 
 129. Kidney. \WX*A*. Transection. Miiller's fluid ; par- 
 affin ; sections 0.6 ft. Stain with hematoxylin and eosin. 
 
 Kxamine the gross preparations of kidney for (a) the form and 
 color of the organ, () the unilobular (apparently) and multilobular 
 kidney ; compare the kidney of the child with that of the adult, 
 and with the kidney of the ox. In the transected and longisected 
 organs, recognize if possible (#) the cortex (superficial zone) and 
 (#) medulla, (c) the papillae. Some of the kidneys have one, others 
 several papillae ; note in which animals each of these conditions oc- 
 curs and correlate lobules and papillae. 
 
 In studying the section recognize the following regions : {a} 
 cortex and () medulla ; in the cortex, medullary rays and laby- 
 rinth, and in the medulla, the pyramids and the columns of Bertini. 
 Study carefully these four regions, identifying the structures that 
 should be present in each. Understand the course the uriniferous 
 tubule would pursue from its beginning (Bowman's capsule) to the 
 opening of the collecting tubule into the pelvis of the kidney and 
 the regions in which its successive parts are found. Again examine 
 the longisected kidneys ; recognize the regions and note the struct- 
 ural appearance of each, for which you have the explanation in this 
 preparation. 
 
 130. Injected kidney. Cat. Transection. Blood vessels 
 injected with carmine gelatin mass ; collodion ; sections ' $ft J*. 
 
6i 
 
 This preparation will illustrate the relations of the blood vessels 
 within the kidney, and should be studied in connection with 129, 
 which it supplements. Understand the regions in which the fol- 
 lowing occur and the courses they pursue : (a) the renal arteries, 
 (6) "interlobular" arteries, (c) afferent arteries, (d) glomeruli, (e) 
 efferent vessel, (/) capillaries, (g) interlobular veins, (/*) renal 
 veins, and (i) the arteriae and venae rectae. Identify them in the 
 preparation. 
 
 131. Bowman's capsule. Cat. Demonstration. Injected 
 kidney ; paraffin ; sections ~ /*. Stained with hematoxylin and 
 picric alcohol. The injection has caused a transudation of fluid into 
 Bowman's capsule ; this fluid has taken the hematoxylin stain, thus 
 illustrating the relation of the glomerulus to Bowman's capsule and 
 the beginning of the urinary tubule. 
 
 132. Papilla of the kidney. () Horse. Transection. 
 paraffin ; sections /Q IJL. Stain with hematoxylin and picro- 
 fuchsin. From your study of 129 and 130 identify the tubules and 
 vessels shown in this section and note their grouping in relation to 
 each other. 
 
 133. Same ; () Homo. Miiller's fluid ; paraffin ; sections 
 /o }*. Stain with hematoxylin and eosin. Compare this with 
 
 132. 
 
 URETER. 
 
 134. Ureter. VH, . Transection. Picric alcohol ; col- 
 
 lodion ; sections %_n jn. Stain with hematoxylin and picrofuchsin. 
 
 In addition to the recognition of the three coats and the struct- 
 ure of each, note well (a) the character of the lining epithelium and 
 compare it with the stratified squamous epithelium (10, 99), and (b) 
 the direction of the muscular fibers in the two (or three) muscular 
 layers, and compare with the condition in the digestive tract, e. g., 
 intestine. Compare the epithelium of the ureter with that of the 
 pelvis of the kidney (129). 
 
 BI, ADDER. 
 
 1 35. Bladder, distended. C^Jk . < Vvv Jkr r O^UL ^iJWfct coll - 
 dion ; sections X^ /*. Stain with hematoxylin and picrofuchsin. 
 
 Note the three coats, and their structure. Can you recognize 
 three layers in the muscular coat ? What is the course of the fiber 
 
62 
 
 bundles? Recognize blood vessels, nerves (and ganglia ?). Study 
 well the epithelium and compare it with the epithelium of the ureter. 
 
 136. Bladder, collapsed. VAX/A*. JU*WX. ; (collodion ) 
 . sections 10 jit. Stain with hematoxylin and V >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 (<z) the theca and its layers ; () the membrana 
 granulosa surrounding the cavity of the follicle and bearing on one 
 side an eminence containing the ovum, the discus proligerus. 
 
 Study carefully the structure of the ovum and its parts, identi- 
 fying it as a single cell, and. comparing it with other cells. Under- 
 stand how the ovum may escape from the ovary by the rupture of 
 the Graafian follicle. Compare i. 
 
 (19). Ovary. Cat, new-born (1-2 weeks). Assigned for 
 study. 
 
 This preparation illustrates especially (a) the germinal epi- 
 thelium, () the egg-nests or egg-tubes, and (V) early stages in the 
 formation of Graafian follicles. Examine the germinal epithelium 
 to find here and there the large sexual cells. Can you find inter- 
 
66 
 
 mediate steps between them and the young ova ? Remember the 
 difference of opinion as to the origin of the follicle cells. 
 
 (18). Ovary. Cat (young). Preparation previously made 
 (18). 
 
 In this will be found stages in the formation of the Graafian 
 
 follicles. Note the layer of young ova under the tunica albuginea, 
 Graafian follicles with one layer of follicle cells surrounding the ova, 
 follicles with a several-layered follicular epithelium, and older follicles 
 in which the cavity of the follicle has appeared. 
 
 146. Corpus luteum. Section of the ovary of a pregnant 
 ; collodion ; sections yu. Stain with 
 
 In studying this preparation understand when and how the 
 corpus luteum is developed. Note its structure, observing the poly- 
 hedral cells, the connective tissue, and the blood vessels. Is there 
 any trace of the original cavity of the follicle ? 
 
 FAI^OPIAN TUBE. 
 
 147. Fallopian tube. . ; paraffin ; sec- 
 tions //. Stain with hematoxylin and picrofuchsin. 
 
 Identify the coats, noting their structure, the extensive folds of 
 the mucosa, the character of the lining epithelium, the presence 
 of a muscularis mucosae (?) the arrangement of the fibers in the 
 muscular coat, the character and location of the blood vessels. Are 
 all the cells of the epithelium ciliated ? 
 
 UTERUS. 
 
 148. Uterus. . Transection. ; 
 collodion ; sections /*. Stain with hematoxylin and eosin. 
 
 The same coats are present as in 147, but note the differences, 
 especially (#) the character of the lining epithelium, (b) the pecu- 
 liar structure of the mucosa, (c) the glands contained in the mucosa, 
 (d) the thickness of the innermost muscular layer and the direction 
 of its fibers, (e) the veins. 
 
 VAGINA. 
 
 ( I 37)-. Vagina. . Transection. Preparation already 
 
 made (137). 
 
 Recognize the three coats and study the structure of each, not- 
 ing (a) the character of the lining epithelium, (#) the structure of 
 the mucosa, (c) the course of the fibers of the muscular coat. Are 
 ganglia recognizable in the muscularis or mucosa ? Note 'the num- 
 ber and size of the blood vessels. 
 
6 7 
 THE SKIN AND ITS APPENDAGES. 
 
 LABORATORY WORK FOR THE -My.- WEEK, 
 References : CJU . x v (** ^^jL^oo^^^Ur ^ 
 Points for Quiz : 
 
 Laboratory Report : ^&^* V^V \ \> 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 <iAwY.<jv/<d, . 
 paraffin ; sections *0 /*. Stain with hematoxylin and eosin. 
 
 This section is to be compared with 157, the identity of struc- 
 
 TOPICS FOR QUIZ i7th Week. 
 
 THE SKIN AND ITS APPENDAGES. 
 
 1. What is the skin and what its appendages ? 
 
 2. Compare the skin of the palm or sole with that of the general surface 
 of the body. 
 
 3. Hairs. What are hairs, where are they present and where absent ? 
 
 4. Nails, claws and hoofs. In what animals? Structure. 
 
 5. Glands of the skin. Relations with the hairs. 
 
 6. Hair muscle. 
 
 7. Section of a lip. This shows a transition from skin to mucosa. How 
 
70 
 THE CENTRAL NERVOUS SYSTEM. 
 
 "W*. 
 LABORATORY WORK FOR THE- J- WEEK. *cj - 
 
 References : CA/v . > 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, (<r) the direct cerebellar tract, (d) 
 ascending root of the Vth, and (<?) the nucleus of the Xllth, (/) the 
 cuneate nucleus (and tract). Has the gracile nucleus disappeared ? 
 Note the (dentate) olivary nuclei causing the prominences upon 
 the lateral aspects of the oblongata (the olives). Are the fibers of 
 the Xth or Xllth nerves shown ? Note the extent of the cavity 
 (metacoele) ; the membranous roof (metatela) has been removed. 
 
 167. Pons. Transection of the oblongata 
 through the pons. Weigert's hematoxylin method ; sections 
 
 /t. Now in alcohol. Clear and mount in alkaline balsam. 
 
 Identify in the section (a) the pyramidal tracts, () the lem- 
 niscus, (c) the ascending root of the Vth, (d) the posterior longi- 
 tudinal fasciculus. Does the section pass through the nuclei of the 
 Vlth, Vllth or Vlllth nerves t Are any of these nerves shown t 
 What has become of the restiform body t Note well the fibers 
 forming the mass of the pons, their decussation, relation to the 
 medipeduncle of the cerebellum ; the nuclei of the pons. 
 
 MESKNCKPHAL. 
 
 Gross preparations. In the region cephalad of the cerebel- 
 lum and pons, the mesencephal, identify upon the dorsal aspect the 
 pregemina and postgernina (corpora quadrigemina, anterior and pos- 
 terior); upon the ventral aspect, the crura (cerebri) and the Hid 
 cranial nerve. 
 
 1 68. Crura and Gemina (Pre- or Post-). . Tran- 
 section through the mesencephal. Weigert's hematoxylin method ; 
 sections // ; now in alcohol. Clear and mount in alkaline 
 
 balsam. 
 
 In this section identify (a) the pyramidal tracts (the crusta), 
 
 (3) the fillet, (<:) the posterior longitudinal fasciculus. Note also 
 the central cavity (mesocoele) with the lining endyma, the central 
 cinerea surrounding it, the nucleus of the Hid nerve. Locate the 
 following parts: (/) the crusta, (2) the substantia nigra, (j) the 
 tegmentum, (4} the prepeduncles of the cerebellum, or their decus- 
 sation. Does the section include the red nucleus? Is the Hid 
 nerve shown ? 
 
74 
 THE CENTRAL NERVOUS SYSTEM. 
 
 LABORATORY WORK FOR THE--'-^-- WEHK. 
 
 References: IV . >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 : <sJ^ -XVIM * t V 
 
 ^ Xx * ** \ b U- 
 
 Points for Quiz : 
 
 Laboratory Report : . _ 
 
 Obtain from the delivery desk (a) paraffin sections 187, 188, 
 (b) collodion section 186. 
 
 THE EAR. 
 
 186. Middle ear, inner ear, semi-circular canals. 
 
 ; collodion ; sections yw. Stain with 
 
 hematoxylin and eosin. 
 
 Study this preparation for (a) the mucosa of the tympanum 
 and the character of the epithelium, () the bony labyrinth, mem- 
 branous labyrinth, perilymphatic space of the inner ear. Does the 
 section include one of the maculae or cristae of the membranous 
 labyrinth? Is the utricle sectioned as well as the semicircular 
 canals ? Note carefully the structure and relations of the parts. 
 
