HX64098842 QP44 .H99 Outlines of expenme RECAP Columbia ©nttiersiit!) int^fCitpofl^fttigiJrk College ot 3^f)v^icim& anti burgeons: Digitized by the Internet Archive in 2010 with funding from Open Knowledge Commons http://www.archive.org/details/outlinesofexperiOOhyde Outlines of Experimental Physiology ii IDA H. HYDE, Ph. D. Professor of Physiology of the University of Kansas I.AWRENCE, KANSAS 1905 C?A-«?M 4^U^C>^^&iJXji.^\ affi- le fi COPYRIGHT. 1905. By Ida H. Hyde. preface * * it This pamphlet contains the directions for the laboratory work required of the medical students in the Department of Physiology of the University of Kansas. While man}' of the experiments are original, the author has also freely used the work of others where it seemed best suited for the special subjects of investigation. For these hearty acknowledgments are extended to the authors. Lawrence, Kansas, 1905. Table of Contents Page Aconite, Effect 44 Accommodation 86 Amoeboid Movements 10 Anode and Cathode Poles 73 A stig-matism 84 Blood, General Directions 18 Blood, Globulicidal Action 17 Blood, Laking- 17 Blood Microscopic Study of 12 Blood Pressure 47 Blood Opacity - . 17 Blood Reaction II Blood, Spectroscopic Study of 30 Blood, Transfusion on Pressure 49 Blood, Pigment Test 30 Blind-spot Mapped 89 Carbonic Acid and Oxygen in Air 59 Cardio Pneumatogram 59 Capillary Electrometer 63 Carbon Monoxide Test 31 Cardiac Nerves . . , 38 Carotid Pulse x 41 Circulation Time 50 Circulation Scheme 32 Chromatic Aberration 85 Circulation in Frog's Web 16 Ciliary Motion . . 36 Conjugate Foci 82 Contraction of Human Muscles '. 78 Conductivity Changes '75 Constant Current on Muscles 70 Complementary Colors 92 Coagulation 16 Comparison of Make and Break 70 Constant Current on Irritability 74 Constant Current on Excitability 74 Color Vision Test 91 Construction of Lense Images 83 Curare Effect 47 Current Variations by Rheostat 63 Demarcation in Muscle 77 Diffusion 87 Differences of Potential in Muscle 77 Digitaline on Heart 43 Drug's Action on Heart 42 Electrotonus 73 Electro Magnetic Induction 66 Page Electro-physiolog-ical Apparatus 60 Estimation of Ked Corpuscles 19 Estimation of White Corpuscles 21 Eye as a Camera 88 Exclusion of Make and Break 70 Focal Distance Estimated 82 Frog's Brain 79 Galvanometer 62 Galvanis' Experiment 68 Gradual Changes in Stimuli . . 72 Haematolog3' 12 Haemaglobin in Blood 24 Haemorrhage on Pressure 50 Hardy's Experiment on Colloids 35 Hearing 92 Heart Action 39 Heart Sounds 41 Heart Transfusion of Solutions 44 Human Muscle Curves 72 Hypermetropia 85 Identical Points 87 Induction Currents 65 Inductorium 66 Inhibition of Heart 43 Irritability and Conductiviy of Nerves 75 Isotonic Solutions 23 Isometric and Isotonic Curves 72 Keys or Switches 62 Kymograph 37 Laboratory Work Directions 9 Laboratory Book Criticisms 9 Lack of Oxygen and KCn 36 Larjnx .... 93 Law of Muscle Contraction 76 Liquif action 15 Lines of Force 65 Liquifaction by Irritants 16 Lung Capacitj' 55 Macula Lutla 87 Milammeter 63 Morphine on Heart ^. 43 Muscle Fatigue — Work 71 Muscle Reaction , 69 Muscle Twitchings . 69 Myopia 84 Near and Far Point- 87 Negative After Image 91 Nerve Muscle Preparation 39 Nerve Stimuli 68 Osmosis 13 Page Osmotic Pressure 14 Ophthalmoscope 89 Perimetry 88 Physostigma 44 Phag-ocy tosis 11 Pithing a Frog 39 Plasmol^'sis 15 Polar Stimulation of Heart . 73 Positive After Images 91 Previous Stimuli 71 Precipitin Test 31 Pressure Pulse 41 Pulse Volume 42 Purposive Character of Reflex 80 Purkinji Sanson Figures 88 Purkinji Sanson Images 83 Radial Pulse 42 Reflection from Concave Mirrors 81 Refraction by Convex Lenses 82 Refraction by Concave Lenses 83 Refraction by Segments of Lenses 84 Reflex in Man 8i Reflex Inhibition 80 Reflex Action of the Spinal Cord 79 Reflex Time 80 Reaction Time 81 Respiratory Movements 54 Respiratory Pressure 56 Respiratory Mechanics of 60 Respiratory Chemistry of . . . . 60 Rheostat in Series 64 Rheostat in Parallel 64 Sciatic Action on Heart 41 Scheiner's Experiment 86 Semipermeable Membranes 14 Specific Gravity of Blood 24 Skin as a Sense Organ 92 Stethogoniometer , 58 Strychnine Action ... 46 Suprarenal on Heart 51 Suprarenal on Blood Pressure 44 Summation of Stimuli 71 Supplies .* 7 Surface Tension 10 Systolic Phase 41 Sympathetic Nerve 38 Sympathetic on Heart 40 Taste . 93 Thoracometer Records 57 Valves of the Heart 31 Veratrine Effect 47 Voltmeter 62 Volume of Corpuscles and Plasma 22 Vagus Action on the Heart 40 student's Supplies. 1. A set of dissecting instruments, one small scissors, one small forceps, a metal seeker, and a medium sized surgeon's needle. 2. Four camel's hair brushes. 3. Note book containing heavy drawing paper and note paper 7^ by 9^, cover 8 by 10. (No. 4.) 4. A small cbservation note book. 5. A padlock with two keys. 6. Two towels. 7. Slides and cover glasses. 8. Two pipettes. g. A tube of paste. 10. India ink and a fine pen. 11. A spool of waxed linen thread. 12. A spool of white silk and a paper of pins. 13. The Physiological Laboratory Note-book. 14. Students using more than the average number of animals, will be charged ten cents for each additional frog and twenty-live cents for each tortoise. 15. The cost of cleaning, repairing, or replacing articles which have become damaged will be charged to the students to whom they were issued. Directions for Laboratory Note Books. 1. --Use 'pen and ink for writing all notes. Number each page of notes in the upper right hand corner and write on one side only. 2. Place all drawings and explanations of the same on the left pages on heav}' paper; th^e observations and conclusions on the right. Number the plates in the upper left hand corner with Roman numerals. 3. Illustrate rather than explain in writing. The different parts of the drawings are to be labeled with small letters in definite order. These letters are to be explained on the same page. The heading of the drawing is to be placed above it. 4. Place the drawings near the center of the sheet, allowing for a wide margin. Never copy a drawing from a book. Put only such draw ings on a page that relate to the same subject. 5. Arrange the notes of an experiment as follows: Head, number and date each experiment conspicuously. 6. State the object of the experiment. Describe the method. 7. Arrange the observations in accordance with the order of the directions. 8. Whenever possible, state 5'our conclusions as a separate note. 9. Seek the laws or applications underlying each experiment. 10.. Work for your own results. Doing is better than seeing. I.*--: .^' Keep an index of notes and one of plates in the front part of the book. 12. Keep a correction sheet at the back of the book for all criticisms. Outlines of Experimental Physiology LABORATORY WORK. 1. Laboratory books must be in Friday before six. 2. Credit is not given for work not in on time. 3. The work must be illustrated in ink or crayon; diagrams should be employed whenever possible. Time should not be spent in needlessly detailed drawings, make all the statements as concise and brief as possible. 4. Laboratory periods are of three and four hours' length. Less time is not credited. Absence from a laboratory period means that the period is not credited and must be made good before the material for the work has been removed. Three absences from recitation and two from laboratory' periods during one term require a special examination at the end of the term. 5. The apparatus is divided into general and private apparatus. For the for- mer, two students are together held responsible and for the latter, each student is held personally responsible. The cost of all broken or damaged apparatus is de- ducted from student's breakage fee. Apparatus left outside of the lockers is collect- ed by the assistants. Laboratory cleanliness and neatness are considered in making up the final grade of the students. 6. Students are urged to select their comrades for themselves. The work should be arranged so that one day the preparation of the frog or other material shall fall to one student, while the arrangement of the apparatus falls to the other; the next day, these duties are exchanged. NOTE-BOOK CRITICISMS. 1. At the close of each week's work, a list is made of all the work required in the note-book for that week. This is placed on the direction file by the instructor, and is to be used by the student as a guide in writing his notes and to the instructor in making corrections. 2. Any required work not in the books even in pencil, is marked missing when the books are corrected. Notes or illustrations marked missing should be added by the time the books are again handed in for correction. 3. An}- notes or illustrations still in pencil at a time when they should be in ink and completed, are marked "not complete." These should be completed before the books are again handed in for correction, and a statement made where the cor- rection is to be found. 4. The corrections, "missing" and "not complete" will always be found on the correction sheet. Criticisms are sometimes made, however, on the notes or drawings themselves. These are always made in pencil so that they may be easily erased, and the notes or drawing is marked "criticised," on the correction sheet. Criticisms should not be erased bj' the students until they are marked O. K. on the correction sheet. 5. No erasures or marks of any kind except the statement under 3 are to be made on the correction sheet. When any criticism has been corrected satisfactorily, the instructor will place the mark "O. K." after it on the correction sheet. 6. The instructors will always be glad to assist the students in any waj- pos- sible in their work in the laboratory or in writing notes on their work. Thej- will also be glad to explain any criticisms not understood by the student. 10 Outlines of Experimental Physiology PHENOMENA RELATED TO THE STUDY OF BLOOD AND CIRCULATION. Spontaneity is the power apparently possessed by living things by virtue of which they carrj"^ on activities independent oi external stimuli or changes in their environment. Some or all so-called spontaneous actions may be referred to responses to stimuli. Manifestations of life as exhibited in the reactions of leucocytes, amoeba, infu- soria and other forms of life, are due to physical and chemical phenomena or stim- uli. A knowledge of these, it Ls believed, will aid us to interpret more correctly the complex functions exhibited in higher forms of life. As an introduction to the study of blood, we begin, therefore, with experiments involving special physico-chemical laws that pertain to the reaction of protoplasm. Expt. I. (A) Amoeboid Movements and Reactions. (1). Place a drop of water containing amoeba on a slide and examine under the high power, (a). Study the movements of the amoeba and of the ectosarc and en- dosarc (b), reactions to light, mechanical stimulations and food particles, (c), note the effect of placing a heated needle point in advance of the amoeba on the slide under low power. (2). Study the currents in a drop of fluid due to changes in surface tension. Mix some India ink or lamp-black with clove oil, — place a drop of the oil on a slide in a mixture of two parts of glycerine -to one part of 95 per cent alcohol. Support the cover slip with a hair or glass fibre. It has been assumed by some authors that amoeboid movements are due to changes in surface tension. Are they alike? Literature. — Jennings, Study of Lower Organisms, 1904; Davenport, Experi- mental morphology; Verworn, General Physiology. (3). Phj'sical Imitation of Reaction to Stimuli. Place a drop of castor oil 1-10 mm. in diameter in alcohol under the supported cover slip. Bring a capillary tube containing 5 per cent. KOH or chloroform under the cover near the drop. The KOH diffuses out against the drop, lowers the surface tension, and the drop reacts by entering the tube. (B) Experiments in Surface Tension. (1) Make a wire ring and to this attach a loop of thread (see model). Dip this ring into a solution of soap so that a film of soap is stretched across the ring. Have the thread-loop float on this film. Notice the shape of the loop. Puncture the film in the loop by means of a hot wire. Effect? Explain. (2). (a). Place a thin rubber band on the surface of the water. Notice its shape. Dip a pencil or wire into alcohol and then into the water within the rubber band. Notice the effect, (b). Place several bits of cork near each other on the surface of the water and then bring a drop of alcohol on a wire into the midst of the particles. Explain, (c). Cover a clean glass plate with a thin layer of water. Dip a glass rod in alcohol and bring it near the center of the wet surface. Explain the resulting phenomenon. (3). In a watch crystal, place about 10 c.c. of 5 per cent. H2SP4. In this place a small drop of mercury and near the Hg a tiny piece potassium bichromate, (b). Hold a nail very near to the Hg. The drop of Hg will change shape (describe and give reason) and will pulsate rhythmically. Place a piece of K2Cr20T very near the Hg and notice the progressive movement of the Hg. K2Cr2 07 oxidizes the Hg when it touches it and lowers the surface tension at the point of contact. Fe carries plus charges; it robs the Hg of some of the negative charges which have Outlines of Experimental Physiology 11 electrostatically formed a double layer around the Hg. What is the result? Hg ions resist gravit}' by cohesion. (4). Draw a needle between thumb and linger to cover it with a thin coat of oil. Hold the needle parallel to the surface of the water and gently place the needle on the surface. Notice that the needle floats. Observe the surface of the water around the needle. Now clear the needle in Na2C03 solution. Does the needle float? Explain this phenomenon fully. (5). Place 3 or 4 drops of water in a watch crystal and on the water place a drop of castor oil. Watch behavior of the oil. Make this same experiment with cedar oil. Why do these two oils behave diff^erently? (6). In a watch glass place 4 c.c. of )4 per cent. NagCOs solution and add a drop of oli%e oil. Place the watch glass on a black surface. Study the spreading of the soap and oil and the formation of processes. Repeat until thoroughly familiar with the phenomena. What becomes of these processes finally? Why? (7). Place a drop of olive oil on a slide and near it place a drop of Nag CO 2 so- lution. Carefullj- bring the two drops together and observe the action. Notice the vortex movements in the oil drop. Repeat several times. (8). Place 4 c.c. of water in a watch glass and carefully place a drop of olive oil on the surface of the water. About 1 cm. from the oil drop, place a piece of the NagCOa crystal. Where does the spreading take place? Why? (9). Place a few bits of camphor gum on warm water. Explain the phenomena. (10). Blow a soap bubble without detaching it from the pipe. Leave the stem open and notice the bubble. Does it retain its first volume? Explain. Fill a pipette to a given point with water. Slowly expel and count the drops. Repeat, filling the pipette to the same point with alcohol. Is there anj- difference in the size of the drops? Explain. (11). Fill a long glass tube drawn out to a fine capillary with water. Does the water issue from the capillary as a steady stream? Explain rhj'thmic phenomena. (12). Secure an air bubble under a piece of glass in a dish of water. Arrange a bent tube drawn into a capillary at one end, the other end dipping into a bottle of alcohol. So arrange the tube that a fine stream of alcohol strikes the center of the bubble. How many times does the bubble beat rhj'thmically and what forces pro- duce the rhjthm? Literature. — Loeb, Phys. of the Brain, p. 21. Expt. II. Phagoc3'tosis. (1). Inject powdered carmin suspended in m | 8 NaCl solution into some grass- hoppers and into the lymph sack of a frog. Examine the blood after 15, 30, and 60 ninutes. The power of taking up foreign or solid particles has been thought due to local iquefaction of the white blood corpuscles. Literature. — Hekton, Pathology 143; Loeb, Studies of General Physiology. Expt. III. Reaction of Fresh Blood. (zi.) Obtain blood as directed under General Directions on page 18. Allow a Irop of violet-red litmus tincture to soak into a porous claj' plate, then place on the pot a drop of blood and wash it off at once. What is the color reaction? (b). The reaction maj* also be shown with red litmus paper that has been Qoistened in sodium sulphate or concentrated magnesium sulphate. Place a drop if blood on the paper and absorb the liquid at once with blotting paper. 