 187. Cochlea. Guinea-pig. Longisection. Zenker's fluid ; 
 decalcified (131) ; paraffin ; sections ^. Stain with hema- 
 toxylin and eosin. 
 
 Study this preparation in connection with figures in your text 
 book, identifying the parts and studying their structure. Is Reiss- 
 ner's membrane intact? 
 
 THE TASTE BUDS. 
 
 188. Papilla foliata. Rabbit. Longisection. Zenker's fluid ; 
 paraffin ; sections //. Stain with hematoxylin and eosin. 
 
 The taste buds will be found in the epithelium in the depres- 
 sions or transverse fissures of the papilla. Examine them carefully, 
 observing their form and structure, relation to the surface, and the 
 gustatory hairs. 
 
TECHNIC. 
 
 i . Very few structures of the animal body can be examined 
 microscopically without being first subjected to a preparatory treat- 
 ment involving in most cases the employment of complicated meth- 
 ods. Increase in our knowledge of the finer structure of the body 
 in the past has been accompanied and made possible by the improve- 
 ments in the methods employed. Advance in the future, likewise, 
 will be dependent on the application of a more exact technic. For 
 those who aim to do work in practical histology and pathology, a 
 mastery of the more important methods is indispensible ; for the 
 student of pathology, also, very desirable is the acquisition of skill 
 in the application of simple methods which require neither expensive 
 apparatus nor expenditure of time, methods which, while they do 
 not advance knowledge, serve to meet the needs of a rapid clinical 
 diagnosis. 
 
 Of the multitudinous methods employed in microscopic work, 
 only those are here given which are necessary for a general working 
 knowledge in histology, or are used in this course. Animal tissues 
 are bulky and unfitted for examination under high powers of the 
 microscope ; examination may be made possible in one of two 
 ways, the elements composing the structure may be separated from 
 each other, or thin slices may be prepared. Furthermore, tissue 
 may be examined in either of these ways, fresh (or alive), or after 
 special chemical treatment. The technic of histology involves 
 then : 
 
 A. Examining fresh, by either B. or C. Advantageous or neces- 
 
 sary when haste is required, or in examining the tissue 
 alive. 
 
 B. Isolation or Dissociation. Separating out the elements compos- 
 
 ing a tissue by (a) teasing or (b) treatment with reagents 
 and teasing. 
 
 C. Cutting thin sections of the tissue or organ. 
 
82 
 
 For C. are generally necessary : 
 
 1. Fixing the tissue ( 18-38). Hardening. 
 
 2. Sectioning by one of the following methods : 
 
 (a) Free-hand, without an imbedding mass, or 
 
 (b) With an imbedding mass, as 
 
 (1) By the Paraffin method ( 42), or 
 
 (2) By the Collodion method ( 50), or 
 
 (3) By the Freezing method ( 61). 
 
 ISOLATION. 
 
 2. One of the simplest ways of examining the structure of 
 a tissue is the separation from one another of the structural elements 
 composing it, thus permitting its analysis. Likewise, for a correct 
 conception of the forms of the cells and fibers of the various tissues 
 of the body, one must see these elements isolated and thus be able 
 to inspect them from all sides. It frequently occurs also that isola- 
 tion is not quite complete and one can see in the clearest manner 
 the relations of the cells or fibers to one another. 
 
 In the employment of this method the tissue may be taken 
 fresh and isolation accomplished by teasing with needles or similar 
 instruments ; or it may be treated with media which will serve to 
 render teasing partially or entirely unnecessary. In such cases 
 simply shaking or gently tapping the preparation will often suffice. 
 In many instances it is desired to examine the tissue while the 
 elements are still alive, as, for example, in the study of ciliated 
 cells, and recourse must be had to some "normal," "indifferent" 
 medium. Best of all is the medium with which they are bathed dur- 
 ing life in the case of tissue from the animal body, blood serum, 
 the aqueous humor of the eye, liquor amnioticus, or, as an artificial 
 substitute often more convenient if reagents are to be used subse- 
 quently, normal salt solution, being a iV~A^ solution of common 
 salt (sodium chlorid) in distilled water. 
 
 The chemical agents or solutions for isolating are, in general, 
 the same as those used for fixing and hardening. But the solutions 
 are only about one-tenth as strong as for fixing and the action is very 
 much weaker and requires from one or two hours to as many days. 
 In the weak solution the cell cement or connective tissue is softened 
 
83 
 
 so that cells and fibers may be separated from one another, and at 
 the same time the cells are preserved. In other words, a weak fix- 
 ing action is retained while the hardening action is reduced on dilu- 
 tion. The time required for the action of the dissociator varies in- 
 versely as the vehemence of the fixer and the density of the tissue, 
 2-3 hours to several days. In fixing and hardening, on the other 
 hand, the cell cement, like the other parts of the tissue, is made 
 firmer. It is better also to dilute the fixing agents with normal 
 salt solution than merely with water. 
 
 3. Of the many dissociators, the following may serve most 
 of the needs of histology : (i) Miiller's fluid dissociator ; (2) for- 
 maldehyde dissociator ; (3) nitric acid dissociator ; (4) caustic pot- 
 ash dissociator ; (5) Ranvier's one-third alcohol ; (6) osmic acid 
 
 (A*). 
 
 4. Miiller's fluid dissociator. Formula: Miiller's fluid, 
 i part ; normal salt solution, 9 parts (i. e., potassium dichromate, 
 2.5 grams; sodium sulphate, i gram; sodium chlorid, 6 grams; 
 water, 1,000 c.c.). 
 
 This is a good general dissociator for epithelia, including glands. 
 Dilution decreases the hardening action of Miiller's fluid as is 
 shown especially on the cell-cement, hence its dissociating ac- 
 tion. Considerable latitude in time is allowed in the use of this 
 dissociator ; 12 hours being often sufficient, although a stay of sev- 
 eral days in the dissociator usually does no harm. 
 
 5. Directions for use. In the employment of this fluid for 
 the isolation of epithelial cells, proceed as follows : 
 
 Place the tissue covered with the epithelium which it is desired 
 to isolate in the dissociator in a shell vial or dish, where it may 
 remain from 2-3 hours to 2-3 days ; for the epithelium of the tra- 
 chea, intestines, etc., the action is sufficient in 2-3 hours, although 
 good preparations may be obtained after two days or more. For the 
 stratified epithelia, like those of the skin, mouth, etc., it may require 
 1-3 days for the most satisfactory preparations. After the tissue has 
 remained in the dissociator a sufficient time, scrape the epithelial 
 surface gently with a scalpel and place the scrapings on a slide in a 
 drop of the dissociator ; cover and examine. If one proceeds after 
 two hours or so, probably most of the cells will cling together, and 
 
8 4 
 
 in the various clumps will appear cells on end showing the tops or 
 bases, and other clumps will show the cells in profile. 
 
 Tap the cover gently with a needle-holder or other light object 
 in order to separate the cells from each other more completely. 
 Many fully isolated cells as well as cells in groups will be seen. 
 Examine carefully. 
 
 6. Staining. Scrape gently the epithelial surface in a fresh 
 spot and place the scrapings on the slide in a drop of eosin ( 94) or 
 a mixture of eosin and methylgreen ( 92). Mix well so that the 
 stain can penetrate. If for temporary examination, cover immedi- 
 ately and examine as before. 
 
 7. Permanent preparations. If a permanent mount is de- 
 sired, (a) allow the above mixture to stand for 5-10 minutes ; drain 
 off the stain, collecting the fragments carefully at one side ; add a 
 small drop of glycerin or glycerin-jelly ( 104, 105) ; cover and 
 examine, tapping the cover gently as before, if necessary, in order 
 to separate the cells more thoroughly. Unstained preparations may 
 be mounted in the same manner. 
 
 () The following method will also give satisfactory stained 
 preparations. Place a small mass of cells in a drop of alum carmine 
 and eosin glycerin ( 156) ; cover and isolate the cells by tapping 
 gently on the cover-glass as before. Clean and seal the preparation 
 ( no). 
 
 If it is desired to make a permanent mount of a preparation 
 already covered and examined, place a drop of glycerin at the edge 
 of the cover, apply to the opposite side of the cover a piece of blot- 
 ting paper or filter paper, and by absorbing the fluid dissociator or 
 stain under the cover, cause the glycerin to flow under to take its 
 place. 
 
 Seal the preparation ( no) ; it is usually better, however, to 
 allow a glycerin or glycerin-jelly mount to stand for a day, being 
 careful that it is not touched, and then seal. 
 
 8. Formaldehyde Dissociator. Formula : 40% formal- 
 dehyde (formalin), 2 c. c. ; normal salt solution, i,oooc. c. (i. e., 
 .08% sol. of formaldehyde in normal salt solution). This is a good 
 general dissociator and as such maybe employed instead of Miiller's 
 fluid dissociator. It is especially serviceable in the isolation of the 
 nerve cells of the brain and spinal cord. 
 
5 
 
 9. Directions for use. Employ this fluid for the isolation of 
 the nerve cells of the spinal cord and of the cerebral cortex, pro- 
 ceeding as follows : 
 
 Split the spinal cord along its median plane, separating thus 
 the two halves, and place it in an abundance of the dissociating 
 fluid. The cerebral cortex should be cut into small pieces by sec- 
 tions vertical to the surface. Allow it to remain in the dissociator 
 from 2-24 hours ; for the best results a stay in the fluid of more 
 than 24 hours is not so satisfactory ; although isolated cells are 
 readily obtained their processes are broken off much nearer the cell 
 body. 
 
 ro. Place a fragment of the cinerea of the spinal cord or the 
 cortex of the cerebrum on a clean slide in a drop of T ^% eosin in 
 formaldehyde dissociator ; with the blade of a scalpel crush the tis- 
 sue, grinding it thoroughly with a rotary movement, which will re- 
 duce it to small pieces. Gather the debris together, drain off the 
 fluid, and add a drop of glycerin containing ^% of eosin. Cover 
 and examine, tapping the cover sharply with the handle of the 
 scalpel to shake out the processes of the cells and free them from 
 surrounding matter. Examine, searching for cells with many and 
 long processes. If a satisfactory preparation, seal according to 
 
 II0 - 
 
 11. Nitric acid dissociator.* Formula : Strong nitric acid, 
 20 c.c. ; water, 80 c.c. This fluid is employed in the isolation of 
 muscle fibers, both striated and plain. The nitric acid acts upon the 
 connective tissue surrounding the muscle fibers, softening and gela- 
 tinizing it so that the fibers may be quite easily separated from one 
 another. 
 
 12. Directions for use. Place in the nitric acid dissociator 
 the fresh striated muscle, gland or organ containing the muscle, 
 (plain or striated,) that it is desired to isolate. If it is the inten- 
 tion to work out the anatomy of the muscle or the relation of the 
 muscular coats in an organ, the entire muscle or organ should be 
 taken ; otherwise, portions will suffice. At the ordinary tempera- 
 ture of the laboratory the dissociating action will have been sufficient 
 
 * A more detailed discussion may be found in the original paper : " Stain- 
 ing and Permanent Preservation of Histological Elements Isolated by means of 
 Caustic Potash (KOH) or Nitric Acid (HNO 3 )," by Simon H. and Mrs. Susan- 
 na Phelps Gage. Proc. Am. Soc. of Microscopists. 1889 : pp. 34~45- 
 
86 
 
 in from i to 3 days ; test at intervals with needles to ascertain 
 whether the fascicles and fibers can be easily separated ; or frag- 
 ments may be shaken in a test tube or vial with water in order to 
 separate the fibers. 
 