12 Outlines of Experimental Physiology NORMAL HAEMATOLOGY. The examination of the blood, like that of the urine, gives a positive diagnosis in a number of diseases. The examination of the normal blood consists of an actual study of the bloed by use of the microscope and the determination of many of its properties by the use of various instruments. The accurate use of the instruments can be learned onlj^ b)' experience. While the instruments are delicate and easily broken, j^et the technique of their use is easily mastered by the student if he is care- ful, accurate, and persevering. The technique once acquired can be quickly re- gained in later years although it may apparently be forgotten for the time being. Speed in the tests can be obtained only by continuous practice. Theoretically all these instruments are accurate, but because of the minute quantity of blood used, slight inaccuracies vpill be multiplied in the final results and may be large or small according to the experience and carefulness of the observer. By knowing where these errors are possible and avoiding them by the best-known methods, and by adopting a definite method of use of each instrument, these inaccuracies can be largely eliminated and good comparative results obtained. In the use of blood instruments the observer must constantly avoid manufacturing results. There is always a tendency to read into a test a preconceived result. This is best governed by control tests and by repeated tests. When one can repeat a test three or four times with the same individual's blood and obtain approximately the same results he is quite proficient. Reference Books: Clinical Examination of the Blood, by Cabot. Clinical Pathology of the Blood, by Ewing. Clinical Haematology, by Da Costa. Histology of the Blood, by Ehrlich and Lazarus. Text-book, on Physiology, by Hall. Works on Histology and Physiology. Expt. IV. Microscopic examination of blood fresh and stained is of great clinical importance. In some diseases, it gives a specific diagnosis which could not otherwise be gained. Study the red and white blood corpuscles of man, frog, sheep, or other animal. Draw surface and side views. Appliances: — Microscope, eyepiece micrometer, white ground glass slides, cover glasses, Paccini's Fluid. — HgCl2 1 gram, NaCl 2 grams, Glycerine 13 grams. Dis- tilled water 325 grams. The micrometer is a small piece of glass on which there is a scale marking off equal spaces. This is placed on the diaphragm of the eyepiece of the microscope and put in focus by pushing it up or down as needed. The scale is then compared with a stage micrometer marked in microns, and the value of the eyepiece scale thus determined. Preparation. Wash the cover glasses and slides with soap and water and then "thoroughly rinse in clean, warm water. Polish the glsses with a clean, soft towel. When handling the slides or cover glasses, hold them always by their edges and never touch a flat surface. Success in preparing fresh blood specimens depends largely upon the absolute cleanliness of the glasses used. Before using the glasses pass them through the Bunsen or alcohol flames six or eight times while holding them with the fingers, then they will not be broken. Put the glasses down in a clean, safe place, with the heated side up, as this is the side to be used. Technique. Obtain the blood as before, using the second or third drop. Bring one of the previously heated cover glasses underneath the drop of blood and allow it Outlines of Experimental Physiology 13 to jvist touch lightly the centre of the glass; then quickly place the cover glass, blood side down, upon a glass slide. If the glasses are clean, the blood fresh enough, and of the right amount, the blood will spread out into a thin layer, in which the corpuscles lie on the flat surface in a single layer. Around the margin the cells will be more or less grouped together. Protect further with vaseline around the edge of the cover. The specimen should then be jilaced under the microscope and studied at once. (a) Mix a small drop of blood with a small droj) of water; (6) another with a drop of Paccini's Fluid, placed on the skin, which is then punctured, letting the blood flow into the fluid. Put a drop of these on a slide and cover with a cover glass and examine, (c) Smear a drop of blood on a slide by drawing another slide over it. cover one with a glass, and {d) make another and leave uncovered and examine. Precautions. It is very important that the slides and cover glasses should be kept perfectly clean and dry. If alcohol is used an alcoholic residue is left upon the glass and often interferes with the examination. Touching a glass surface with a freshly cleaned finger will leave enough f;it and dirt to prevent the blood spreading. The blood must be transferred to the gL-isses and covered quickly, or it will partially clot and prevent spreading. The drop must be large enough to give a clean field of at least one-half inch in diameter, but it must not be so large that the blood cannot spread out into a thin film between the glasses. The spreading must take place entirely by capillary attraction; pressure must never be used to continue or cause spreading. The glass must touch only the tip of the drop while obtaining the blood; if it touches the ear the blood will not spread. QUESTIONS— Red Cell. 1. Describe a red cell, shape, dimensions, and variations? 2. What are the maximum and minimum dimensions? 3. What percentage are large, normal or small? 4. How are the red cells arranged? 5. What causes crenation? 6. What causes vacuolation? 7. How large are blood-platelets? 8. What happens to the red cells in the presence of water? • 9. What happens to the red cells when drying in the air? 10. What happens to the red cells when spread by pressure? 11. Of what does a red cell consist? 12. Are red cells nucleated? White Cell. 1. Describe a white cell, its dimensions and nucleus? 2. Are there any variations in the size of a white cell? 3. What are the percentages of the various sizes? 4. Do they float readily under the cover glass? 5. What becomes of the white cell in the presence of water and while drying in the air? 6 Of what does a white cell consist? Semi -Permeable Membranes and Osmosis. Expt. V. All substances in solution tend to dift'use from regions of higher to regions of lower concentration. The pressure to which this movement is due is the 14 Outlines of Experimental Physiology osmotic pressure; that pressure is the same as would be exerted by the dissolved substance if it were in the form of a gas of the same temperature and volume as the solution. (Law of van't Ho£f.) If solution and solvent, e. g- , water, are separated by a membrane which is permeable to the solvent, but not to the dissolved substance (semi-permeable membrane), the effect of the osmotic pressure is seen in an increase in the volume of the solution due to the passage of the solvent into it through the mem- brane. Semi-permeable membranes are of universal occurance in. living organisms and are represented by the cell walls of animal cells and the plasma membranes (pri- mordial utricles) of plant cells (A) Artificial Scmi-Permeabic Membranes. (1). (a). Using a fine pointed pipette, attached to a supported burette, introduce carefully a drop of m/5 CUSO4 solution beneath the surf ace of an m/7 K4Fe (Cng) solution in a watch glass. Note that the two solutions do not mix. Why? Note size of the CUSO4 drop; set aside and note if its size changes in the next hour or so. Measure by placing a paper scale under the dish. (b). Repeat using ^/z CUSO4. Set aside for one hour and note effect, (2). Ascertain if- the semi-permeable membrane of Cuo Fe (Cng) is permeable to other substances; e. g., sugar. Prepare a large globule of the ^/z CUSO4 solu- tion; then inject into it with a fine long nozzled pipette an ^/i solution of cane sugar. If carefullj' done, the sugar solution will form a separate layer at the bottom of the globule next the membrane. Does the sugar solution flow out? What do j'ou con- clude? (3). Good artificial membranes are also made as follows: Use a 10 per cent, gelatine solution that has been boiled till dt no longer gelatinizes (eight hours). Beneath the surface of this solution, introduce a drop of a second solution containing equal parts of 5 per cent, tannic acid and m/ cane sugar. Note the character of the membrane formed. What is the reaction? Try tannic acid M'ithout the sugar. Any difference? Why? Note the changes in the size of the globules after varying intervals of time. Also color the tannic acid solution with Congo red. Note if the color passes the membrane or not. What do you conclude as to the permeability of the membrane ? The osmotic pressure of CuSO 4 is less than that of K4Fe (Cng). 1 per cent, cane sugar=47 cm. of Hg pressure. 1 per cent K2S04=193 cm. Hg. 1 percent. KN0 3=178cm. Hg. (B) Natural Semi-Pcrmeabic Membranes (a). Cut a cylinder of beet 3x1 cm. and wash in several changes of water for an hour. Then place in a beaker and cover with 100 c c. distilled water. At the next laboratory period concentrate the 100 c. c. to 4 c. c and test for sugar. Result? (Boil about 10 min. with 25 per cent. HCI, then use Trommer or Fehling's test). (b). Obtain a solution of the beet pulp by cutting the cylinder lengthwise, scrape out the pulp and grind in a mortar with sand and 100 c. c water. Press out the liquid through cheese cloth and evaporate to 4 c. c. Does this solution give the reaction for sugar? What are your conclusions? (4). Obtain 3 sets of osmometers, three long glass tubes and three pieces of membrane. Parchment paper is not a true semi-permeable membrane, but approxi- Outlines of Experimental Physiology 15 mates such a membrane. Soak the parchment paper in water, then fasten it over the osmometers. Test by placing- it under water and blowing- into it. Use rubber or water proof cement, vaseline and rosin. Fill one of the osmometers with an m ,^ cane sugar solution, a second with an ^/C NaCL solution and a third with a solu- tion of albumen. To fill the osmometers, use a small pipette. Now attach to the nipple of each of the osmometers a long glass tube, b}' means of a short piece of rub- ber tubing; (or, better, with glass directly, using waterproof cement). Support the osmometers on j-our desk in a large dish of distilled water, or in three small dishes containing enough water to just cover the osmometers, label and date each After 24 hours, note the difference in the height of the fluid in the three tubes, Explain. What is osmotic pressure? (5). Use an egg membrane, seal a capillary tube to a punctured side and re- move the shell from the other wider end, which dip in water. C. Plasmolysis of Plant-Cells and Blood Corpuscles. (1). Mount some Spirog3'ra fllaments on a slide in a drop of water and familiarize yourself with the structure of the cell. Note the spiral bands containing- chloroph}-! (chromatophores), the nucleus and the thin laj-er of protoplasm just in- side the cell wall. Sketch. (Stain with carmine to bring out the nucleus). (2). Place a few of the Spirogyra filaments on a slide in a drop of m/i cane sugar solution (by M is denoted the Mol or gram-molecule-liter solution), support the cover glass on bits of paper and study the changes that take place in the cells (plasmolj'sis of the cells). Sketch, comparing it with its appearance before placing- in the solution. (3) Repeat, using an ^^A, ^^./lo, and an -^1/20 solution of cane sugar. Note the effects produced in each case. (4). Repeat the above experiments, using instead of cane sugar a 10 per cent, solution of either gelatine, egg albumen, or urea ^^ / 20- Why does not this solution acton the cells in the same waj' as the cane sugar solution? (5). Repeat the above experiments on Vorticella or Paramoecium, using- sugar solutions of 2M, ^^/i, and "^1/20 strength. What strength of solution is practically isotonic with the cell? Does plasmolysis result with strong solutions? (6). Collect a small drop of blood upon the slide, according to the directions on page 18 and cover it with a cover slip. Note carefully the size, contour and behavior of the red blood corpuscles for a few minutes. Prepare a second slide in the same way, only immediatelj' after covering, permit a drop of distilled water to flow under one edge. Upon a' third slide let a drop of M sugar solution run under the coverslip. Watch and compare the appearances of the red corpuscles in the three experiments. Draw. Explain. (7). Find the sugar solution which affects the corpuscles least, i e., is isotonic with them, Expt. VI. (A). The corpuscles develop the property of adhering to the walls of the vessels in so-called inflammatory states It may be due to changes which induce liquefaction. The latter is produced bj' lack of oxygen, action of OH and H ions by replacing the insoluble Ca by Na, Li, K, by certain drugs and by heat. (a). Place a crustacean (amphipod) in a hanging drop in an Engelmann gas chamber. Observe the circulation in the leg-s, note the behavior of the corpuscles. Pass hj'drogen through the chamber until changes in the circulation are noticed ; now- pass oxygen through and observe effects, then carbon dioxide and note the changes. 16 Outlines of Experimental Physiology Revive with oxygen. Note in each case the changes produced on the v? alls of the vessels and in the blood. (b). Liquefaction and differences of osmotic pressure of different solutions. Place corpuscles, also Paramoecia or Vorticella, on plane or cell slides under covers supported by hairs, (The animalcules are obtained by filtering the infusion containing them through a tiny paper filter and the apex of this with the animal- cules in, is then placed in a small dish containing a little fresh water. From this supply material, the Infusoria may either be put on a slide or in a small watch crystal). Allow a drop of NaCl m/jg, KCl m/jg, CaClg ^/w, MgClg t^/iq, NaOH iq/800, HCl Di/800to flow under the coverslips and note changes in the cor- puscles or Infusoria. (c). Repeat, keeping the corpuscles and Infusoria in the various solutions 24 hours. Study the effects of the chemicals on the protoplasm. {d). Liquefaction by heat. Place a slide with a hanging drop containing the material experimented with in (6) on the warm stage, heated to about 38 = C, and note the effect. Microscopic Study of Circulating Blood and Liquefaction by Irritants. Expt. VI (B). Place a small frog with a bit of cotton moistened with a drop of ether under a tumbler until the frog is partially anaesthetized. It must still respond to eye reflex and just attempt to turn over if placed on its back. Lay it covered with moist paper and the bit of ether cotton at its nares on the cork frog plate kept in place by the microscope clips, and pin either the tongue or web over the glass cover. Tie a thread to 2d and 3d toes, the web between these, not too tightly stretched, is pinned down. (a). Study with both the high and low power the velocity and flow in the axis and periphery of the blood stream in arteries, veins, and capillaries. How can you distinguish these? ib). Is there a pulse? in which blood vessels? Is there a disappearance and appearance of capillaries? Can you detect white and red corpuscles? Are there evidences of elasticity and flexibility? of diapedesis of corpuscles? Note the width of the vessels in a special area to compare with (2). (2). {a). Put a piece of ice on the foot, and after a few minutes note the effect (a) on the flow of blood and the width of the above noted vessels. (b). Place a bit of cotton heated with water to about 38= C on the same area; again note the flow and width of the vessels. (c). Put a drop of NaCl, 8 per cent , on the web; note results; wash off; effects? (d). Put a drop of chloroform on a fresh area of the web. Effect? Wash off thoroughly with water. (e). "When the circulation is about normal put on a drop of turpentine. Effect? Wash off with water. (/). Place a small drop of oil of mustard on the web. Note effect. Wash off with water. State your observations regarding the influence of toxic or irritating substances upon the inflammortj' state, the flow and behavior of corpuscles? Coagulation and Fibrin Ferment. Expt. VII (a). Place a drop of blood on a slide, expose it to the air. Soon filam- ents radiate from the centres, apparently blood-platelets. A small aperture diaphr- gam and little light make them plainer. Their importance is, that under certain pathological conditions, the fibrin network is much, increased and helps in diagnosis. Outlines of p]xperimental Physiology 17 It is important, therefore, to be familiar with ordinary net work in normal blood. Compare either with frog^'s or with fellow student's blood. Coagulation of Normal Blood. The coagulation of normal blood is a phenomenon that lakes place quite constantly in from three to five minutes. But in disease, this time ma3' be prolonged indefi- nitelj'. The coagulation ma}- be approximately' tested by taking- a large drop of blood on a warm slide and while holding it in the hand, draw through the drop a needle or a straw everj' minute and note when a clot follows the straw out of the drop. Wrig-ht's instrument for testing- coagulation is slightly more accurate. Are there any variations in the time of coagulation among the individuals in j-our section? Other Experiments on Coagulation. Expt. VIII. (a). Opacity of Blood.— Smear a little fresh blood on a glass slide and laj' the slide on some printed matter. It will not be possible to read it, be- cause the light is reflected from the corpuscles in all directions and little of it passes through. (). Laking of Blood. — Put a little fresh blood in three test tubes, A, B and C. Dilute A with an equal volume, B with two volumes and C with three volumes of distilled water and repeat (a). The print can now be read, probably, through a laj'er of B and C, since the haemoglobin is dissolved out of the corpuscles by the water and goes into solution, the blood becoming transparent or laked. That the difference is not due merel}' to dilution, can be shown by putting an equal quantity of blood into two test tubes, D and E, and gradually diluting D with distilled water and the other with a 0.9 per cent, solution of NaCl, but use more of NaCl solution than 3'ou do distilled water for D. Print can now be read through the first (D) with a smaller degree of dilution than through the second (E). Whj-? Examine the laked blood with the microscope for the ghosts, or the shadows of the red cor- puscles. The addition of methylene blue will render them somewhat more distinct. Take a drop of D and E and see the great difference between the corpuscles under the high power of the microscope. Conclusion? Globulicidal Action of Serum. Expt. IX. (a). Make a careful study of the frog's and calf's blood before bringing them together. Note the size with the micrometer scale; the form and ap- pearance of the red and white corpuscles. {b). Place a small drop of frog's and one of calf's blood on a slide not quite in contact with each other. Put on a cover glass so that the two drops just touch. Examine at once with high power. Note the changes produced in the size, form and appearance of the frog's blood acted upon by the calf's serum, and the calf's acted upon by the frog's serum. {c). What would ^-ou infer regarding the osmotic pressure of the different sera? (d) Heat some of the calf's serum to about 55- C for from a '4 to '2 hour and repeat {b). What is the result? What change was produced in the calf's corpuscles after they were heated? Literature — Iminune Sera, Wassermann ^lnd Boldnar. Blood Immunit\', Nut- tall (2). Haemolvsins and Agglutins. 18 Outlines of Experimental Physiology GENERAL DIRECTIONS. All blood instruments must be perfectly clean and dry if the best results are to be obtained. The various pipettes are cleaned by the use of hydrog-en peroxide and distilled water; they are then dried by the use of alcohol to remove the vs^ater, followed b}' ether, which will evaporate quickly and remove the alcohol. Hydrogen peroxide oxidizes organic matter; alcohol and ether coagulates. The cleaning fluids (hydrogen peroxide and water) are used by filling the pipette and rolling it for a few minutes between the thumb and fingers and then blowing or drawing the fluid out. In the use of drj'ing fluids (alcohol and either) do not blow the fluids out of the pipettes, as the moisture of the breath will defeat the object which one is seeking. Having filled the pipette with alcohol or ether, draw it into the rubber tube, replace it upon the pipette, then draw air through the pipette. After using alcohol in this way, followed by ether, one may be assured that the pipette is absolutely dry. For the student's work, secure the blood from the lobe of the ear or the side of the tip of the thiid finger. The ear is better, as it contains fewer nerves, gives more blood and will continue to bleed for a longer time. The ear or finger should be lightly washed with a towel moistened with distilled water, then dried with the towel to remove any dirt or loose epithelial cells. The needle used should be a fair sized glover's needle. It is a three-sided needle, the sides of which are so ground that each has a fine saw-edge, and will not cut the tissues as a saddler's needle will. The needle should be kept clean with distilled water and hydrogen peroxide, and sterilized with alcohol. A lancet may be used instead of a needle. The puncture should be made by holding the lobe of the ear between the thumb and finger and pricking lengthwise of the ear in its lowest part The needle should enter about one-quarter of an inch, and should be thrust in quickly while the thumb and finger hold the ear, and when withdrawn it should be given a half-turn and be quickly removed. The first drop of blood should always be wiped away to moisten the skin with blood, and also because it clots quicker than the following drops. The blood should gradually ooze out of itself It should never be forcibly squeezed by pinching, as that will give an abnormal specimen; but the ear may be gently pressed an inch or so above the puncture, to make the blood flow more freely To fill a pipette by suction, take the lobe of the ear between the thumb and finger of the left hand, standing behind and to the right when using the right ear and in front and to the left when using the left ear. Place the tip of the pipette upon the thumb that is behind' the ear, hold the pipette with the right hand near its upper extremity, with the marks showing in front; then, by turning the thumb, insert the capillary point into the drop of blood and do not allow it to touch the skin of the ear; the column of blood drawn into the capillary must be accurate and com- plete. It must not remain short of or go beyond the mark desired, and air must not be allowed to enter the pipette. If any of these errors take place the pipette must be recleaned, dried and filled again When properly filled, any blood adhering to its outer surface mu.-^t be completely removed before proceeding further. It is better to have a large drop of blood at first than to use two or three small drops, as there is less liability of getting air into the capillary and of the blood clotting. Be sure to clean the apparatus before putting it away. Outlines of Experimental Physiology 19 Expt. X. Introduction. — In health the number of red cells in the blood is quite constant. The variations that occur are quite small and are due to normal pro- cesses. In the male there are about 5,000,000 red cells in each cubic millimetre. In the female there are about 4,500,000 cells. Any deviation from normal health quickly causes a diminution in the number of red cells In fact, simply unhyf^ienic surrounding-s or habits are sufficient to speedily reduce the number of red cells without other demonstrable pathological conditions. The life of the red cell is probably of about two weeks duration. There are approximately in the normal male's blood 200,000,000,000 red cells. Then according to the length of the lifetime of the cells, about 14,000,000,000 cells die and must be disposed of each day. A cor. responding number must be manufactured each day in order to keep the number within its normal limits. It will be readily seen that such an immense process, which depends upon perfect elimination as well as assimilation, can be disturbed very easily. It is important that this fact about the blood be thoroughlj' under- stood. Even though the phj-sician may not estimate the number of red cells in every case, yet he must recognize the fact that every disturbing element in the normal body must disturb the number of red cells contained therein. There are, then, two objects to be gained by actually counting the I'ed cells and estimating their number. First, to gain a clear idea and understanding of the number of red cells in normal blood; and second, to be able readily and accuratel}' to estimate the number of red cells per cubic millimetre in any given clinical case. There are three methods of estimating the number of red cells per cubic milli- metre: 1. The Thoma Haemacytometer. An instrument by which, with accurate dilution, the corpuscles may be actually seen and counted in a known space. 2. The Oliver Haemacytometer. This instrument depends upon the transmis- sion of a transverse line of light from a candle through a flat glass tube. The blood stops this light until a certain dilution is obtained. The tube is graduated to read the number of cells per cubic millimeter of the blood used according to the dilution. 