 When the dissociation is sufficient pour off the acid and wash 
 the muscle gently but thoroughly with water. If the tissue is to be 
 stained with hematoxylin or carmine, or kept for any length of 
 time, drain off the water and add a half- saturated solution of alum. 
 For permanent storage, pour off the alum solution and place suc- 
 cessively in 67% and 82% alcohol. 
 
 For temporary examination, tease out a portion of the muscle in 
 water, separating the fibers carefully by means of needles ; cover 
 and examine. 
 
 13. Permanent preparations, (a} unstained. After teasing 
 out with the needles drain off the water and add a small drop of 
 glycerin or glycerin jelly ; cover, and seal after first properly clean- 
 ing ( no, in). (^) stained. Either before or after the final 
 teasing stain with picric alcohol, picrofuchsin (for relation of muscle 
 fiber to tendon), or after the alum solution with hematoxylin or car- 
 mine. Wash away the staining fluid with water and mount (a) in 
 glycerin or glycerin jelly ( 104, 105), or (V) dehydrate, clear, and 
 mount in balsam ( 107 + ). 
 
 14. Caustic potash dissociator.* Formula: Caustic pot- 
 ash, potassium hydroxid (in sticks), 3540 grams ; distilled water, 
 65 or 60 c.c. This solution will be used for the isolation of cardiac 
 muscle cells, although it may be used for striated or plain muscular 
 tissue, or as a general dissociator. It may also be employed for iso- 
 lating the cells of hair, horn or nail, either full strength or diluted. 
 
 15. Directions for use. Place in the fluid small pieces of the 
 heart muscle of a fetal, new-born or young animal ; after 10 or 15 
 minutes, the tissue should be tested with needles at intervals of 
 about five minutes, so that the action may not be too prolonged ; 
 probably 15-30 minutes will suffice. As soon as the elements sepa- 
 rate readily, pour off the caustic potash solution and add an abund- 
 ance of 60% solution of potassium acetate (potassium acetate, 60 
 grams ; distilled water, 40 c.c.). Take small fragments and tease 
 them in this solution, or shake them in a vial, until the cells are sep- 
 arated from each other. 
 
87 
 
 For temporary examination, cover in a drop of the potassium 
 acetate solution. For permanent preparations, drain off the potassi- 
 um acetate solution and add a small drop of glycerin or glycerin- 
 jelly. 
 
 Stained preparations. Pour off the potassium acetate solution 
 and add a half saturated solution of alum, letting it remain for 24 
 hours or longer. Tease in water, stain with hematoxylin or carmine, 
 wash away the stain with water, and add a drop of glycerin or gly- 
 cerin-jelly. Cover and seal ( no). 
 
 If a large amount is desired, the tissue may be carried through 
 the various steps in a vial. 
 
 16. Ranvier's one-third alcohol. Formula : 95% alco- 
 hol, t part, water 2 parts. This is an excellent dissociating fluid 
 for epithelia. An action of 24 hours is generally sufficient. A 
 weaker solution is often advantageous. 
 
 17. Osmic acid. A -fa% solution of osmic acid is a valua- 
 ble dissociator, especially serviceable in the isolation of nerve-fibers, 
 myelinic and amyelinic, and when fat is present, since fat and the 
 myelin of myelinic nerve-fibers are blackened by it. 12-24 hours 
 generally affords sufficient time for it to act. 
 
 FIXATION. 
 
 1 8. lyiving tissue, when allowed to die and remain undis- 
 turbed, gradually loses the structural features it had, in life and 
 undergoes disintegration and decay. A fixer is a fluid (or gas) into 
 which the living, or at least very fresh, tissue is placed in order to 
 preserve the structure of its elements as nearly as possible as in life. 
 
 The action of the fixer is a chemical action in which the living 
 substance is coagulated (fixed) by the reagent, which in some cases 
 forms a chemical union with the organic substance. The chemical 
 action is generally attended with a more or less marked mechanical 
 distortion, as shrinking or swelling, to obviate which chemicals of 
 opposite tendencies are, in the best fixers, combined with each other 
 in proportions intended to neutralize such effects. The chemicals 
 of most service as fixers are (i) osmic acid, (2) platinic chlorid, (3) 
 picric acid, (4) acetic acid, (5) chromic acid, (6) mercuric chlorid, (7) 
 nitric acid, (8) ethyl (or methyl) alcohol ; also, (9) potassium 
 dichromate, (10) sodium sulphate, (n) copper sulphate. The last 
 
three are not as strictly fixers as hardeners. The following solu- 
 tions containing these salts, combined or uncombined, are used as 
 fixers in this course : (i) Osmic acid, aqueous solution ; (2) Her- 
 mann's fluid ; (3) Flemming's fluid (stronger formula) ; (4) Picric 
 alcohol ; (5) Mercuric chlorid, in aqueous solutions ; (6) Picro- 
 aceto-sublimate (vom Rath's fluid) ; (7) Zenker's fluid ; (8) Miil- 
 ler's fluid; (9) Brlicki's fluid; (10) Potassium dichromate in 
 aqueous solutions; (n) Chrome-oxalic; (12) Alcohol (absolute 
 67% strength) ; (13) Formaldedyde ; also, as embryological fixers, 
 (14) Perenyi's fluid ; (15) Picro-sulphuric ; (16) Picro-nitric ; (17) 
 Nitric acid. 
 
 19. The following general rules should be regarded in the 
 fixation of tissues and organs : 
 
 1 i ) The volume of fixing-fluid used should be large, exceed- 
 ing the volume of the tissue at least thirty times. The less ener- 
 getic the action of the fixer, the greater the amount of fluid to be 
 employed. When the fluid becomes turbid, it should be changed to 
 fresh at once. 
 
 (2) Fix only as small pieces of tissue as possible, or as practi- 
 cable with the results in view. The block of tissue should not be 
 more than i c.m. in any diameter, and, if possible, let one of the 
 diameters be much shorter, only one- fourth or one-half of a centi- 
 meter. This is desirable for the rapid -and complete penetration of 
 the fixer. Of course, in the case of entire organs, it may not be pos- 
 sible to comply with the conditions. 
 
 In addition to these two general principles, there are four points 
 to be carefully considered, upon which the excellence in the results 
 attained depends ; they are (a) the fixer chosen, (&) the time of fix- 
 ation, (c) the washing out of the fixer, and (d) the subsequent hard- 
 ening in alcohol. 
 
 (#) The choice of a fluid into which the tissue is placed 
 should be made dependent on ( i ) a consideration of the degree of 
 excellence of fixation that is desired or necessary, whether, in other 
 words, details of cell structure or the structure of the tissue in terms 
 of cells, be sought ; (2) the penetrating power of the fixer and the 
 size of the piece of tissue that it is necessary to have ; and (3) the 
 stain that is desired subsequently, which is largely affected by the 
 fixer employed. For example, Hermann's fluid is possibly the finest 
 
8 9 
 
 fixer known, but its penetrating power is weak, and it gives good 
 results only when very small pieces of tissue are taken ; furthermore, 
 the number of stains that give good results after its employment is 
 limited. On the other hand, such fluids as Miiller's fluid or Er- 
 licki's fluid should be avoided when the preservation of finer nuclear 
 structures is desired. Within these limits, the fixer that will 
 give the best results in a special case must be determined by experi- 
 mentation, or the experiments and results of others accepted and 
 their methods applied. 
 
 (b) The time a fixer is allowed to act must be considered in 
 connection with the character of the fluid and the tissue. Often the 
 exact limitation of time is a matter of secondary importance ; in 
 other cases, however, its disregard affects the results seriously, and 
 as a general rule, there is a minimum and a maximum time and be- 
 tween them an optimum time that should be adhered to. 
 
 (r) After the tissue has been in the fixing fluid a proper length 
 of time, it is necessary that it be washed thoroughly to remove the 
 fixer from it. This should be done by means of water or alcohol or 
 both. In general, fixers containing salts insoluble in alcohol, or but 
 slightly soluble, as osmic acid, chromic acid, potassium dichromate, 
 etc. , should be thoroughly washed in water. Fixers containing pic- 
 ric acid should always be removed by alcohol ; mercuric chlorid may 
 be washed out by either water or alcohol. 
 
 Inadequate washing out of the fixer may either seriously affect 
 the cutting quality of the tissue if it is to be subsequently imbedded, 
 the ease with which it can be stained, or there may be formed pre- 
 cipitates in the tissue, giving illusory effects, distortions, or at least 
 a dirty appearance to the preparation. Time in properly washing 
 out a fixer is always well spent, as it is a matter for serious attention. 
 
 20. Resume. In brief, then : In fixing, take relatively 
 large amounts of fluid and small pieces of tissue, choose the fixer 
 well with a view to the tissue and the results desired, regulate the 
 time carefully, and wash out thoroughly. 
 
 HARDENING. 
 
 21. Each fixer has also more or less of a hardening action 
 upon the tissue. Some fluids spoken of above as fixers are preemi- 
 nently hardeners, such as Miiller's fluid and Erlicki's fluid ; while 
 
go 
 
 with others the hardening action is a minimum, e. g., aqueous solu- 
 tions of picric acid. The hardening action of the fixer is generally 
 supplemented by the subsequent use of alcohols of increasing 
 strengths (50% to absolute), as well as in preparation for the paraffin 
 and collodion methods of imbedding. In fact, with modern meth- 
 ods of imbedding excessive hardening of the tissue is not necessary 
 and indeed often should be avoided as affecting the cutting quality 
 of the tissue. Tissue after fixation has been completed may be 
 stored in 82% or 95% alcohol, or (better) imbedded at once. 
 
 FIXERS. 
 
 $ 22. Osmic acid. A very useful as well as expensive reagent and some- 
 what difficult to use. It is generally employed as a fixer in conjunction with 
 other reagents, as in the mixtures below ($$ 23 and 24). When used alone as 
 a fixer weak solutions are generally best T$~ I %- ^ penetrates slowly 
 and it "over-fixes" cells very easily, obscuring detail and giving the parts a 
 homogeneous, glassy appearance. Over-fixed cells cannot be stained, or with 
 great difficulty. More or less blackening of the protoplasm also occurs. It 
 will be used in this course chiefly to demonstrate fat, which is blackened by 
 it, and the zymogen granules of pepsin and trypsin, which it preserves and 
 browns slightly. 
 
 Fix small (about % c. c.) pieces of tissue in i% osmic acid for 6-12 hours, 
 wash well in water (running or changed frequently) for 12-24 hours, and place 
 in 67% and 82% alcohols. It is somewhat difficult to prevent pure osmic acid 
 of this strength from over-fixing the tissue, and cell detail is generally lost, 
 though the form of cells is well preserved. 
 
 23. Hermann's fluid. Formula: \% aq. sol. platinic chlorid, 15 parts ; 
 2% aq. sol. osmic acid, 4 parts ; glacial acetic acid, i part ; or you may take 
 10% aq. sol. platinic chlorid, 3 parts ; \% aq. sol. osmic acid, 16 parts ; glacial 
 acetic acid, 2 parts ; water, 19 parts. This is generally recognized as the finest 
 fixer known, and it is also the most expensive. The form and structure of 
 cells are well preserved. It should only be employed, however, with very small 
 pieces of tissue, and is to be used especially when cell structure is to be 
 studied. Fat and the myelin of nerve fibers are stained black. 
 
 Fix in this 1-24 hours (or longer days or weeks are used by some), wash 
 well in water (running or frequently changed) 2-24 hours, and then place in 
 67% and 82% alcohols, 12-24 hours in each. In using this fluid, the smaller 
 the pieces taken the better the fixation will be, and in order that it be possible 
 to obtain a good stain afterwards tissue should not be over-fixed and the fixer 
 should be thoroughly washed out. If there is a blackening of the tissue, or a 
 precipitate in it, both may be removed by treatment of the sections on the 
 slide with a 10-20% solution of hydrogen dioxid in 67% alcohol. Employ 
 
after Hermann's fluid, as stains, Heidenhain's iron hematoxylin, Delafield's 
 hematoxylin, safranin (as a red stain), or gentian violet (as a blue stain). 
 
 \ 24. -Flemming's fluid (Chrome-aceto-osmic). Formula: i% aq. sol. 
 chromic acid, 15 parts; 2% aq. sol. osmic acid, 4 parts; glacial acetic acid, i 
 part; or, ro aq. sol. chromic acid, 3 parts ; i% osmic acid, 1 6 parts ; glacial 
 acetic acid 2 parts ; water, 19 parts. This is a fine fixer and in most cases gives as 
 good results as Hermann's, and is not as expensive. It browns tissue less, and 
 while it blackens fat, does not blacken the myelin of myelinic nerve fibers 
 as does Hermann's. It should be employed in general in the same cases and 
 in the same way as Hermann's fluid. 
 
 Fix tissue 1-24 hours (or longer); wash well in running water 2-24 hours ; 
 place in 67% and 82% alcohols, 12-24 hours in each. Bleaching of the sections 
 may be necessary, as with Hermann's fluid. Take only very small pieces of 
 tissue. Employ the same stains as with Hermann's fluid. 
 
 \ 25. Picric alcohol. Formula: Distilled water, 250 c. c. ; 95% alcohol, 
 250 c. c. ; picric acid, i gram. A delicate fixer, penetrates well, and does not 
 make the tissue brittle or tough. It may be used with most tissues and organs. 
 
 Allow it to act 1-3 days, changing to fresh each day if the bulk of tissue 
 is large, transfer to 67% alcohol for 1-2 days, 82% alcohol several days, chang- 
 ing several times. If the paraffin method of imbedding is to be employed it 
 is best to leave the tissue in 82% alcohol until no more or very little color 
 comes away from it, as otherwise it affects the cutting quality of the tissue. 
 Most staining methods may be employed. Staining is good, though not quite 
 as brilliant as with mercuric chlorid fixers. 
 
 % 26. Mercuric chlorid. One may employ (a) a saturated solution in 
 water, or better, (b) a saturated solution in normal salt solution with 1-5% gla- 
 cial acetic acid. Water will dissolve about 5%, normal salt solution about 12% 
 of the mercuric chlorid. This is a good fixer, especially when the piece is 
 small. It fixes as soon as it penetrates and is apt to make tissue brittle if it is 
 left too long. Staining after it is brilliant. The larger percentage of acetic 
 acid is, perhaps, to be preferred for most histological objects. 
 
 Fix the fresh tissue ^-24 hours according to the size of the piece. Re- 
 move to 67% alcohol for 1-2 days, 82% alcohol several days, changing often. 
 The 82% alcohol should contain enough tincture of iodin to give it a yellow 
 color, and fresh tincture added or (better) the alcohol changed when the yel- 
 low color of the iodin in the alcohol is lost. As long as the alcohol is decol- 
 orized, washing should be continued, since it is important that the mercuric 
 chlorid be all removed from the tissue ; otherwise precipitates will form in the 
 preparation after it is mounted or before, and spoil the result. Wash out in 
 alcohol thoroughly and carefully. Almost any stain may be employed after a 
 mercuric chlorid fixation. 
 
 g 27. Picro-aceto-sublimate. (vom Rath' s fluid). Formula : Saturated 
 aqueous solution of picric acid, 50 c.c. ; saturated aqueous solution of mercuric 
 chlorid, 50 c.c. ; glacial acetic acid, i c.c. (1-5 c.c. according to the organ). 
 This is quite a satisfactory fixer if care is taken in its employment, combining 
 
9 2 
 
 to a certain extent the advantages of picric acid and mercuric chlorid. A 
 stronger percentage of acetic acid (4 or 5%) is to be preferred with organs con- 
 taining a large amount of connective tissue. 
 
 Fix the object for 1-24 hours ; place in 67% alcohol for 1-2 days, 82% alco- 
 hol for several days, changing several times, whenever the alcohol has become 
 colored yellow. The tissue should be left in the 82% alcohol until almost all 
 the picric acid has been washed out and the alcohol no longer becomes colored, 
 or but slightly. Tincture of iodin may be used as with mercuric chlorid 
 tissue, but is not necessary. Stain as you like. 
 
 $28. Zenker's fluid. Formula: Potassium dichromate, 2.5 gr. ; sodium 
 sulphate, i gram; mercuric chlorid, 5 gr. ; water, 100 c. c.; and add before 
 using, glacial acetic acid, 5 c. c. This is a fluid used quite generally of late. 
 The potassium dichromate seems to check the brittleness that the mercuric 
 chlorid would cause, otherwise its advantages and faults are those of mercuric 
 chlorid ; staining after it, however, is apt to be a little more difficult and not 
 as brilliant as with mercuric chlorid fixation. Its penetration is surprisingly 
 good. 
 
 Fix in Zenker's fluid 12-48 hours, wash well in water, running or fre- 
 quently changed, 12-48 hours, to remove the dichromate ; transfer to 67% alco- 
 hol for 1-2 days, 82% alcohol for several days, keeping in the dark while in 
 the alcohol. To the 82% alcohol add a drop or so of tincture of iodin, adding 
 fresh iodin or changing the alcohol when the color is lost. This should be 
 continued as long as the iodinized alcohol is decolorized in order to avoid a 
 precipitate of the mercuric chlorid in the tissue. Avoid, however, adding an 
 excess of iodin, -since it will affect the s taining quality of the tissue. Stain as 
 you wish. 
 
 $ 29. Miiller's fluid. Formula : Potassiurii dichromate, 2.5 grams; sodium 
 sulphate, i gram ; water, 100 c.c. This fluid is more of a hardener than a fixer ; 
 it should be avoided (as likewise Erlicki's fluid and potassium dichromate) 
 when the preservation of nuclear structure is desired. Staining after its use is 
 sometimes difficult. It is, however, useful for general work. 
 
 Place the object in an abundance of the fluid and harden for from 1-8 
 weeks, changing the fluid at first each day. In general, 10-14 days will be suf- 
 ficient. Wash in running water for 24-48 hours or longer, remove to 67% alco- 
 hol for 1-2 days, 82% alcohol several days. Keep in the dark while in the alco- 
 hols, and change to fresh when the fluid is colored yellow. Tissue hardened 
 in Miiller's fluid cuts well, and it is useful in preparing sections of large organs, 
 or organs with much connective tissue. 
 
 % 30. Erlicki's fluid. Formula : Potassum dichromate, 2.5 grams ; copper 
 sulphate, i gram ; water, 100 c.c. This is quite similar to Miiller's fluid in its 
 action and results, save that its action is more rapid and stronger. Therefore, 
 it had better be employed with smaller objects, and allowed to act only 2-14 
 days. Otherwise, employ like Miiller's fluid. 
 
 \ 31. Potassium dichromate. 2%, 3%, and 5% aqueous solutions. 
 
93 
 
 This is quite similar to M tiller's fluid in its action, and may be employed in the 
 same cases. It is generally used for the central nervous system. 
 
 Harden in an abundance of the solution for 2-8 weeks, beginning with the 
 2% solution for 2-6 days, 3% solution 1-3 weeks, 5% solution 1-3 weeks. 
 Wash out in running water 24-48 hours. Place in 67% and 82% alcohols sev- 
 eral days, keeping in the dark meanwhile, changing when the alcohol is 
 colored. 
 
 I 32. Chrome-oxalic (Graf). Formula: 8% aqueous solution of oxalic 
 acid, 4 parts ; 95% alcohol, 3 parts ; i% chromic acid, 3 parts. Mix the solu- 
 tions in the order given. 
 
 Fix tissue in the solution 1-6 hours, wash in running water >-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 (<r). 
 
 (a) Albumin fixative and ^ % collodion. Place upon the 
 slide a small drop of albumin fixative, spreading it out with a clean 
 finger into a very thin, even layer. Place the paraffin section in 
 position upon the slide and with a clean finger, press the section 
 into the albumin fixative, beginning at one edge of the section and 
 by a rolling motion of the finger, ironing out any wrinkles there may 
 be. With a camel's hair brush and by a single sweep, spread over 
 the section a thin coating of y^% collodion. Allow it to dry in the 
 air for a minute or so and then place the slide in a jar of benzin. Be 
 sure that the y% collodion in the bottle has not thickened, and 
 spread as little over the section as possible, otherwise a much longer 
 time in the benzin will be necessary. 
 
 (b) Albumin fixative and heat. The employment of y\% col- 
 lodion causes the benzin to dissolve out the paraffin from the sections 
 slowly, and the collodion also stains intensely with certain dyes, 
 such as safranin, gentian violet, methylgreen, methylene blue, iron 
 hematoxyline, etc. When these stains are to be used, fasten to the 
 slide in the manner following : Prepare the slide with albumin fixa- 
 tive and press the section to the slide as directed above. Look upon 
 the reverse side of the slide to see if the section really adheres to the 
 albumen fixative, as in some cases it does not. Heat the slide gently 
 and slowly over a small flame until the paraffin melts and begins to 
 run away from the specimen. Keep the paraffin just melted for a 
 minute or so, and then transfer to the benzin. A minute or two in 
 benzin will suffice. Should the paraffin section not adhere to the 
 albumin fixative when well pressed down, it can in many cases be 
 made to do so by briskly rubbing the reverse side of the slide with a 
 woolen or silk cloth. 
 
 (c) Extending sections with warm water. If the sections are 
 wrinkled, as they often are, remove the wrinkles by extending the 
 sections on warm water, as follow T s : Place the sections upon a slide 
 prepared with a very thin, even layer or film of albumin fixative 
 ( 67, ), and add at the side enough water to float them. Warm 
 the slide carefully and slowly over a small flame until they begin to. 
 straighten out. Do not let the water become warm enough to melt 
 
no 
 
 the paraffin. When the sections have become quite flat and smooth, 
 drain off the water and allow the slide to stand 2-3 hours, or better 
 over night, when a thin coating of Y\% collodion is given ( 67, a} 
 and they are placed in benzin. 
 
 If the sections are large and thin, the coating with Y\% collo- 
 dion may be omitted ; in this case, let the sections stand 12-24 hours 
 after spreading, or for 2-3 hours if left in a warm place, as in an 
 incubator. 
 