3. The Haematocrit. B}' this instrument is obtained the volume of the cor- puscular elements in the blood by centrifugation. From this the number of red cells per cubic millimetre may be estimated, except in some special cases. A. To Count the l^cd Blood Corpuscles. Appliances— Microscope; Thoma corpuscle counter, consisting of the ruled counting slide and the diluting pipettes; three small dishes; 3 per cent NaCl solu- tion. Technique. — Having prepared the apparatus and solutions, make the puncture and fill the pipette by gently sucking a continuous column of blood up to the mark "05" or "1" on the pipette, which is near the bulb. Wipe the end of the pipette free from blood with a clean cloth, but do not allow any blood to be drawn out by the capillary attraction of the cloth. As soon as possible now insert the point of the pipette into the diluting solution and suck up a continuous stream of solution until the mark "101" above the bulb is reached. Roll the bulb between the finger and thumb as the blood enters the bulb. If there is not blood enough to reach the mark "1," draw it only to mark "0.5" and proceed in the same manner. Now hold the pipette in the horizontal position 20 Outlines of Experimental Physiology with ends free and roll it back and forth for three minutes to thoroughly mix the blood and solution. When thoroug-hly mixed blow out the contents of the capillary below the bulb and then place a small drop on the marked plate of the counting- slide, putting just enough of the mixture on it to fill the space between the marked plate and the cover-glass, and being careful not to allow any of the mixture to get into the moat. Adjust the cover-glass, and be careful not to allow any of the mixture to get into the moat. Adjust the cover-glass over the drop, quickly and carefully, by placing one-edge of cover glass in contact with the slide and letting the opposite edge down gently with a needle. Place the counting slide, when properly filled, under the microscope and find the upper left-hand corner of the marked area. Wait until the corpuscles come to rest upon the surface of the marked plate, then begin the actual estimation by count- ing all the corpuscles in the first marked space, including those that are on the upper and left-hand lines of the space. Then count those in the space to the right, including the corpuscles on the upper and left-hand lines as before. Continue counting each space to the right until six spaces are counted; then drop down to the next space below and count each space to the left until six spaces in the second row are counted. Then drop down the next row of spaces and continue counting back and forth in the same manner until six rows of six spaces each are counted. Place the results of counting of six rows in the same relation to each other as the spaces that were counted. Having made the count, the slide and cover-glass should be cleaned as previously described. The pipette, which has been left in a horizontal position in a safe place, should be rolled again for three minutes. Fill the counter and adjust the cover- glass carefully as before; count another group of thirty-six spaces and record the results obtained. If the averages of the two counts differ more than one, the same procedure must be carried out the third time, and the average of the two fields nearest alike taken and the estimate made. To compute the number of corpuscles per cubic millimetre, find the average number of cells for each space and multiply this by 4000, as each space is >^ o mm.x ^0 mm. x /lO rnm. This will give the actual number of cells per cubic millimetre in the diluted blood. Then make the correction for the dilution of the blood by multiplying by 100 or 200, and the result will be the number of red cells per cubic millimeter in the specimen of blood taken. Precautions. — The cement used on the counting slide is dissolved by alcohol or ether; so these liquids should not be used on the plate. Roll the filled pipette be- tween the thumb and finger, and do not shake the pipette, as some of the solution is' sure to be lost. A common source of error that can easily be detected, but which is often overlooked, is the unequal distribution of cells on the marked plate. As soon as the drop is placed on the marked plate the cells begin to settle and, of course, most of them settle where the drop is thickest, that is, in the centre. This can be avoided by getting the cover-glass in place quickly and making the whole drop of an even thickness. Each specimen, before being counted, should be tested to see that the corpuscles are evenly distributed over the whole drop. For the same reason the filled counting slide should be kept in a horizontal position. Theoretically, counting the cells in one small space should be sufficient, and it would be, if the measurement and dilution of the blood and the distribution of the cells were all perfectly accurate. This is impossible, and the errors are mostly Outlines of Experimental Physiology 21 eliminated by the methods given. It is best for beginners always to make three counts of 36 spaces each from the same pipette and take the average. Questions— 1. Why is alcohol used to dry and not to clean the pipette? 2. Why is hydrogen peroxide used? 3. Why should the marked plate be dried without friction? 4. Why wipe away the first drop of blood? 5. Why wipe the end of the pipette before putting it into the diluting solution? 6. Why blow out a few drops before putting a drop on the slide? 7. Whj' draw air through the pipette after the ether is drawn out? 8. What kind of a solution should be used to dilute the blood; that is, what properities should it have? 9. Why are there 101 parts in the pif)ette instead of 100? 10. Is there any appreciable variation in the number of red cells in normal individuals? 11. If there is a variation, give some of the reasons. 12. Account for the variations observed in members of your section? B. To Count the White Blood Corpuscles. A Yi per cent solution of acetic acid in distilled water will destroy the red cells and render them invisible, while it will not destroy the white cells, but make them show more plainly. The diluting pipette holds solutions diluted 10 to 100, instead of 1 to 100. Technique — The technique of obtaing the blood and filling the pipette is the same as with the red cells, except that the capillary tube is so lai-ge that we must have more blood. For this reason, for beginners, we recommend that only a half- part of blood be taken at first. More accurate results can be obtained by using one part, but much time and practice are necessary to fill the tube easily. .The capillary is so large that solutions Will not stay in it, but run out quickly when the tube is out of the horizontal position. First, then, we must have more blood; usually two or three good-sized drops are sufficient. Second, the tube must be held hori- zontal or the blood and solution will run out. As the capillary tube is large it is very easly cleaned and dried. Roll the pipette as before, when filled, and in a few moments the mixture will turn quite dark; when it no longer changes color it is ready to be counted. Allow a few drops t© flow out of the tube as in the case of the red-cell pipette, then place a small drop from the end of the pipette on the ruled plate. It is not necessary to blow the fluid out; it will run out. Take the same precautions in fill- ing the counter and adjusting the cover-glass as before, except that there is no need of haste in placing the cover-glass, because the white cells are lighter. Here, because we have a clear field with little in it, and the cells are quite large, we can use a lower power of the microscope and see a whole square milli- metre at once. Place a square aperture card in the ocular tube to aid in defining the field. Begin at the upper left hand corner and count the cells in each space 1 mm. square, and observe the same method in keeping the record as when counting the red cells. Clean the counting slide, roll the pipette for a moment, and refill the marked plate and count the nine spaces again, keeping the records as before. Do this at least three times, so that the area counted will be 27 spaces, each 1 mm. 22 Outlines of Experimental Physiology square. The more cells counted the more accurate the results should be, but the three fields should be sufficient. To estimate the number of cells per cubic millimeter in the blood specimen used, add tog-ether the number of cells and divide bv the number of millimetre spaces counted. Each space is /lo mm. x 1 mm. xl mm., or /lo cmm. Now multiply the averag-e number of cells in each space by 10 to find the number of cells in the diluted blood, and then by 10 or 20, according- as the blood was diluted, and that will give the number of white cells per cubic millimetre in the blood specimen. Question. — 1. What is the number of white cells per cubic millimetre in the blood in the normal individual? 2. What is the normal variation? 3. What are some of the causes of the variations? (A.j To Determine the Relative Volume of Red Corpuscles and Plasma. Expt. XI (a). Appliances. — Electric haematocrit; small rubber tubing to fit capillary tube; needle; white paper; fine wire for cleaning- tubes. Reagents. — Distilled water, hydrogen peroxide, alcohol, ether, and lYz per cent potassium chromate. Preparation. — Adjust rubber to capillar^' tube. Put empty tube in one arm of cross-piece to preserve balance. Use fine wire to remove blood from the capillary tube, then clean and drj' as other tubes. Put vaseline on peripheral rubber pad to prevent blood being drawn out. Technique. — Obtain blood from the lobe of the ear as heretofore described. Re- move the rubber tube by pushing it off and not by pulling, while the vaselined finger is held over the other end of the tube. Remove any blood from the Outside of the capillary, and make a record of the amount of blood in the capillary. Place the tube in the cross-piece of the instrument as quickly as possible and centrifugalize at least three minutes at the rate of 2,000 to 10,000 rotations per minute. Take out the tube and lay it on a piece of white paper to read the divisions. Each degree of the scale is estimated to contain about 100,000 cells; hence, a tube in which the red colum stands at 50 would indicate about 5,000,000 red corpuscles per cubic milli- metre. The use of this instrument is designed, however, chiefly to show the volume of red corpuscles rather than the number. (b). When the determinations cannot be made at the bedside. Adjust the rubber tube to the capillary. Put an emptj' tube in one arm of the cross-piece to preserve the balance and vaseline the peripheral pad of the other arm. Draw a definite volume of a 2}i per cent solution of potassium chromate into the tube and then draw in blood from the lobe of the ear to fill the tube. Determine accurately the volume of blood in the tube, holding a vaselined finger over the free end of the tube before pushing (not pulling) off the r\ibber. Place the tube quickly in the cross-arm of the centrifuge with the blood end toward the centre, so that the blood is forced through the solution, then proceed as in (a) to ascertain the volume of plasma and corpuscles. Precautions. — Do not displace the rubber pads in the outer ends of the rotating arm, as the blood will be thrown out of the tube and necessitate the repetition of the test. Before starting each test see that the pads are in place. Outlines of Experimental Physiology 23 If the tube is not adjusted in the apparatus and set to rotating- within a few seconds after the blood is drawn, coag^ulation will set in and hinder the complete separation of the corpuscles from the plasma. Should separation not be complete in three minutes the test should be repeated. The instrument should be started and stopped gradually, as the sudden starting- and stopping- injures it. Questions — 1. Determine the volume percentage of red blood corpuscles in a number of normal individuals. 2. bo apparently normal individuals have the same or approximatelj' the same volume percentage of red blood corpuscles ? If not, seek for causes of the variations in different individuals. 3. Does the same individual have the same volume percentage of red blood cor- puscles all the while ? (a). If there is a variation, is there anj* periodicity to be observed ? {b). Seek for causes of any variation in the same apparently normal individual. 4. The volume percentage as recorded by the haematocrit varies with the pro- duct of two factors: the average volume of the individual corpuscles by the number of corpuscles per unit volume. (V=vxn). {a). Is the average volume of the individual corpuscles (v) necessarilj^ constant ? {b). If it is not constant, would one be justified in drawing conclusions regard- ing the number of corpuscles per unit volume (n) after observing the volume percent- age (V) with the haematocrU ? 5. What variation of the observation, as above made, would enable one to deter- mine with reasonable accuracy the number of corpuscles per cubic millimetre ? 6. If the tube were onlj' partl3' filled at first, could one make an accurate test ? If so, tell how to proceed. The importance of some of the above questions will be better understood in the following experiment. B. The Determination of an Isotonic Solution and the Relative Osmotic Pres- sure of Dift'erent Salt Solutions. Literature. — Cohen, p. 153. (a). Mix2c.c.. 2/>^MKCl, 2/^ M NaCl, and 2/ g M CaClg each with 2 c.c. of defibrinated blood and bj' means of the fine pipette fill three of the haematocrit tubes with these mixtures. Place the tubes in the cross arm and centrifugalize for exactlj- 2 minutes. Note the relative height of the blood corpuscle column in each tube. Repeat the experiment using >sM NaCl, >s M KCl, >s M CaClo and then '. 16 M NaCl, ^s M KCl, >s MCaClo. Note the relative height of the blood cor- puscle column in each of the series of solutions and then compare the series with the other series. Why is the serum colored red in the third series? Do blood corpuscles follow the laws of osmotic pressure? One experiment must be performed showing the relative volume of plasma and corpuscles of the blood alone, as a comparison control for the determination of the relative pressure of the dift'erent salt solutions. The isotonic solution is different for different kinds of blood. In order to get good results in these experiments the following precautions must be observed: Have all your apparatus perfectly clean, and after washing with dis- tilled water, rinse in js M NaCl. Ahuays add the blood to the sohitions. The cen- trifugal tubes after being washed in physiological salt solution must be rinsed with a specimen of the blood to be centrifugalized. 24 Outlines of Experimental Physiology Expt. XII. The Estimation of the Percentage of Coloring- Matter in the Blood. The estimation of the coloring matter in the blood is based on the supposed fact that a normal individual under normal surroundings has a normal amount of color- ing matter, and that is called 100 per cent. The instruments that have been devised for making the estimation are numerous, and all, while theoretically correct, practically are liable to a greater or less error according to the experience and carefulness of the observer. Thej^ are, howrever, in a skillful and conscientious operator'.s hands, quite accurate, and are especially so when used to compare the tests of the same patient's blood, week by week. The haemoglobin contains practically all the coloring matter, and it constitutes 90 per cent of the solids of the red cell. The haemoglobin consists of 96 per cent globu- lin and 4 per cent haematin. In the haematin is the iron of the corpuscles ; the coloring matter of the blood varies as does the amount of iron. Theoretically, the most accurate way to test the haemoglobin would be to measure the amount of iron in a certain amount of blood. But the chemical extraction and weighing of so small an amount of iron is too difl&cult and tedious. Because of this, other tests have been devised, which depend upon the observer's eye to detect the likeness of shades of red as rep- resented by the blood and colored glass, solutions or paper. Again, the specific gravit}' of the blood except in rare cases depends upon the amount of iron in the red cells, and varies as the iron does. Then we can estimate the percentage of haemo- globin by finding the specific gravity of the blood. The principal tests may be classified as follows: 1. Estimation of iron in the blood. JoUes' ferrometer. 