 The sections may also be straightened out by placing them on 
 a dish of water, such as the waste-jar, and hot water added slowly 
 until the sections extend, when, after the water has cooled, they 
 may be transferred to albuminized slides and carried on as before. 
 
 PREPARATION FOR STAINING. 
 
 68. Benzin. Both paraffin and collodion sections are trans- 
 ferred to benzin, which in the first case dissolves out the paraffin, in 
 the second serves to remove the clarifier still present in the tissue 
 and collodion. 
 
 Leave paraffin sections in benzin until the paraffin is entirely 
 dissolved, requiring from a minute or so to 10-15 minutes, if $% 
 collodion was used in fastening the sections to the slide. 
 
 Collodion sections should remain in the benzin 5-15 minutes or 
 longer, according to the thickness of the sections. Benzin may be 
 omitted and a proportionately longer time in the alcohol given. 
 
 Sections may be left in benzin over night, and 2-3 days even 
 might do no harm, but it is better to proceed at once to the other 
 operations. 
 
 69. Alcohol. Transfer from benzin to 95%alcohol to re- 
 move the benzin, leaving the section in the alcohol 5-10 minutes ; 
 or, if you wish, shorten the period to a minute or so by waving the 
 slide gently to and fro in the alcohol. A stay of a day or so in alco- 
 hol does no harm. Collodion sections generally require a longer 
 period than paraffin sections because of the collodion mass present 
 and their greater thickness. 
 
 70. Water. Remove the 95% alcohol from the section by 
 plunging the slide into a jar of tap water and gently waving it 
 about in it. Should the stain to be employed be an alcoholic solu- 
 tion this step may be omitted. 
 
Ill 
 
 STAINING. 
 
 71. While staining has a value in histology in coloring the 
 preparation as a whole and thus making its parts stand out more 
 sharply, the aim is to employ such stains as by their selectivity give 
 different parts or structures different colors or intensities of color, 
 thus differentiating them the better. 
 
 In histology, selective stains are employed (a) to color the 
 nucleus differentially, thus distinguishing it sharply from the cell 
 body, and by means of the nucleus setting apart groups of cells ; 
 (b) to distinguish between different kinds of tissue, as, for example, 
 connective tissue and muscle. 
 
 72. The following two general rules may be given : 
 
 1 i ) The preparation should be transferred to the stain from 
 the fluid in which the staining principle is dissolved. The majority 
 of the stains employed are aqueous solutions ; some, however, such 
 as hydrochloric acid carmine, are alcoholic ; with these alcoholic 
 stains the last preparatory step ( 70), removal of the alcohol by 
 water, should be omitted. Many stains may be used either in 
 aqueous or in alcoholic solution. 
 
 (2) The stain should be washed out with the solvent of the 
 dye, water or alcohol as the case may be. 
 
 With many aniline dyes ( 74) one may, and in many cases 
 should, employ alcohol for washing out, even though the staining 
 solution is aqueous. 
 
 73. Differentiation. To bring out the differential or 
 selective coloring of the dye. This is necessary with many stains, 
 especially anilines, and may often be satisfactorily accomplished by 
 the use of 95% alcohol and made a part of the washing out. Alcohol 
 acidified with hydrochloric acid ( 149) is sometimes used, or a 
 special differentiator is required. 
 
 74. Classification. (#) According to their chemical nature 
 the stains generally used are (i) hematoxylin stains, (2) carmine 
 stains, (3) aniline stains 
 
 (d) According to their selectivity. Stains as used in histology 
 may for convenience be classified as (i) nuclear, (2) general, and 
 (3) special. These are often used in combinations of two or three, 
 either together in the same solution or separately in solutions used 
 
112 
 
 successively. Double and triple stains are usually combinations of 
 nuclear with general or special stains. Quadruple stains are less 
 serviceable ; see, however, p. 68, 154. 
 
 (c) According to the mode of employment, staining may be 
 either (i) in toto, or (2) section staining. When in toto stain- 
 ing is employed the piece of tissue is stained entire, and imbedded 
 and sectioned afterwards. In this case the tissue should be stained 
 before the process of imbedding is begun, after the washing out of 
 the fixer has been completed ( 19, 21). But a single stain may be 
 given, and the one chosen is generally a nuclear one hematoxylin 
 or carmine. 
 
 (2) Section staining, i. e., after the tissue is imbedded and sec- 
 tions cut. This is more serviceable, especially if highly differential 
 results are desired. 
 
 75. Impregnations. In addition to the typical methods of 
 coloring tissue by means of stains there is a group of methods in 
 which the coloring matter is deposited in the cell or tissue that it is 
 desired to differentiate, in the form of a precipitate. These are 
 known as impregnation methods and are of great value, especially 
 as applied to nervous tissues. A hard and fast line, however, can- 
 not be drawn between true staining methods and impregnations. 
 Silver nitrate and gold chlorid are the substances most generally 
 employed in the impregnation of tissue ( 146). 
 
 76. Choice of stains. Remember that the staining is 
 greatly affected by the previous treatment ; brilliancy or selectivity 
 in the result being in many cases dependent on the fixer employed 
 or impaired by the improper or incomplete washing out of the fixer. 
 
 In staining, therefore, consider three things, (a) what it is de- 
 sired to bring out, what kinds of stains you need to employ, (ft) 
 the mode of fixation that has been employed, and (V) the imbedding 
 method must also be considered in the choice, since if collodion is 
 employed certain stains that color it deeply should be avoided. 
 
 77. The time of staining. Although in general certain 
 time limits can be given to the period during which a stain should 
 be allowed to act ; with most stains, especially those with which no 
 differentiation is needed, such as hematoxylin, and most carmines, 
 the correct intensity of color should be determined by examining the 
 preparation with the microscope. One soon becomes able to judge 
 
of the right stain in this way better than if a given time were ad- 
 hered to. 
 
 In the use of stains requiring a subsequent differentiation, the 
 rule is to over^stain and watch the differentiation carefully with the 
 microscope, stopping it when sufficient. In this case it is the differ- 
 entiation and not the staining that should be carefully regulated. 
 
 78. In this course are employed : (i) Chloral hematoxylin, 
 (2) Ehrlich's acid hematoxylin, (3) Delafield's hematoxylin, (4) 
 Weigert's hematoxylin, (5) Borax carmine, (6) Alum carmine 
 (Grenacher's), (7) Paracarmine, (8) Hydrochloric acid carmine, 
 (9) Picro-carmine, (10) Safranin, (n) Gentian violet, (12) Methyl- 
 green, (13) Ehrlich's triacid mixture, (14) Eosin, (15) Erythrosin, 
 (16) Picric alcohol, (17) Picro-fuchsin ; also (18) Heidenhain's 
 iron hematoxylin, and special stains given subsequently under 
 "Special Methods." 
 
 79. The combinations most employed are (a) hematoxylin 
 and eosin (or erythrosin), and (^) hematoxylin and picrofuchsin, 
 the former a double and the latter a triple combination. 
 
 The following scheme indicates the manner of their employment : 
 
Paraffin or Collodion Sections 
 (66 and 67) 
 
 v 
 Benzin 
 
 95 % Alcohol 
 
 Water 
 
 v 
 Hematoxylin, 10-30 min. 
 
 v 
 Water 
 
 v 
 
 Picrofuchsin. eosin 
 or erythrosin, ^2-2 min. 
 
 v 
 Distilled water or 67 % alcohol 
 
 v 
 Dehydrate (95-99 % alcohol) 
 
 Clear 
 
 (carbol-xylene) 
 
 y 
 Mount in Balsam 
 
 STAINS. 
 
 | 80. Chloral Hematoxylin. (Proc. Am. Micr. Soc., vol. XIV, pp. 125- 
 127). Formula: (A) Hematoxylin crystals, -fa gram, dissolved in 10 c.c. of 
 95% alcohol ; (B) distilled water, 100 c.c., potash alum, 3^ grams ; boil for 5 
 minutes in an agate-ware or glass vessel and add sufficient boiled water to bring 
 the volume back to 100 c.c. When cool, mix (A) and (B) and add chloral hy- 
 drate, 2 grams. Allow the solution to ripen for a week or two in the air and 
 sunlight, or add 1-2 c.c. hydrogen dioxid, when it is ready for use. Its staining 
 quality improves up to an optimum and then begins to deteriorate. Old 
 hematoxylin generally contains a precipitate and should be filtered often or 
 before using. 
 
 Stain sections from water for 5.30 minutes according to the age of the solu- 
 tion, the character of the tissue and the fixation employed. After staining, 
 
wash well with tap water to give a blue tone to the stain. Counter-stain as 
 desired. 
 
 $ 81. Ehrlich's acid hematoxylin. Formula: Water, looc.c. ; 95% alco- 
 hol, loo c.c. ; glycerin, 100 c.c. ; glacial acetic acid, 10 c.c. ; alum in excess. 
 L/et the mixture ripen in the light until a dark red. Sections stain in this hem- 
 atoxylin in a short time, generally 5-10 minutes. Wash with water after 
 staining. 
 
 $ 82. Delafield's hematoxylin. Formula : Saturated aqueous solution of 
 ammonia alum, 200 c.c. ; 16% alcoholic solution of hematoxylin crystals, 12^ 
 c.c. (hematoxylin crystals 2 grams, 95% alcohol 12% c.c.). Allow the mix- 
 ture to stand in the light and air in an unstoppered bottle for 4 or 5 days ; filter 
 and add glycerin, 50 c c., and methylic alcohol, 50 c.c. Permit it to stand for 
 a week or so to ripen ; filter and keep in a stoppered bottle. The staining power 
 increases for several months. In using, dilute 3 or 4 times with distilled water. 
 
 Stain sections from water ; 4-5 minutes will generally be sufficient. Wash 
 well with water after staining. 
 
 This is a very strong hematoxylin stain and may be used to advantage with 
 tissues that stain with difficulty. It is likewise a more diffuse stain than either 
 chloral or Ehrlich's hematoxylin, staining cell-body as well as nucleus, a fea- 
 ture having its advantages and disadvantages. Old solutions (several months 
 to a year ) should be filtered before using. 
 
 $ 83. Weigert's hematoxylin. Formula : Hematoxylin, i gram, dis- 
 solved in 95% alcohol, 10 c.c. ; distilled water, 90 c.c. ; saturated aqueous solu- 
 tion of lithium carbonate, i c.c. This stain may be used at once or (better) be 
 allowed to ripen for 2-3 days. 
 
 Tissue should be stained from water and rinsed in water after staining. 
 This is a powerful stain and is used in Weigert's method of staining myelinic 
 nerve fibers ( '4. 1 36 ) . 
 
 \ 84. Borax carmine. (Grenadier). Formula: Borax, 4 grams; car- 
 mine, 3 grams ; water, 100 c.c. ; allow the mixture to stand for several days, 
 shaking occasionally when most of the carmine will have dissolved ; filter and 
 add 100 c.c. of 70% alcohol. Let the mixture remain for several days, filter 
 again again and the solution is ready for use. 
 
 This is a good carmine stain for in toto staining. Stain objects in toto for 
 one to several days, according to size; remove to 67% (jo%) alcohol, acidu- 
 lated slightly with hydrochloric acid, (4 drops in each 100 c.c.), for a day and 
 then remove to 82% alcohol. It affords a bright red stain that is quite trans- 
 parent. 
 
 g 85. Alum carmine. (Grenacher). Formula: Carmine, I grm ; potash 
 (or ammonia) alum, 4 grams (1-5 grams) ; water, 100 c.c. ; boil for 15-20 min- 
 utes, allow it to cool and filter. 
 
 A good carmine stain for sections. Stain 5-30 minutes, or longer if neces- 
 sary ; rinse off the excess of stain with distilled water. The stain is a purple- 
 red. 
 
u6 
 
 \ 86. Paracarmine (Mayer). Formula: Carminic acid, i gram ; alumi- 
 num chlorid, .5 gram ; calcium chlorid, 4 grams ; 70% (67%) alcohol, 100 c. c. 
 Allow it to stand a day or so, shaking occasionally until the carminic acid has 
 quite dissolved, and then filter. 
 
 This is an excellent carmine stain for in toto staining. The tissue may be 
 stained i-several days (i week), then washed in 67% and 82% alcohols to re- 
 move the excess of staining fluid. A red nuclear stain, more opaque than 
 borax carmine. It does not over-stain readily, and since it is an alcoholic so- 
 lution (70%) it is quite penetrating and may be allowed to act for a greater 
 length of time, being thus suited for staining in toto objects of considerable 
 size. 
 
 \ 87. Hcl. carmine. Formula : Carmine, 2 grams ; concentrated hydro- 
 chloric acid, 3 c. c. ; 70% alcohol, 100 c. c. Boil gently for 15-20 minutes to 
 dissolve the carmine ; cool and filter. 
 
 This is a strong carmine stain, quite suitable for sections, especially such 
 as stain with difficulty. It may also be employed for staining in toto. Stain 
 sections from alcohol or water for 5-15 minutes ; rinse away the superfluous 
 stain with 67% (70%) alcohol and differentiate for a few seconds to a minute 
 with acid alcohol (95% alcohol looc. c. , concentrated hydrochloric acid i c. c.). 
 Wash away the acid alcohol with ordinary 95% alcohol. If a pure nuclear 
 stain is not desired the differentiation may be omitted. 
 
 Picric alcohol may be used as a counter stain, and in that case differentia- 
 tion is ordinarily not required. 
 
 \ 88. Picro-carmine. Employ a %% aqueous solution of the dry picro- 
 carmine, made according to Ranvier's formula. Stain sections J^-i hour or 
 longer and rinse well in water. If the sections are over-stained they may be 
 differentiated by means of acid alcohol as used with Hcl. carmine. 
 
 While it is itself a good stain, it may be used with hematoxylin as a coun- 
 ter-stain with good results. It is an alkaline solution, and the hematoxylin 
 stain is intensified. As employed in this way, stain in hematoxylin the usual 
 length of time, rinse in water and stain 2-3 hours in the picro carmine solution ; 
 rinse in water ; dehydrate, clear, and mount in balsam. As used in this way, 
 there is afforded an excellent stain for developing bone. 
 
 \ 89. Safranin. Formula (Babes') : Concentrated aqueous solution of 
 safranin, i part ; concentrated alcoholic solution of safranin, i part. 
 
 Stain sections 1-4 hours, or over night ; wash away excess of stain with 95% 
 alcohol, differentiate with acid alcohol (95% alcohol, 100 c.c., hydrochloric 
 acid, rV c.c. ) for a few seconds, rinse with 95% alcohol and clear in carbol- 
 xylene or bergamot oil. If a pure nuclear stain is not required, the differentia- 
 tion may be omitted. This gives a good stain with tissue fixed in Hermann's 
 or Flemming's fluid. It is a brilliant, transparent red. (See \ 99). 
 
 \ 90. Gentian violet. Formula : A concentrated solution in distilled 
 water. Stain (paraffin) sections from water for 5-10 minutes, rinse in water, 
 dehydrate and differentiate with 95% alcohol and complete the differentiation 
 with clove oil. When the differentiation is sufficient, clear with bergamot oil 
 
and mount in balsam. This may be used alone to give a blue stain with tissue 
 fixed in Hermann's or Flemming's fluid. 
 
 $ 91. Methyl green. This is an nuclear stain of much value, besides 
 being an important ingredient of triple stains (e.g., Ehrlich's triacid mixture). 
 In very dilute solutions it is serviceable in staining the nuclei of fresh tissue 
 and of isolated cells (formula below). A i% aqueous solution is used with 
 hematoxylin and picrofuchsin in differentiating the structure of the hair folli- 
 cle. (See 1 54). 
 
 92. Methyl green and eosin. Formulas: (a) Aqueous solution. i% 
 aqueous solution of methyl green, \y 2 c.c. ; %% aqueous solution of eosin, i 
 c.c. ; normal salt solution, 100 c.c. 
 
 (b) Glycerin solution (for mounting). i% aqueous solution of methyl 
 green, 2 c.c. ; V 2 % aqueous solution of eosin, I c.c. ; glycerin, 100 c.c. 
 
 These solutions may be used for staining isolated cells ; formula (a) for 
 temporary examination, formula (b) as a mounting and staining medium 
 combined. 
 
 $ 93. Ehrlich's triacid mixture. Formula : Saturated aqueous solution 
 of orange G, 12 c. c. ; saturated aqueous solution of acid fuchsin, 8 c. c.; sat- 
 urated aqueous solution of methyl green, 10-12 c. c.; distilled water, 30 c. c. ; 
 alcohol (95% -j- ), 18 c. c. ; glycerin, 5 c. c. Mix the orange G and acid fuchsin 
 and add drop by drop, or a few drops at a time, the solution of methyl green, 
 stirring or shaking between each addition ; then add the alcohol, water, and 
 glycerin. Shake thoroughly and allow the mixture to stand for 24 hours. Do 
 not filter or shake, but take the stain from the bottle by means of a pipette. 
 
 Stain sections for 10-15 minutes, rinse off the superfluous stain with dis- 
 tilled water and dehydrate and clear as quickly as possible ; this is necessary 
 since the alcohol washes out the methyl green (nuclear stain) very rapidly. 
 This stain colors collodion very deeply, hence it cannot well be used with col- 
 lodion sections. It affords a good and often valuable stain. The sections 
 should, however, beihin, and dehydration must be rapid. For the use of the 
 mixture in staining blood films, see \ 125. 
 
 $94. Eosin. Formulas: (a) y 2 % aqueous solution; (b) 2% aqueous 
 solution ; (c) ^% solution in water or 50% alcohol. Formula (a) is prefera- 
 ble for most work; (b) affords a stronger and (c) a weaker stain. This may 
 be used as a counter-stain with hematoxylin to differentiate nucleus from 
 cell-body. Stain sections after hematoxylin for 10-30 seconds, wash away the 
 excess of stain with distilled water or 6j% alcohol. Since alcohol tends to 
 wash out the eosin, unless the color is too strong it is advisable to hasten the 
 process of washing out and dehydration. 
 
 ^95. Erythrosin. Formulas: (a) l / 2 -i% solution in 67% alcohol, (b) 
 y 2 -\% aqueous solution, ( c) ^% aqueous solution. This is a general stain sim- 
 ilar to eosin in its staining properties, but gives a redder color. Formulas (a), 
 and (b) may be used with sections and in the same way as eosin. Formula (c) 
 is used with tissues dissociated in formaldehyde dissociator. 
 
 \ 96. Picric alcohol (acid). Formula: Picric acid, .2 grams; distilled 
 
u8 
 
 water, 500.0. ; 95% alcohol, 50 c.c. A good counter-stain with hematoxylin 
 or carmine. Stain in hematoxylin or carmine, rinse in water (or alcohol if Hcl. 
 carmine is used), and stain with picric alcohol 10-30 seconds ; wash off with 
 67% alcohol, etc. Since picric acid tends to wash out the nuclear stain, it is 
 best to over-stain somewhat with the nuclear dye and regulate carefully the 
 time of staining with the picric alcohol. 
 
 | 97. Picro-fuchsin. Formulas: (a) General stain, 1% aqueous solu- 
 tion of fuchsin acid, 10 c. c.; saturated aqueous solution of picric acid, 75 c.c. ; 
 distilled water, 25 c.c. (b) For nervous tissue, i%aqueous solution of fuchsin 
 acid, 15 c.c. ; saturated aqueous solution of picric acid, 50 c.c. ; distilled water, 
 50 c.c. This is a valuable counter-stain to hematoxylin, especially serviceable 
 in the differentiation of white connective tissue fibers. The nuclei are a pur- 
 plish brown (hematoxylin stain), the connective tissue red, cell bodies and 
 muscle yellow-orange. In special cases the relative amount of fuchsin acid 
 may be decreased or increased, thus giving a preponderance to the yellow or 
 red in the general stain. 
 
 Stain well with hematoxylin, rinse in water, and stain with the picro-fuch- 
 sin 15-30 seconds ; wash away the excess of stain with distilled water or 67% 
 alcohol. Picro-fuchsin will gradually wash out the hematoxylin, therefore 
 stain strongly with hematoxylin and regulate carefully the time of staining 
 with picro-fuchsin. Picro-fuchsin is quite sensitive to alkalies, so that tap-water 
 (unless slightly acidulated ) should not be used for washing out, and the mount- 
 ing medium should be slightly acid or neutral, not alkaline. 
 
 $ 98. Heidenhain's Iron Hematoxylin. Three steps are necessary, (a) 
 mordanting, (b) staining, (c) differentiating. 
 
 1. Mordant sections 1-2 hours in a 4% aqueous solution of ferric alum 
 (iron-ammonium-persulphate), rinse in water a few (5-10) minutes, and 
 
 2. Stain for 1-3 hours in a Yz% aqueous solution of hematoxylin ( form- 
 ula : 1 6% alcoholic solution of hematoxylin 30. c., water 97 c. c. , chloral hy- 
 drate 2 grams), rinse in water, and 
 
 3. Differentiate by dipping the slide into the mordant for a few seconds 
 and then rinsing in tap-water, repeating the operation until the correct differ- 
 entiation has been attained, as ascertained by examination under the micro- 
 scope. The chromatin (nucleus) should be a deep blue or blue-black, the 
 cytoplasm ( cell-body ) gray or light blue. After differentiating it is necessary 
 to wash the section well in running water for about 20 minutes to ensure the 
 complete removal of the ferric alum. The above times are for tissue fixed in 
 Flemming's or Hermann's fluid ; shorter times may be given for tissue fixed 
 by most other methods. 
 
 A counter-stain is ordinarily not needed, but, if desired, the sections may 
 be stained for 10 seconds to 2-3 minutes with a y 2 saturated aqueous solution of 
 orange G. 
 
 This is a valuable stain in all cytological work, and for all muscular tissue. 
 
Iron Hematoxylin 
 
 Any Fixation desired 
 
 v 
 Paraffin Sections 
 
 v 
 Benzin 
 
 95.% Alcohol 
 
 
 Water 
 
 I 
 
 v 
 
 Mordant 
 1-3 hours in 4 % ferric alum 
 
 Wa V ter 
 
 v 
 
 Stain 1-6 hours 
 in V<2 % aq. hematoxylin 
 
 Water 
 
 V 
 
 Differentiate, by dipping alternately in 
 
 mordant and in the tap-water, 
 
 a second in each 
 
 I 
 
 v 
 
 Water (running) 
 15 minutes 
 I or 
 
 v 
 
 Orange G. 
 sat. aq. sol.) 
 
 v 
 Water (rinsed) 
 
 > 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. 
 