2. Estimation of percentage of coloring matter bj' color tests. A. Fleischl's haemometer. B. Gower's haemoglobinometer. C. Dare's haemoglobinometer. D. Tallquist's haemoglobinometer. 3. Obtaining the specific gravity of the blood by Hammerschlag's method. (A). Fleischl's Haemometer. Appliances. Fleischl's haemometer; needle; pasteboard tube two inches in diameter; artificial light; small beaker; a dark room. Fleischl's haemometer consists of a sliding colored-glass wedge which moves in a standard underneath a cylindrical metallic cup, and a capillary tube. This cup is divided into two equal compartments and has a glass bottom and a detached glass top. The capillary tube is verj' small and is held by a small metallic band on a handle. The glass wedge and the capillar3^ tube are the important parts of the instrument and are made to be used together. There is a number on the handle of the capillar}' tube, indicating its capacity, and this same number is stamped on the top of the standard, also a number is placed on the end of the sliding frame that holds the glass wedge, and the same number appears on the base of the standard of the instrument to which it belongs. Reagents. Distilled water and hydrogen peroxide. Preparation clean metallic cell or well with water and dry with a cloth only when it needs it. The capillary tube should be cleaned with water and hydrogen peroxide, and then with water again, by waving it back and forth in the solutions for a moment or two. Then carefully dry the tube by blowing air through it, hold- Outlines of Experimental Physiology 25 ing- the tube about two inches from the mouth so as to avoid the moisture of the ex- haled air. Fill each side of the metallic cup about three-fourths full of distilled water. Prepare the needle and tlie ear or fing-er as in other tests. Technique. Obtain the blood in the usual manner. Hold the lobe of the ear with the thumb and fing-er. Use the second drop. Hold the capillary tube horizon- tally and Ciirefully touch the drop of blood with the end of the tube only. If the tube is clean it will fill rapidly by capillary attraction. If there is any blood on the outside of the tube or air-bubbles inside, it must be cleaned, dried and refilled properly. If the capillary is overfull, remove the excess by touching- the tip to a cloth or filter paper. Then quickly put the capillary tube into the water in one compartment of the metallic cell and wave it back and forth or up and down, and the blood, if fresh enough, will readily mix with the water; then allow a few drops of water from the medicine dropper to fiow through the capillary into the same com- partment to wash the blood thrtt sticks to the tube. Now fill each compartment al- most full with distilled water, taking care that the contents of either compartment does not flow into the other. Take the handle of the capillary and stir the one that contains the blood so as to make the mixture complete. Now carefully' slide the thick cover-glass over the compartments and gradually fill each cell with water as the cover-g-lass is put on until there is no air left in either cell. Exclude daylight by use of a dark room and adjust the reflector so that the artificial yellow light is thrown up through the^diluted blood and water from the side of the instrument, thus placing both cells in the same relation to the reflector and the light. While making the test always shade the eyes from the light by placing some thick paper or a pasteboard tube, that reaches from the instrument to the forehead, before the eyes. It is better to use only one eye at a time, and look only for a few seconds each time, giving the eye a rest and a chance to regain the ability to distinguish tints. Stand at one side of the instrument or turn the instrument so as to face the light and to bring the two cells into similar relations with the eye. Begin with a glass of a lighter color than the blood, and move the colored-glass slide by quick turns about i one-fourth of an inch each time until the color or tint of the diluted blood appears to I be the same as that of the colored slide; then make the reading. Next turn the ( colored glass on until it is darker than the diluted blood and do the same as before, except in the opposite direction, turning the slide until the color of the glass and j blood are the same, and then make the reading. Usually the first reading will be [ too low and the second too high. The difference will usually be about 10 per cent. The correct result will be between these two readings which can now be obtained In carefully moving the glass back and forth or by taking the middle point between the two readings. It is almost impossible to make the reading accurately and honestly unless great care is taken. The writer has found the method given to produce by far the best results. This method should be practiced again and again I and done with care. A hasty reading is rarely correct. Repeat the whole test until you can obtain the same result each time with the same individual's blood. Precautions. If the capillary tube is not perfectly clean it will not take blood by ca.pillary attraction. While cleaning the tube always test it by touching a drop of water, when it should fill immediately. This will save time and ensure quick work. The amount of blood taken is so small and this is diluted so much that the i;ast error is multiplied man}' times. We can expect accurate results on!}' when 26 Outlines of Experimental Physiology foreigTi matter in it, the tube will not hold the right amount of blood and the result will be too small. The blood must be obtained and mixed in the metallic cup with the water very quickly, or it will clot and stick in the capillary, or if it does leave it it maj' remain as a clotted thread of blood in the bottom of the cell. It takes a little practice to learn to wave the capillary in the small space of the cell. Very gentle constant waving back and forth, or into the water and out is the most effective in getting the blood out of the tube. Too vigorous movements are liable to break the glass tube. When completing the filling of the cells with water, fill the cell con- taining water only first, and then there is no danger of getting any of the diluted blood into the water compartment. If you neglect to stir the blood and water just before adjusting the glass cover the blood will remain in the lower part of the cup with the water on top, and it will have a darker color than it should because the blood reall3^ is not as dilute as necessary. This v»^ill give a higher reading than is accurate. The glass cover should always be used; it not only makes the amount of dilution accurate, but it gives an even surface for the transmitted light rays. With- out the glass the surface of the water is either concave or convex. The metallic cup should not be taken apart unless it is very dirty. If the glass is clean, that is sufficient. As a laboratory precaution, where several instruments are in use, alwaj^s compare the markings to be sure that you have the capillary tube that goes with the glass wedge that you have. Questions. 1. Why use distilled water to dilute blood and not a saline solu- tion similar to the plasma of the blood. 2. Can different individuals make approximately the same reading of the same test? 3. Does everj^ individual in ordinar}^ health have the same percentage of haemoglobin ? 4. How would you explain the variation, if any ? 5. Do individuals who have a low percentage of haemoglobin have a corres- pondingly lessened number of red cells per cubic millimetre? 6. Is the reverse of the above true ? (B). Gowers' Haemoglobinometer. Gowers' haemoglobinometer consists of three pieces: a capillary measuring pipette, a graduated tube containing a standard colored solution. The standard colored solution represents the color of 1 per cent, solution of normal blood. The graduated tube is marked in 100 or more parts, and each part represents 20 c.mm. The capillary pipette holds 20 c.mm. up to the mark on the tube. If the blood is normal it will be necessary to add water to the hundredth mark in order to make the colors correspond. If the blood is not normal the percentage can be read off the graduated tube at the top of the diluted blood when the colors correspond. There are two kinds of instruments: one for use with daylight, which has a white sub- stance in the sealed end of the tube containing the colored solution; the other is for use with artificial light and has a black substance in the sealed end of the tube. Reagents. Distilled water, hydrogen peroxide, alcohol and ether. Preparation. Clean the instruments in the usual manner. The capillary pipette should be cleaned with the same care and in the same way as the diluting pipette, being careful to first clean and then to dry the pipette. Fill the graduated tube to the mark 20 or 30 with distilled water; prepare the needle and ear as usual. Outlines of Experimental Physiology 27 Technique. Obtain the blood in the usual way except that a larg^er quantit}- of blood is needed than for the preceding- instrument. Hold the ear in the same way and fill the pipette as when obtaining blood in the pipette for counting corpuscles. Wipe away the first drop of blood. Touch the tip of the pipette to the drop of blood, resting- the pipette on the end of the thumb, which is behind the ear, and slowly suck the blood up to the mark on the pipette, but do not allow the blood to go beyond that point. If the drop is not sufficient, quickly obtain the second drop by gentle pressure high above the wound in the ear. If there is any excess of blood on the point or sides of the pipette, quicklj' wipe it away. Insert the pipette into the tube almost to the water and SI0WI3' blow the blood, drop by drop into the water. Now immediately shake the tube to mix the water and blood; this is to prevent the blood clotting or remaining as a thread in the bottom of the tube. Blood still remains in the capillary on its sides; so till the pipette with distilled water and blow this into the tube, three or four times. Then thoroughlj' mix the blood and waiter by shaking or rolling the tube gently. Do not place the thumb over the end and shake, as an appreciable amount of color will be lost and a foam is formed that delays the reading. • Now place the tube in a rubber block beside the tube containing the standard solution and add distilled water, drop bj' drop, to the diluted blood, always shak- ing the tube between the additions to keep the blood and water well mixed. Con- tinue this until the color of the blood solution is not darker or lighter than the stan- dard solution. The comparison of the colors is made either b^- transmitted or re- flected daylight. The eye will be assisted by placing the tube behind a piece of white paper and holding them toward the window light. The reading is made directly from the graduated tube, in percentage of haemoglobin when the color of the diluted blood is the same as the standard solution. Repeat the test until the same result is obtained continuall3% Precautions. If air bubbles are drawn up into the capillar}', or if it is either over or underfilled, the tube must be recleaned and dried and the test repeated until done accurately. If the pipette contains moisture or foreign matter the measure- ment will not be accurate. Always remove any blood that may happen to get on the outside of the pipette, as it will increase the result. It is a good plan to have a large drop of blood read}' before you begin to fill the pipette, rather than to take two or three small drops. Because of the time consumed to obtain the amount of blood needed there is liabilitj* of the blood clotting and sticking in the capillary. When only partially clotted the blood is blown into the graduated tube and remains as a clotted thread in the bottom. Gently striking the finger against the tube or shaking the tube sidewise is sufficient to mix the blood and water, and is far better than placing the thumb over the mouth of the tube and shaking it up and down. If this latter method is used it will make a difference of 5 to 10 per cent, in results. Alwajs be sure to wash the capillar}' out a number of times and place the washings in the graduated tube, or the result obtained will be less than the test should show. If a tube has been partiall}- or improperlj- filled, do not leave it so; always blow out the blood before it can clot, and much time will be saved. A reading should be made each time and recorded before more water is added, for if one should dilute the blood too much and had no record of the last reading the test is spoiled and the work lost. 28 Outlines of Experimental Physiology (C). Dare's Haemoglobinometer. Preparation. Dare's instrument estimates the percentage of haemog-lobin by comparing- the color of a thin film of blood of a certain thickness with a revolving colored, wedge-shaped disk of glass. The onlj^ preparation necessary is to clean and polish the glass plates that hold the blood, and adjust them in their holder. Technique. Obtain a good-sized drop of blood in the usual manner. Touch the edge of the plates to the drop of blood, and the space between them will be filled with blood by capillary attraction. Place the holder in its socket adjust the tele scopic tube and the lighted candle, and make the reading in the same manner as with the Fleischl instrument. A dark room is not necessarj', but it is well to hold the instrument toward some dark object as a background. (D). Tallquist's Haemoglobinometer. Tallquist's haemoglobinometer consists of a chart or a paper on which are twelve oblong, red-colored stripes, ranging from, 10 to 120 per cent in degree "of color. The color of the stripe marked 100 is supposed to be the same color and shade as that of a piece of filter paper in which there is normal blood. The other stripes vary from this as the numbers indicate. Preparations. ' Take a large piece of light yellow-colored paper and cut an ob- long hole in its centre, not quite as large as one of the colored stripes on the chart. Take a piece of the filter paper, at least twice the size of the colored stripes, and cut a straight edge on one side of the paper. Prepare the needle and ear as usual. Technique. Obtain the blood in the usual manner. Take the prepared piece of filter paper and allow drop after drop of blood to be absorbed into the paper until it is covered with blood over an area as large as one of the colored stripes of the chart. Put on just enough blood to saturate the paper, no less and no more. If there is too little blood on the filter paper it will "be white still on the under side. If there is too much blood on the paper it will have a glistening surface, and later will clot upon the paper. This must be prepared quickly and verj' evenly and then compared with the stripes of the chart at once. Itwill be noticed, when the blood is first put upon the filter paper and is still fresh, that it has a glistening appearance, but that it soon loses this and appears dull red for a few moments, and then it takes on a darker red appearance of clotted blood. The time to take the reading is while it has the fresh, dull-red color, just after the glistening surface has disappeared and before the dry, darker red color comes. This gives only a few moments in which to make the reading. Place the perforated paper on the colored chart and place the filter paper with the blood right next to the oblong perforation. The ex- aminer must control his inclination to manufacture results. This is best accom- plished by using the same method as with the Fleischl instrument. Do not allow the numbers to show. Begin the comparison with a colored stripe much lighter than the blood specimen and move up one stripe at a time until the colors appear the same; then make a reading. Next begin with a stripe of a darker color than the blood and compare colors in the opposite direction until they appear the same, and then make a second reading. The correct result will be between these two readings and usually the two readings will be 10 per cent or more apart. The test is made by reflected daylight. It is well to have a good bright light, although direct sun- light is not good. Do not let the wet blood paper touch the chart as it will destroy the color. Outlines of Experimental Physiology 29 (E). Estimation of Percentag-e of Haemog-lobin of the Blood by Finding ihe Specific Gravity. The Specific gravity of the blood can be obtained direct from a quantity of blood as with other solutions. This is not necessary, because when a drop of any fluid is put in another fluid of the same specific gravity that the drop does not mix with, it will go to the center of the latter fluid and remain there. If it is lig-hter it will come nearer surface, ane if it is heavier, it will sink. There area number of solutions that might be used. One of the most accurate is sodium sulphate in solution, placed indifferent cylinders in different strengths. The specific gravity of the blood, except in some cases, as in leukaemia and dropsy, varies as the amount of iron in the corpuscles. It must therefore be evident that the specific gravitj- of the blood varies as the percentage of haemoglobin varies. 'By consulting the table of Hammerschlag, given below, the percentag-e of haemo- globin can be read for the specific gravitj'of the blood at once. The most practical solutions to use for finding the specific gravity- of the blood are benzole and chloroform, because of the ease and the speed with which they may be used. TABLE OF HAMMERSCHLAG. Specific gravity. Hjiemoglobin. Specific gravity. Haemoglobin. 1.033-1.035 = 25-30 per cent. 1.048-1.050 = 55-65 per cent. 1.035-1.038 = 30-35 per cent. 1 050-1.053 = 65-70 per cent. 1.038-1 040 = 35-40 per cent. 1.053-1.055 = 70-75 per cent. 1.040-1.045 = 40-45 per cent. 1.055-1.057 = 75-85 per cent. 1 045-1.048 = 45-55 per cent. 1.057-1 060 = 85-90 per cent. Appliances. Specific gravity bulb or hydrometer; a quadrilateral or cylindri- cal graduated glass tube about six inches high; a pipette or pointed '"-lass rod- a stirring rod, and a glover's needle. Reagents, Those for cleaning capillary pipette, graduated tube, and needle also benzole (sp. gr. 0.879) and chloroform (sp. g-r. 1 060.) The h3'drometer is a glass tube containing mercurj- and air, and o-raduated so that when placed in distilled water at room temperature it reads 1.000. Preparation. Clean all apparatus as usual, and make a mixture of benzole and chloroform in the glass tube of a specific gravity of about 1 060. Technique. Secure the blood in the usual vvaj'. Suck at least three goodsized drops of blood into the pipette. Now before the blood clots insert the point of the pipette into the solution and blow out one or two drops of blood, but no air If the drop of blood goes to the center of the mixture and remains there after the mixture is well stirred, then the specific gravity of the blood is tlie same as that of the mixture. If the drop comes to the top it is lighter than the mixture, and benzole must be added and stirred in. If the drop goes toward the bottom it is heavier than the mixture, and chloroform must be added. Add just a few drops of benzole or chloroform at a time and stir well and test before adding more. The quickness with which the test is performed depend upon the carefulness in adding the benzole or chloroform and in keeping the mixture stirred. Repeat the test until the same result is easily and quicklj' obtained. 30 Outlines of Experimental Physiology Precautious. Everything- must be clean and dry. The blood will stick to what, ever it comes in contact with, the sides of the graduated tube, the stirring rod, or the specific gravity bulb, if they are not clean and dry There is danger, when the pipette is used to obtain the blood, of blowing small air-bubbles into the drop as it is put into the solution, which will cause it to float. If the mixture is lighter than the blood, the drop will go straight to the bottom and adhere with the force of the fall. The difficulty with the pipette can be overcome easily by using a pointed glass rod. Secure the drop of blood on the point of the rod and shake it off into the solution. The benzole and chloroform evaporate very rapidly and change the speci- fic gravity of the mixture. The two liquids do not stay mixed, but need stirring frequently. Do not attempt to work with the same drop of blood more than two min- utes. Take a fresh drop and continue. Make the specific gravity of the mixture as near that of the blood as possible before adding the second drop. One or two drops will always determine approximately what the specific gravity of the blood is; then take a third drop and prove it exactly. The solution of benzole and chloroform can be put into a glass-stoppered bottle and used again; so there is little waste except from evaporation. This is one of the best tests for obtaining the percentage of hae-, moglobin, as the personal equation is largely eliminated and the burden of accuracy is placed upon the instrument. QUESTIONS. 1. Why make the mixture 1.050 to begin with? 2. What is the specific gravity of benzole? Of chloroform? 3. Why are they better than other solutions for a quick test? Expt. XIII. Microscopic Test for Blood Pigment. Put a drop of blood on a slide and let it dry. Add a drop of strong glacial acetic acid. Add a grain of NaCl put on a cover glass and heat gently till the liquid begins to boil. Cool and exam- ine with the high power. Small brownish black crystals of haemin will be seen. This is an important test in medico-legal cases. Apply, this test to blood stains from a piece of cloth. Soak the cloth in a small quanity of NaCl solution. Evapo- rate to dryness and put on acetic acid and proceed as above. Expt. XIV, Spectroscopic Examination of Haemoglobin and its Derivatives. With a Spectroscope look first at a bright part of the sky. Focus until the num- erous fine dark lines (Frauenhofer's) running vertically across the spectrum are seen. Narrow the slit by moving the milled edge till the lines are sharp. Note especially the lines D in the orange, E and b in the green and F in the blue. Always hold the spectroscope so that the red is in the left of the field, (a) Now moisten a plati- num loop with H2O, dip in NaCl and hold in a fishtail flame. Exam- ine with the spectroscope. A bright yello;v line will be seen occupying the posi- tion of the D line in the solar spectrum. This is a convenient line of reference in the spectrum and in studying the spectra of haemoglobin and its derivatives, the po- sition of the D line with regard to the absorption bands, should always be noted. The dark lines in the solar spectrum are due to the absorption of light of a definite range of wave lengths by metals in a state of vapor in the sun's atmosphere, and of course no dark lines are seen in the spectrum of a gas flame. Arrange the spectro- scope, flame and test tube on a table. Half fill a test tube with defibrinated blood. Outlines of Experimental Physiology 31 Nothing- can be seen with the spectroscope till the blood is diluted. Pour a little of the blood into another test-tube and g-oon diluting until, on focusing-, two bands of oxyhaemoglobln are seen. Draw. Dilute more, — which of the bands first disap- pear? How dilute was the blood? One drop of blood to 50 c. c, HoO=l:750. (b). Make a solution of blood which shows the oxyhaemog-lobin bands sharply. Add 20 drops of strong- ammonium sulphate solution to reduce the oxy haemoglobin. Heat gentl}' to the bodj' temperature or add a few drops of freshly prepared Stokes fluid (ferrous sulphate and tartaric acid, each the size of a pea, dissolve in H2O, make alkaline with ammonia hydroxide,) gives a clear green solution; now shake it so as to mix it with air and note the oxyhaemoglobin band appear. Why? U). Carbonic oxide haemoglobin. Pass coal gas through blood for some time. Dilute and examine. Two bands almost in the position of the oxyhaemoglobin band are seen but no change is caused by the addition of ammonium sulphide, since car- bonic acid haemoglobin is a more stable compound than oxyhaemoglobin. Now shake it with air. Does the oxyhaemoglobin band appear? (d). Methaemoglobin. Put some blood in a test tube, add a few drops of strong potassium ferricyanide and heat gentl}'. Dilute, examine. A well marked band will be seen in the red. On addition of ammonium sulphide, this band disappears; the oxyhaemoglobin bands are seen for a moment and then g-ive place to the reduced hiiemoglobin band. Shake in air. Results? {c). Dilute a solution of eosin or picro carmine with water until it has the shade of blood that gives the two ox3iiaemoglobin absorption bands. If the two bands ap- pear, test with Stokes fluid or strong NH 4 S. Result? Mix with air. Result? Literature. — Ziemke and Muller, Archiv fur Anat.-Phys. 1901, Sp. Bd, 177. Test for Carbon-monoxide Blood. Expt. XV. (I), Kunkel's test. (a). Mix carbon-monoxide blood with four times its volume of water. To a measured quantity of this mixture add an equal volume of 3 per cent tannin solution. Note the change in a few hours. (b). Repeat (a) with normal blood; let stand a few hours and compare with (a). Result? Precipitin Test for Human Blood, Medico Legal Test. Expt. XVI. See Nuttall. Blood Immunity, 1904. The Action of the Valves of the Heart. Expt. XVII. From the mammalian heart, note the form, structure and attach- ment of all the valves in the heart. Observe the difference between the walls of the veins and arteries, the left and right auricles and ventricles. (a). Illustrate on the mammalian heart, the position of the blood vessels semi- lunar and auriculo-ventricular valves on a median section of a heart or show dia- grammatically the action of the valves and the course of the blood through the heart after having studied the following experiment (b). Tie a wide glass tube (A) lOcm. long in the left auricle and one (B) 5Ucm. long into the aorta. Ligate all the other blood vessels of the left side of the heart. Connect A with a faucet by rubber tubing and place the heart in a large dish. Let water flow into the heart and support B by a clamp to a stand. When the water 32 Outlines of Experimental Physiology stands on a level in both tubes, compress the ventricle. In which tube does the water stand higher? Relax the pressure. What is the effect? Why? By alter- nately compressing- the ventricle and allowing- it to relax, water can be sent into the aorta tube to overflowing into the left auricle, imitating- one-sided circulation. (c). Pour water into the pulmonary artery and note the semilunar valves. Make an incision into the right auricle and one in the right ventricle so that you can see the tricuspid valves; study its action by pouring water into the ventricular incision and also into the auricular. The Circulation Scheme. Expt. XVIII. With this apparatus the physical phenomena of the circulation may be thoroughly studied. Describe (1) the conversion of the intermittent into the continuous flow. (2). The relation between the rate of the flow and the width of the bed. (3). The relation of peripheral resistance to blood pressure. (4). The period of outflow from the ventricle. (5). The pulse waves. (6). Compare venous with arterial pressure. The flow of blood through the arteries, veins and capillaries follow the laws which govern the flow of any liquid through a system of tubes, so that we are able to illustrate many features of blood flow by means of an artificial arrangement of tubes drawn up in imitation of the circulatory mechanism. The rubber bulb pro- vided with valves which permit the flow of the fluid in one direction only, represents the heart. (Left). One manometer is situated in the course of the arterial system* represented by rubber tubing connected with the bulb at the end having the out- flowing valve. The other manometer is beyond the two branches that anastomose to form one vein. The tube filled with wood fibre represents the capillaries; the tubing from there on, the veins. (b). When a pump forces water or any other incompressible fluid through tubes with rigid walls, the inflow and outflow are equal and in the same time. The outflow ceases the instant the inflow ceases. The same is true in a system of elastic tubes so short and wide that friction between the liquid and the walls causes prac. tically no'resistance to the flow. Here the quantity received from the pump can still escape from the distal end of the system during the stroke of the pump. When this resistance is increased bj' narrowing the tubes or increasing their length, or in both ways, not all the liquid received from the pump can pass by the resistance during the stroke of the pump; the remainder must pass during the interval between one stroke and the next. This portion which cannot pass during the stroke, finds room between the pump and the resistance by the dilation of the containing vessels. To efl'ect the dilation, the force or pressure transmitted from the pump presses out the vessel walls until this pressure is held in equilibrium by the elastic -reaction of the walls As the pressure from the pump wanes, the energy stored by it in the tension of the vessel walls is reconverted into mechanical motion and the walls return toward their original position, driving the liquid out of the tube, past the resistance- In using this scheme, only slightly compress the bulb at each beat. Watch the arterial manometer and be careful not to force the mercury out of it. • 1. Open the side branch by unscrewing the pressure , clip. See that the tubes are free from air and well filled with water. To get rid of the air hold the tubes up with the right hand while filling with water. Make a single, brief, gentle pressure Outlines of Experimental Physiology 33 on the bulb. (Note 1) That practically all the liquid driven out by the stroke escapes throug-h the side branch in which the resistance is low rather than throug-h the hig-h capillary resistance. (2). Only a portion of the liquid escapes throughout the stroke. (3). The portion which cannot escape by the resistance during the stroke finds space in a very evident dilation of the tubes nearer the pump; i. e., between the pump and the principal resistance. The membrane manometer shows a sudden rise and fall indicating a sudden rise and fall in the intraventricular pressure. Fill the bend of the manometers with mercury and the whole apparatus with water, all air being expelled. Open the clip opposite the capillaries. You will now study the flow along a closed system of elastic tubes without peripheral resist- ance, if the extremity tubes are connected. Now imitate the beating of the heart by rhythmical pressure of the bulb, — gradual!