 (<r) Staining with (#) Eosin and Hematoxylin, (b} Hhrlich's 
 'Triacid Mixture, or (c) Methylene Blue. 
 
 124. Eosin and hematoxylin. Stain the fixed blood films 
 for 2-3 minutes with a l / 2 % aqueous solution of eosin, rinse with 
 water and stain for 10-15 minutes with hematoxylin, rinse again 
 with distilled water and allow the film to dry. When dry, warm the 
 cover gently to remove the last traces 'of moisture and mount in 
 balsam. This gives a good general stain of the blood corpuscles, 
 both red and white. Red corpuscles will be stained red or pink, 
 the nuclei of the white corpuscles will be blue, and their cell bodies 
 but faintly stained ; eosinophile granules, a bright pink. 
 
 125. Ehrlich's triacid mixture ( 93). Place a drop or 
 two of the mixture upon the film side of the cover. Stain for 10-15 
 minutes, rinse in distilled water, dry, and mount in balsam. 
 
 If the stain is successful, red corpuscles will be orange-yellow, 
 nuclei of white corpuscles, blue, cell bodies, pale pink, eosinophile 
 granules, copper red. This stain is used a great deal in the clinical 
 examination of blood and is a valuable one. Should the red corpus- 
 cles be colored red instead of orange-yellow, it means that the fixa- 
 tion has fjeen insufficient ; if ether-alcohol was used for fixing, the 
 time was too short ; if heat was employed, the degree of heat was 
 insufficient or the time too short. The right time of fixation and 
 staining: must be, in many cases, determined by experiment. 
 
129 
 
 126. Methylene blue. Formula : Methylene blue, satu- 
 rated alcoholic solution, i part ; distilled water, 2 parts. 
 
 This is for staining " basophile " granules in mast cells. Prep- 
 arations fixed by heat or ether-alcohol are stained 15 minutes or so, 
 washed with water, dried, and mounted in balsam. Nuclei and bas- 
 ophiie granules stain blue ; all else is colorless, or nearly so. This 
 stain has but little value in the study of normal blood, since baso- 
 phile leucocytes are of rare occurrence. 
 
 FINE INJECTION. 
 
 For the purpose of examining microscopically the finer arteries 
 and veins and the capillaries in a tissue, and their relation to the 
 other parts, it is necessary to fill them with some colored injection 
 mass, or otherwise stain or color them. The masses employed for 
 the fine injection of tissue in this course are Carmine Gelatin Mass 
 (red) and Berlin Blue Gelatin Mass. 
 
 127. Carmine gelatin mass. Formula : Dry gelatin, 75 
 grams ; carmine (No. 40), 10 grams water, 90 c.c. ; ammonia, 10 
 c.c. ; acetic acid, q. s. ; chloral hydrate, 10 grams. 
 
 Soak the gelatin in water until it is soft ; pour off the superflu- 
 ous water and melt it (in an agate or porcelain dish) over a water 
 bath. Grind the carmine to a paste with water ; add all the am- 
 monia and water ; filter, warm to 80 or 90 C. , and add to the warm 
 gelatin. Then add slowly the acetic acid diluted with an equal vol- 
 ume of water, while constantly stirring the mass, until the mass 
 smells very slightly of the acid. Filter through fine flannel. If the 
 mass is acid, the chloral hydrate may be safely added (as a preserva- 
 tive) ; if any ammonia is present it will decompose it forming chlo- 
 roform and a granular precipitate. If too much acid is added, the 
 gelatin will not set. 
 
 128. Berlin blue injection mass. Formula: Dry gela- 
 tin, 75 grams ; saturated aqueous solution of Berlin blue, 150 c.c. 
 chloral hydrate, 10 grams. Prepare the gelatin in the manner given 
 above ( 127) ; warm the Berlin blue solution (to 80 or 90 C.), 
 and add it to the hot gelatin. Heat the mixture for 10 minutes or 
 more, stirring it occasionally, and filter it through fine flannel and 
 add the chloral hydrate. 
 
130 
 
 129. For securing the best results in injecting the following 
 conditions should be observed : ( i ) A young but nearly mature, 
 lean animal is to be preferred. (2) Kill the animal with an anes- 
 thetic (chloroform; and leave it in the anesthetic at least half an 
 hour before beginning the injection ; do not, however, wait until 
 rigor mortis sets in. (3) Inject only the part desired, tying all an- 
 astomosing vessels and all vessels to other parts. Inject into the 
 artery of the part, leaving the vein open until nearly pure injection 
 mass escapes, then tie it and continue the injection until the part 
 feels hard and is the color of the injection mass. (4) When the 
 injection is finished cool the part injected by means of cold water, 
 ice, or snow. 
 
 (5) Harden the injected tissue i or 2 days in 50% alcohol, 2 
 or 3 days in 67% and 82% alcohols. The acidity of the alcohols 
 should be insured by adding to the 50% alcohol a few drops of acetic 
 acid. The tissue may be .stored in 82,% alcohol until ready for sec- 
 tioning. For sectioning the collodion method is usually preferable. 
 
 130. Silvering blood vessels. Silver nitrate may be used 
 for coloring blood vessels, and thus differentiating them. See 147. 
 
 CALCIFIED STRUCTURES, BONE AND TOOTH. 
 (A). Decalcification. 
 
 131. For the purpose of investigating the soft structures of 
 tissues containing lime salts, such as bone, teeth, and calcified carti- 
 lage, it is necessary to remove the lime salts before sections can be 
 prepared in the usual way by a process known as decalcified tion. 
 Solutions of a large number of acids, combined or uncombined with 
 other substances, may be used as decalcifiers. Very satisfactory 
 are: (i) nitric acid, 3 c.c. ; 70% (67%) alcohol, 97 c.c., and (<?) 
 nitric acid, 5 c.c. ; saturated aqueous solution of (potash) alum, 50 
 c.c. ; water, 50 c.c. In the first formula the alcohol, in the second 
 the alum acts as a restrainer of the nitric acid. The first of these 
 formulas is, perhaps, better for bone ; the second has a more rapid 
 action and is possibly a better decalcifier for teeth. 
 
 132. Directions for use. The tissue to be decalcified must 
 be first thoroughly fixed and hardened by one of the approved 
 methods, picric alcohol is quite satisfactory, and should be in 
 
82% alcohol. In fixing, structures not needed should be removed, 
 muscles trimmed away from the bone, etc. Bones or teeth should 
 be opened with nippers or a saw, so that the fluid may reach the 
 marrow or pulp cavity. 
 
 Place the hardened tissue in the decalcifier, where it should 
 remain until the lime salts have been entirely removed, as may be 
 ascertained by inserting a fine needle ; if any calcified matter remains 
 there will be a gritty feeling on using the needle. The time neces- 
 sary for complete decalcification will depend upon the size and den- 
 sity of the calcified tissue, and will vary from 3 to 15 days or longer. 
 The decalcifier should be changed after the first day, and if the tis- 
 sue is large it is best to change it subsequently two or three times at 
 intervals of two or three days. 
 
 When decalcification is complete rinse the tissue well in water 
 for a few minutes and place it in 67% alcohol for one or two days 
 and then in 82% alcohol for several days, or until ready to imbed. 
 The 82% alcohol should be changed once or twice in order that the 
 nitric acid may be well washed out. Although paraffin in many 
 cases may be employed for imbedding, the collodion method is gen- 
 erally more satisfactory. 
 
 Hematoxylin with eosin, hematoxylin with picrofuchsin, and 
 hematoxylin with picrocarmine afford good stains ; by staining 
 thoroughly with hematoxylin a differential staining of bone and 
 cartilage may be obtained. 
 
 (/?). Sections of Dry Bone or Tooth. 
 
 133. Though the general structure of bone and teeth is 
 shown moderately well when the tissue has been decalcified ( 131), 
 the Haversian canals, canaliculi and lacunae of bone and the denti- 
 nal tubules of the teeth are shown much better in sections of dried, 
 non-decalcified tissue, rendered sufficiently thin for microscopic ex- 
 amination by grinding or filing. 
 
 134. Directions for procedure. Prepare thin transverse 
 sections of dried bone in accordance with the directions below. 
 Longitudinal (radial) sections and tangential (surface) sections may 
 also be prepared in the same manner, the former to show the Haver- 
 sian canals and their anasomoses, the latter to indicate the shape of 
 the lacunae as seen in a different plane. 
 
132 
 
 1. Sawing the section. Make an exact transection of a part of 
 the shaft of a long bone. The section should be about i cm. long 
 and include the thickness of the shaft from the surface to the medul- 
 lary cavity. Make the sections about i mm. thick. 
 
 2. Grinding the sections. Place the piece of bone on a cork or 
 piece of soft wood and wet it with water. File it on one side until 
 smooth and then turn it over. Continue the filing till the piece is 
 from .05 to .10 mm. thick, using the cover-glass measurer to deter- 
 mine the thickness. In the beginning one can press quite hard in 
 filing ; as the section thins, more care should be exercised and the 
 pressure should lessen. 
 
 3. Washing and drying the section. When the section is thin 
 enough, rinse it and dry it with lens paper. 
 
 4. Mounting the sections in hard balsam. To prepare the bal- 
 sam, put two or three large drops on the middle of a slide and heat 
 the slide in some way to drive off the volatile constituents. Do not 
 heat the balsam hot enough to produce bubbles. When the balsam 
 chips after cooling, it is ready for use. 
 
 In mounting, have the section and a clean cover so placed that 
 they may be easily and quickly grasped. A cork somewhat smaller 
 than the cover-glass should be within reach, and also a stone or 
 piece of glass upon which to quickly cool the specimen as soon as it 
 is mounted. 
 
 Heat the slide until the balsam is well melted. Put the slide 
 upon a piece of paper, grasp the piece of bone with the forceps and 
 plunge it into the melted balsam ; put on the cover as quickly as 
 possible and press it down with the cork ; finally put the slide on 
 the stone or glass to cool the balsam quickly. All of this should be 
 done as rapidly as possible, and if done rapidly, the air will be 
 retained in the lacunae and canaliculi, and cause them to stand out 
 as black spots and lines. If soft balsam were used it would soon 
 drive out the air, and being of nearly the refractive index of bone, it 
 would obliterate the lacunae and canaliculi. Further, if the hot bal- 
 sam were not cooled quickly, the air would be driven out and balsam 
 would take its place in the spaces. 
 
 CENTRAL NERVOUS SYSTEM. 
 
 135. There are two special groups of methods employed in 
 the microscopical examination of the central nervous system, (a) 
 
133 
 
 Weigert Hematoxylin Methods, for differentiating my clinic nerve 
 tracts, and () the Chrome- silver Impregnation Methods, to bring out 
 the form of the cells and differentiate the amyelinic nerve-fibers. 
 
 These have their greatest value when used in conjunction with 
 more general staining methods, which supplement the first and give 
 a basis for the correct interpretation of the appearances produced by 
 the second method. In addition, there are other stains helpful in 
 bringing out the finer structure of the cells. 
 