}' increase the rate. Study the move- ment of the pulse and note the following. (1). Compare the movements of the two manometers in time and amplitude. (1). Note the effect of the vein tube and the manometers when the bulb is filling (diastole). (3). Close the clip, thus interposing high resistance, and pump fluid through the system as before. Note the effect of the systole on both manometers. What is the effect of diastole on both manometers? What is the result if only one emptying of the bulb is carried out — i. e., after ocilliations have ceased? What is the result if, before the pressure becomes equalized, the bulb be once more emptied, before the second effect has passed off? What is the effect on the manometer of about 20 strokes at a definite rate? Does the rise in pressure occur at the instant the bulb is compressed? Can you feel a pulse or pressure wave? Is there a nega- tive pressure formed and where? What important condition found in the circulation is produced by this scheme? How could the scheme be improved? B. The Conversion of the Intermittent Into the Continuous Flow. (1). With the side branch open, compress the bulb rhythmically, gradually in- creasing the frequenf'y of the stroke. It will be found that with few strokes the stream will be intermittent. As the interval between the strokes is shortened, the liquid received from the pump in any one stroke cannot all escape by the resist- ance during the stroke and the succeeding interval. The next stroke comes before the outflow from the preceeding stroke is finished, and the stream becomes remit- tent. Still further increase the frequency of the stroke. A rate may be reached at which the intermittent will be converted into the continuous flow. Observe that the duration of the interval is greater than the duration of the . pump. Thus the greiiter part of the tim2, the circulation is carried on, not by the direct stroke of the pump, but the energy stored up by the pump in the elastic walls of the vessels. Note that the arterial pressure remains low evea after this stream becomes continuous. An increase in the frequency of the beat has little influence on the blood pressure when the peripheral resistance is slight. Record pressure in millimeters of mercury. (2). Close the side branch, not completely, but so that the liquid must pass through a high peripheral resistance Compress the bulb at such a rate that the outflow shall be continuous. Be careful not to force the mercury out of the arterial manometer — Compress the bulb only partially. Compare the frequency required to 34 Outlines of Experimental Physiology- make the flow ccntinuous now, with that required when the peripheral resistance was low. N. B. The capillaries sometimes becom'e clog-g-ed up with particals from the water. If that happens, speak to the instructor. (3). The relation of the peripheral resistance to blood pressure. Compress the bulb at such a rate that a continuous flow will be produced. With each success- ive stroke the portion of liquid unable to pass the resistance during the stroke and the succeeding interval, is added to that left behind from the preceding strokes. The arteries become more and more full. The arterial manometer registers a higher and higher pressure. At length the mercury ceases to rise. The mercury remains at a mean level broken by a slight oscillation at each stroke. The pump now merely re' tains the constant high arterial pressure. This pressure suffices to drive through the resistance during each stroke and the succeeding interval, all the liquid received from the pump during the stroke. Is the venous pressure high or low? Why? Is there a pulse on the venous side of the resistance? Why? (D). Inhibition of the ventricle. While the arterial pressure is at a good height (120 mm Hg) arrest the ventricular stroke. (The s'entricle in animals may be thus inhibited b}' stimulation of the vagus nerve.) Observe the arterial manometer and explain. Resume the ventricular beats Explain the effects on pressure. The pulse curve. Cover the thistle tube of the sphygmograph (thistle tube) with thin sheet rubber and cement a bone button to the center of the membrane. Connect the tambour thus formed with a recording tambur. Bring the writing point against a slow moving, lightlj' smoked drum. While the pump maintains a moderate pres- sure (about 50mm Hg), adjust a button on the aorta and then (not sooner), close the side branch of the sphj-gmograph tube. Record a series of pulse curves. Note a quick up-stroke corresponding to the quick distention of the artery by the emptj'ing of the ventricles and the gradual down-stroke, corresponding to the gradual emptying of the artery through the resistance during the diastole or interval between two beats. The down-stroke is broken by several small waves caused by the oscillation of the mercury in the arterial manometer. Near the apex of the more delicately written curves, may be seen a slight depression, the dicrotic notch, — this wave is more easily studied in human pulse curves. Low Tension Pulse. Open slightly the side branch that permits the liquid in the arterial tubes to flow out without passing through the resistance. The arterial pressure will fall in con sequence of the diminished peripheral resistance. Normally, this effect is produced by dilation of the smaller arteries. Let the arterial pressure sink to about 20 mm Hg. Record a series of pulse curves. The oscillations of the mercury column are much lighter than with normal pres- sure (120—150 mmH'^). Feel the pulae with the finger. At each beat the artery quickly expands and as quickly relaxes The artery is softer than usual. High Tension Pulse. Close the side branch almost entirely so that all the water has to pass through the porous wood. What is the effect on arterial pressure? Be very careful not to compress the bulb too hard in this experiment as the mercury will otherwise be forced out of the manometer. Outlines of Experimental Physiology 35 Hardy's Experiments on Colloids. Expt. XIX. Put some of the prepared acid heat-modified proteid into one of the U tubes supported on the stand, tilling,'' it to within an inch of the top. Adjust the platinum electrodes so that they are immersed in the solution and connect with six batteries placed in series or electric lifj;-hting-. using- g-round g-lass and carbon re- sistance = .000001 C Prepare another tube of alkaline proteid in the same way a third of neutral and a fourth of undialyzed albumen and introduce into the same circuit. Watch the experiment through the day and ag-ain the next day if uo defi- nite collection and precipitation of proteid has occurred. Where does the proteid collect? and why? The heat-modified proteid is prepared by diluting- white of eg-g- with nine vol- umes of distilled water, filtering and boiling. The resulting liquor must be blue and transparent The first portion of the boiled proteid is white, owing to the sur- face action of the glass and should be thrown away. The colloidal solution is then dialyzed seven days in a cool place, covered and boiled to remove the salts. Those who wish can prepare some ot this proteid as an extra experiment. Acid or alkaline proteid is made by adding a trace of acetic acid or KOH Dip a needle into KOH per cent, wash this in cc distilled water and from this take a needle full. (2). Colloidal Ferric Hydrate is prepared as follows;— To a fairly dilute solu- tion of Ferric chloride, ammonium hydrate is added until a slight permanent pre. cipitate of Ferric Hydrate is formad. This is redissolved by adding a little Ferric Chloride to the solution. This mixture is subjected to a prolonged dialysis of seven weeks in distilled water to remove the salts in the mixture The solution is neu- tral. Put some of the prepared Ferric hydrate in a U tube, subject it to the electri- cal current of 105 volts for 24 hours Note the region of coagulation. (3). Test coagulation of ferric hydrate solution by ions. Prepare test tubes, each containing 4c. c. of the salt solutions provided. All are m/100 solutions. Have also a tube of distilled water as a control. To each tube add three to five drops of the ferric hydrate solution. With which salts does precipitation take place at once? Which precipitate on warming? Repeat with those that do not precipi- tate at once, letting the tube stand without warming till the end of the day. Try solutions of cane sugar of various strengths. Ascertain the weakest solution of MgS04 that will cause the immediate precipitate (say in two minutes) of ferric hy- drate. Repeat with MgCU, NaCl, Na2HP04, AloiSOi)^, CuSO*, CuClg, K3SO4, CaCls, Na2S04. (4). Test the effect of the salts employed in (3) on acid heat modified proteid. (5). Test the eflfect of the salts employed in (3) on alkaline heat modified pro- teid. (a) Arrange the results of all the tests in a table, giving results both with and without heat. Literature. Journal of Physiology, Vol. 24, p. 289. Mann p. 37. 36 Outlines of Experimental Physiology Effects of Lack of Oxygen and KCN upon Cells. Expt. XX. (a) Place several drops of Paramoecia or Vorticella culture in an Eng'leman or capillary gas chamber. Cover the edges of the chamber with vase- line and close tightly, making sure that no air can enter. Connect the chamber with your hydrogen generator. Wash the glass as usual and keep up a slow stream through the chamber. From the chamber pass the gas into a breaker of water. Note carefully the behavior of the Paramoecia, and make drawings of the changes produced in the cells. (b) If yoti have time to repeat the experiment, use a hanging drop of the culture and study the changes with the high power, after half an hour. (c) To several drops of Paramoecia culture in a watch glass under the micro- scope, add one drop of an 0.1 per cent solution of KCN. Note the effects and draw. Use the micrometer scale and compare with the normal form. (d) Repeat, using a 0.5 per cent solution of KCN. What conclusions do j'ou draw as to the comparative effects upon the cell of KCN and a lack of oxygen? Explain why KCN is toxic. See Lyon's paper. A. M. Journal of Phys. Vol. VIII. p. 56. Ciliary Motion. Expt. XXI. Cut the frog in two, midway between the fore and hind limbs. Remove the anterior body wall and all the viscera, except the oesophagus and the upper part of the stomach. Remove the arms, cut away the lower and upper jaw on a level back of the eyes, and slit up the oesophagus in the mid-ventral line. Through an opening made between the mucous lining and the skull, pass the glass plate between the oesophagus and the vertebra. Stretch out the oesophagus and the stomach and fix them with pins on the cork each side of the glass plate. (a) Place a small wooden or cork block on the mucous membrane at the end near the head of the frog. It will be carried along towards the stomach. (b). Repeat the experiment, placing a wedge under the cilia board. In what direction do the cilia lash particles? (c). Weight the block with lead weights and determine how great a load can be carried on a level and up an incline plane. The mucous membrane should be wet with normal saline solution. (Avoid excess). (d). Cauterize with a hot wire superficially a small area of mucous membrane in a preparation thinly bestrewn with charcoal. Does the action of the cilia in the neighboring parts change? Are the movements depended upon stimuli from neigh- boring cells? What kind of an area is affected? What does this show? (e). Cauterize two parallel lines (very short) first longitudinally, then across. Effect on the charcoal movements? (/■). Try NaCl (normal) at 35° C on the membrane, noting the time of the move- ment before and after. Expt. XXII. From the edge of the gill of a clam or oyster, snip off a small piece and mount in the animal's own fluid and examine with low and high powers. (a). Make a careful study of the rapid movement. What is its object? Watch the progressive slowing. (b). Tease out single cells from the mass. Does the movement continue? If so, does it differ from that of the mass? Illustrate. Outlines of Experimental Physiology 37 (c). Apply heat with a lamp or the warm chamber of the microscope to the pre- paration and observe the effect. At what temperature is the movement most violent? When do movements betrin to slow?, stop? Apply cold to another specimen. (d). Find a spot in which the movements are very slow and study the action of a single cilium when in the position at rest. What is its path vvhen rapidly moving-? Sketch. Expt. XXIII. The influence of Stimulants