 136. The Weigert hematoxylin methods. These meth- 
 ods all involve (a) mordanting the tissue with a chromium or cop- 
 per salt, () staining (over-staining) with a strong hematoxylin 
 stain, and (r) decolorizing (differentiating) the sections in a 
 bleacher until the myelinic fibers are blue, all else (except blood) 
 white to brown. 
 
 137. The following method is serviceable : Harden the tis- 
 sue in a 3% aqueous solution of potassium dichromate for about 2 
 weeks, and in a 5% aqueous solution for an equal period ( 31). 
 Imbed in collodion ; paraffin may be used if the piece of tissue is 
 small. The sections should not be thicker than 25^. Wash the 
 sections in water ( 68-70) and (a) Mordant them 1-12 hours in a 
 half-saturated aqueous solution of copper acetate ; rinse them 
 well in water and () Stain them for 1-24 hours (until black) in 
 Weigert's hematoxylin ( 83) ; rinse well in water and (r) Differ- 
 entiate in the Weigert's bleacher (Formula: Water, 200 c. c. ; 
 borax, 2 grams ; potassium ferricyanide, 2.5 grams). Watch the 
 differentiation carefully and when the cinerea has become a golden 
 brown and the myelinic fibers a rich blue, stop the action and wash 
 the sections in water, running or changed several times, for at least 
 YZ hour. Dehydrate, clear, and mount in alkaline balsam (best) 
 (158). If the specimens are to be left for some time before mount- 
 ing, place them in 95% alcohol with sodium carbonate added to 
 render it alkaline. 
 
 138. Pal's method may be used if it is desired to stain the 
 nerve cells subsequently. 
 
 Harden the tissue thoroughly in solutions of potassium dichro- 
 mate (3% solution, 2 weeks ; 5,%, 2 weeks or so). Imbed in collo- 
 dion (paraffin, if the sections are small). Stain the sections in Wei- 
 gert's hematoxylin (83) until a blue black ; rinse in tap- water. 
 
134 
 
 Treat for a short time 20-30 seconds with a %% aqueous 
 solution of potassium permanganate and decolorize in Pal's bleacher 
 until the cinerea is grey (white), the myelinic fibers blue. The 
 action will be very rapid and must be carefully watched ; a few .sec- 
 onds will suffice. Wash the sections thoroughly (*4 hour or more) 
 in running water. If it is desired, counter-stain with a red stain, 
 eosin, erythrosin or carmine. 
 
 This method is not as reliable as the method first given ; great 
 care must be exercised in the differentiation with the bleacher. 
 
 139. Chrome-silver impregnation methods. In this 
 group of methods the tissue is hardened a certain length of time in a 
 fluid containing a chrome salt, especially potassium dichromate, and 
 then placed in an aqueous solution of silver nitrate (%% and y^% 
 solutions generally employed). If successful, the cells and their 
 processes, amyelinic and, to a certain extent, myelinic nerve fibers, 
 are outlined by a black precipitate (black by transmitted light ; 
 brown by reflected light). 
 
 140. Success depends on (a) the kind of animal ; different 
 parts and tissues react more satisfactorily in some animals or classes 
 of animals than in others, (b) The age of the animal ; young or 
 fetal animals give better results than do adults, (c) The time of 
 hardening ; it is necessary that the tissue be hardened a certain 
 length of time, constant (relatively) for a certain kind of tissue under 
 the conditions above (a and fr). It is necessary that the correct de- 
 gree of hardening be carefully regulated, (a?) Different organs and 
 regions of the central nervous system vary greatly in the ease with 
 which they can be made to furnish satisfactory impregnations. Al- 
 most certain impregnations of hippocamp can be gained ; cerebral 
 cortex is likewise quite easy to stain. With the olfactory bulb the 
 action is not constant though fairly complete. The optic lobes and 
 retina of birds and large reptiles are more satisfactory than those of 
 mammals. The myel (spinal cord) of embryo birds (7-14 day chick 
 best) is generally more satisfactory than that of mammals ; in any 
 case, fetal or new-born animals should be employed. Difficult are 
 satisfactory impregnations of sympathetic ganglia, organs of special 
 sense and the intrinsic nerves of the viscera. 
 
 141. Golgi's Rapid Method. This is the most generally 
 serviceable of the different methods. 
 
135 
 
 142. Directions for use. Tissue of a (preferably) young ani- 
 mal is placed in a mixture of 4 parts of 3% potassium dichromate 
 and i part of i% osmic acid. The amount of the fluid should be at 
 least twenty times the bulk of the tissue and should be changed as 
 soon as it grows turbid or loses the strong characteristic odor of the 
 osmic acid. 
 
 After the hardening has proceeded to the right degree ( 143), 
 rinse the tissue in water for about 5 minutes and place for 15 min- 
 utes in a l /^% solution of silver nitrate, and then for 2 or more days 
 in a y% solution of silver nitrate, preferably keeping it in the dark. 
 
 Without washing, imbed rapidly in collodion as follows : 
 
 (a) Dehydrate 2-3 hours in 95% alcohol, changed two or three 
 times ; () place in thin collodion for 20 minutes, in thick collodion 
 for 20-30 minutes ; (c) imbed in thick collodion, on a cork or block 
 of wood (best; 55, a) ; (d) harden the mass in chloroform for 
 20-30 minutes, and (<?) place the block in clarifier and cut, sections 
 being 50-100 // thick, according to the nature of the tissue and the 
 character of the impregnation. 
 
 (/) Place the sections in 95% alcohol for a few minutes ; clear 
 in carbol-xylene and mount in balsam by placing the section on the 
 slide, absorbing the clearer thoroughly by means of tissue paper and 
 spreading over it thick xylene balsam. Do not cover. L,ater,when 
 the balsam has hardened somewhat, it may be melted by heat and 
 much of the superfluous balsam drained from the section and scraped 
 away with a knife. 
 
 143. Time of hardening. The following periods will prob- 
 ably be found approximately correct. In general : The best results 
 are to be obtained with kittens 3-20 days old, puppies 2 weeks old, 
 rats 8-10 days, rabbits 8 days, (a) For cerebral cortex (and hip- 
 pocamp) : New-born kitten, 1-2 days ; kitten half grown (3-4 
 months), 3-4 days ; new-born rabbit, 24 hours ; rabbit one month 
 old, 2-3 days, etc. 
 
 (0) For spinal cord : Chick of 5-6 days' incubation, 24 hours ; 
 chick, 14-15 days' incubation, 3 days ; new-born kitten or puppy, 
 2-3 days. 
 
 (c) Cerebellum : New-born kitten, 1-2 days ; kitten half grown, 
 4 days. 
 
 144. Methylene blue. Imbed in paraffin tissue that has 
 been hardened in 95% alcohol ; cut sections rather thick, 15-20 //, 
 
1 3 6 
 
 or even thicker. Fasten the sections to the slide or carry them 
 through in watch-glasses. Stain the sections in a .5% aqueous solu- 
 tion of methylene blue for 5-10 minutes, heating it until it steams ; 
 rinse in water and dehydrate, clear in oil of origanum or cajuput, 
 and mount in balsam. The nerve cells and nuclei will be stained 
 blue, all else colorless. In the cell bodies the corpuscles of Nissl 
 will be stained. Should the stain be not selective enough, differen- 
 tiate for a few seconds before dehydrating with a mixture of aniline 
 oil, i part, 95% alcohol, 9 parts. 
 
 145. General methods, vom Rath's and Zenker's fluids 
 are recommended for the fixation of nervous tissue ; either paraffin 
 or collodion may be used in imbedding ; Delafield's hematoxylin 
 with picrofuchsin (strong formula, b) as counter-stain is recom- 
 mended for staining, though other hematoxylins may be used as well. 
 
 SILVER NITRATE IMPREGNATIONS. 
 
 146. The preparations stained by means of nitrate of silver 
 were prepared as follows : The fresh tissue was washed well for a 
 minute or so in distilled water to remove from the surface all album- 
 inous substance, and then transferred for 2-5 minutes or longer to a 
 y?% aqueous solution of silver nitrate ; it was again rinsed in water 
 and in it exposed to direct sunlight until a light brown. When, by 
 examination with the microscope, the stain was found to be suf- 
 ficient it was again rinsed in water and placed in glycerin or alco- 
 hol. Employed in this manner with fresh tissue, silver nitrate 
 stains the cell cement, affording thus negative images of the cells. 
 
 147. Silvering Vascular Epithelium. In order that the 
 vasular epithelium of small arteries, veins, and capillaries should be 
 well demonstrated, silver nitrate solutions of l / to **/%% strength 
 must be injected into the vessels. 
 
 148. Procedure. Connect a canula with the artery supply- 
 ing the alimentary canal (superior mesenteric) or the brain (carotid) 
 and inject distilled water until the water flows out of the returning 
 vein colorless. Then immediately inject the silver solution until it 
 runs from the vein. After a minute or two follow with distilled 
 water. Place the intestines and mesentery in water and expose 
 them to the light until they become slightly browned. Strips of the 
 muscular coat of the intestines, especially of the rabbit, will show 
 
137 
 
 capillaries well. Veins and arteries side by side may be found in 
 the mesentery. If the brain vessels are injected one can get admir- 
 able preparations showing nuclei as well as cell outline by staining 
 in hematoxylin. Mount in glycerin, or, if desired, dehydrate and 
 mount in balsam. The tissue may be kept in 50% alcohol or in 
 50% glycerin for several months before mounting if it is kept in the 
 dark. 
 
 For large vessels and endocardial epithelium open the vessels 
 or the heart and silver as directed above for mesentery. It may be 
 necessary to make thin free-hand sections so that the preparation 
 will be thin enough for high powers. 
 
 . 
 
 FORMULAS. 
 
 In addition to those given elsewhere; see also The Microscope, 7th ed., 
 | 297-316. 
 
 2 149. Acid alcohol. 95% alcohol, 100 c.c. ; hydrochloric acid, ^ c.c. 
 For Hcl carmine, use 95% alcohol, 100 c.c. ; hydrochloric acid, y 2 c.c. 
 
 2 150. Alcohol, (i) 67%. Take 95% alcohol, 2 parts ; water, i part. 
 
 (2) 82%. Take 95% alcohol, 5 parts ; water, r part. 
 
 3 151. Clarifier. (Castor-xylene). Castor oil, i part ; xylene, 3 parts. 
 
 'i 152. Clearer. ( Carbol-xy lene ) . Melted carbolic acid crystals, i part 
 (by volume) ; xylene, 3 parts. 
 
 \ 153. Normal salt solution. NaCl. (common salt), .6 gram ; distilled 
 water, 100 c.c. 
 
 $ 154. Collodion. ( i ) Thick collodion, 8% solution. Ether-alcohol, 100 
 c.c. ; soluble cotton, 8 grams. (2) 6% solution. Ether-alcohol, 100 c.c. ; sol- 
 uble cotton, 6 grams. (3) Thin collodion, i/4%, ether-alcohol, 100 c.c. ; solu- 
 ble cotton, \y z grams. 
 
 155. Ether-alcohol. Sulphuric ether, i paft ; 95% alcohol, i part. 
 
 2 156. Glycerin, eosin and alum carmine. Glycerin, 85 c.c. ; alum car- 
 mine, 7 >< 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). 
 

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