and Narcotics on Ciliary Movements- Prepare a slide with cilia in a so-called gas chamber; two gas flasks joined each to a faucet and through a T with each other. At the free end of the T attach another T and from each free end of the latter, rubber tubing joins the inlet tube of the gas chamber, while the tubing from the outlet tube ends in a ghiss of water. (To obtain the CO 2 gas, use CaCOs and HCl or obtain it from a CO 2 cylinder). Fill a flask with water and displace with the gas. In the other flask put either oxygen or hydrogen. The flow of gas is regulated by means of the flow of water and clamps, one of which is on each side of the T. Prepare a specimen of cilia for observation with the low power microscope. Bring a good specimen into the field, observe the rate and character of the move- ment. Allow a slow stream of CO 2 to pass in. Observe the effect If no effect oc- curs, repeat the dose of gas. Results? (a). After the effect has become apparent, clamp the tubes and draw in fresh air, thus restoring normal conditions. What is the effect? How many times may cilia be restored to activity by ventilation? after having been completely stopped by CO 2? (c). Try oxygen. (Heat oxygen mixture, MnO^ & KCIO3 for 20 minutes). What happens? (d). Chloroform gas on cilia movements. Take a new preparation of cilia and observe the normal movement. Place cotton saturated with chloroform in a 'flask and allow the gas to pass through the chamber. Note the effect. How many times may the cilia be revived? (e). Use alcohol instead of chloroform. Results? (/). Use 0.4 per cent formole. (^). Tobacco smoke may be tried. (h). Try dilute acids and alkalies, 0.1 per cent each. (/). Dissolve out salts with distilled water }^ hour and revive with NaCl. The Kymograph. Make yourself acquainted with the instrument. Learn how to wind it, not too tightly; how to start and stop the clockwork; how to raise and lower the drum; and how to change the .speed. Always remove the fans by a steady even pull directly in line with the spindle on which they revolve. Never wiggle the fans from side to side on the spindle On the old kymographs, the speed may be regulated by turn- ing the milled head to which the pointer is attached. To put the paper on the drum. Lay the sheet of paper flat upon the table, moisten the gummed end, lay the drum across the paper and with a hand on each side bring the paper up tightly around the drum, lapping the- paper evenly and glu- ing it down. Note that the paper should lap in the direction opposite that in which 38 Outlines of Experimental Physiology the drum will travel, to prevent the writing- point on the lever from catching at the fold. Blackening the drum. Note that the paper is wider than the drum. This is to prevent smoking the drum itself. Hold the drum by the shaft and turn it rapidly in the flame. A thin brown coating is much better than a thick, black one. After blackening the paper, cut away the projecting edges, if necessary, with a scalpel. To remove the paper from the drum. NOTE CAREFULLY. The paper is ex- tra long. Always cut the paper from the drum by inserting a scalpel between the overlapping edges. You thus secure a clean end to handle the tracing by and also avoid scratching the drum itself. Never lay t.he drum down. Denting or scratch- ing or other injury to the drum must be paid for. The facts demonstrated by each tracing should be fully written up in your notes. Put your name and the date on the tracings and fix them by passing them through a solution of shellac. Expt. XXIV. Preliminary Preparation to the Study of the Frog's Cardiac Nerves and Heart Action. Preparation of the Vagus Nerve. Fasten a large, formole-prepared frog on the holder, back down. Pass a glass tube through the eosophagus into the stomach. Remove the muscles lying over the petro-hyoid bone. Lying near the line between the angle of the jaw and the auricle are four nerves; first, the hypo- glossal; this one is superficial. Near their emergence from the skull, it is the lowest of the nerves but later, the uppermost. It crosses the remaining nerves and the blood vessels and passes forward and inward toward the tongue: Second, the glosso-pharyngeal which soon turns forward beneath the hypoglossus parallel to the ramus of the jaw; third, the vagus; and fourth, the laryngeus, the two lying almost parallel in the line between the angle of the jaw and the auricle. The laryngeus rests upon the petro-hyoid muscle and passes upward and inward beneath the arteries toward the larnyx. The vagus runs at first along the superior venacava to the auricle; a branch is given oflf to the lungs. Clear the vagus and tie a silk thread around the nerve. Cut the nerve on the central side of the liga- ture, so that the peripheral stump can be placed on the electrodes for stimulation. Divide the laryngeal branch. Note how near the vagus goes to the eustachian tube and ascertain if stimulation of the tube inside the mouth reaches the vagus. Expt. XXV. Preparation of the Sympathetic Nerve. Cut away the lower jaw of a large frog at the angle of'the mouth a short distance downward, not injurying the vagus. At the junction of the skull and backbone will be seen on each side the L. A. S. muscle. See the chart for the explanation of the parts. OC=Occiput L. A. S.=Levator Anguli Scapulae. Sym.=Sympathetic. Gp=Glosso-pharyn- geal. VS=Vago-Sympathetic. G=Ganglion of the Vagus. Ao=Aorta. SA= Subclavian Artery. (a). Expose the vertebral column where it joins the skull and remove the mucous lining of the mouth and clear away the connective tissue lying over the first cervical vertebra and the sympathetic, with its ganglion, will be seen. The sympathetic is situated directly beneath tbe L. A. S. muscle, which must be carefully removed. The nerve, usually pigmented and lying under the artery, will then be seen. Carefully isolate and put two ligatures around the nerve as far as possible from Outlines of Experimental Physiology 39 the skull (about the level of the larf^e brachial nerve). Cut between the ligatures and isolate carefully to its junction with the vagus ganglion. Note especially whether the sympathetic of both sides can be stimulated with electrodes placed far back in the mouth; how near the wires must be and how far back in the mouth. Illustrate the connection of the vagus and sympathetic with the heart. [b) With the prepared frog (n), follow the directions under Expt. XXVII as far as possible, as a preparation .for future work. (c) With the prepared frog of (b) follow the directions under Expt. XXVI. Draw a dorsal and ventral view of the superficial mucles and indicate the position of the sciatic nerve to its origin. Expt. XXVI. Nerve Muscle Preparation. Divide the body transversely behind the forelimbs. Remove the viscera. Seize the spinal column with the finger and thumb of one hand, and the skin of the back with the other. Draw the hind limbs out of the skin. Cautiously divide the con- nective tissue between the semi-membranosus and the biceps femoris and observe the sciatic nerve and femoral vessels. Clear the nerve from the knee to the vertebral column, using scissors and forceps. The nerve itself should not be touched with the instruments. Divide the spinal column longitudinally and cut away all but a small piece of the bone. (This is left for handling the nerve). Pass now to the leg. Cut through the Tendo Achillis of the Gastrocnemius below the thicken- ing of the heel. Free the muscle up to its origin from the femur, taking care not to harm the branch of the nerve which enters the muscle on its posterior surface near the knee. Cut through the tibia about 1 cm. from the knee joint. Clear away the muscles from the lower end of the femur, avoiding the sciatic nerve. Cut through the femur about its middle. I^ay the sciatic nerve along the gastrocne- mius muscle for safety. Be careful not to stretch the nerve. Keep it moistened with normal saline solution. Expt. XXVII. {a). Pith a frog. Wrap the frog in a cloth, the head out. Hold with fingers of the left hand, pressing down the tip of the frog's nose with the left fore finger. Pass the right forefinger along the middle line of the head. A slight depression will be felt at the junction of the skull andthe trunk Here the cerebro- spinal canal has no bony covering. At this point make a cut li of an inch long through the skin in the mid-line. Thrust the blunt needle vertically through the soft tissues until the point is stopped by the vertebrae. Moving the point of the wire toward the head, push it along the brain cavity, moving it slightly from side to side. Use a piece of a match to stop the bleeding. Place the frog on the table for observation. (b). Compare the reactions with those of a normal frog. Pinch different parts. Place it on its back. Conclusion as to the absence or presence of the brain? Expt. XXVIII. Heart Action. Place the frog, back down, in the holder; make a median cut 1>2 inches long, beginning below the head Avoid the median vessel. Then cut from the middle of the incision from side to side about a half inch Lift the sternal cartilage doing no harm to the deeper part and cut off its tip thus avoid- ing the epigastric vein. Divide the abdominal muscles from side to side in the line of the cross cut. Open the pericardium, turn the frog-holder over to the right nearly perpendicular thus bringing the frog's left side next the table Support the holder 40 Outlines of Experimental Physiology with the pahii of the left hand. With the forceps in the other hand pull aside the chest wall so as to expose the heart, {a). Observe the great veins, auricles, ven. tricles, and bulbus are contracting. The aortae are not. The veins first, then the auricles, ventricles and bulbus contract. Note the color of the contracting ventricle. Make and label an enlarged drawing of both the dorsal and ventral surfaces of the heart, showing the vagus and other nerves near the heart, (Ecker, p. 212. Schenck p. 279). In the frog, the augmentor and inhibitory nerves reach the heart through the splanchnic branch of the vagus. To excite either fibre alone, it is necessary to stimulate the respective nerves above their junction. ib). Place the heart in the heart-holder having its lever, weights, and moisten- ed filter paper in place. Arrange a time-marker for marking seconds on a drum revolving so that the beats shall appear far enough apart to be readily counted. Bring the writing point of the lever and that of the time-marker vertically under each other on the surface of the drum. When the lever rests on the auriculo- ventricular junction, the tracing shows the systole of both. For simultaneous records of auricular and ventricular contraction, special levers are put both on the auricles and ventricle. (a). Which parts of the curve trace the systole and which the diastole? (h). Which is Iwnger, and how much? (c). What is the rate per minute? Expt. XXIX. Action of the Vagus on the Heart. (See Musken, Am. Jour, of Phys. Vol. 1. p. 486 ). Arrange the inductorium for weak tetanizing currents. In the primary circuit place the electro-magnetic signal. Expose the heart, place in a heart-holder. Let the point of the lever write exactly above the signal on a drum revolving so slowly that the individual beats shall appear in the curve very close together and yet far enough apart to be counted. Lay the vagus nerve on the elect- rodes (or use Musken's Vagus Stimulators placed just posterior to the inner opening of the Eustachian tube, pressing the electrodes firmly against the mucous lining of the dorsal side of the roof of the mouth). Start the drum. As soon as good curves are writing, start the inductorium and open the short circuiting key for about 20 sec. The heart will be inhibited. Note that the arrested heart is always re- laxed; i. e., in diastole. The latent period is short (one or two heart beats). A brief after effect is present. If the stimulous is continued, the heart will begin to beat even during stimulation, showing that the inhibitory mechanism can be ex- hausted. The heart beats more rapidlj' and usnally more strongly immediately after inhibition than before. This probably is due to the after effect of the stimula- tion of the augmentor fibres in the vagus trunk. Repeat the stimulation but weak- en the current by moving the secondary farther from the primary coil. With a suitable strength of current, the heart will be slowed but not arrested. The dura- tion of diastole will be less, while the duration of the systole will be changed but little, if at all. A stronger excitation would lengthen both systole and diastole. The diminution in force appears before the diminution in frequency. Compare the curves obtained before, during, and after stimulation. (b). Action of the vSympathetic on the Heart. Arrange the apparatus as in (a). Prepare the sympathetic as previously directed (or use Musken's electrodes placing the lead points close against the sides of the vertebral column, just posterior to the roof of the mouth). Expose the heai-t. Place it in the holder. Obtain a curve, then Outlines of Experimental Physiology 41 stimulate, the sig'tial indicating- the duration period of the stimulation, then obtain a curve after stimulation Compare the frequency and force of the heart's action as indicated by the curves. Expt. XXX. The Influence of the Sciatic on the Heart and Blood-vessels. («)• Destroy the frog's brain in front of the medulla. Expose the sciatic, insulate with rubber, ligate half a centimeter apart Place the heart in the holder, obtain curves, then cut between the ligatures. Obtain a curve while stimulating the peripheral and again after stimulating the central end. Compare. {0) Examine the circulation in the frog's web under the microscope. Note care- fully all the changes produced in the vessels during stimulation of the peripheral end of the sciatic. Has stimulation of the central end any effect? Sounds of the Heart. Expt. XXXI. (a) Study the cardiac impulse in a fellow student, first by direct auscultation, then with {f>) The Stethoscope or Phonendoscope. Make out the sounds and pauses. Which sound is most distinct in this region? (c) With the hand over the radial artery, determine whether the pulse is felt in the period of pause or sound. {d) Place the stethoscope over the junction of the second rib with the sternum on the right side. Compare the relative intensity of the sounds of the heart here with the relative intensity a.% heard over the cardiac impulse or other regions. Expt. XXXII. Time of Systolic Phase. Arrange a tuning fork (100 vibrations per second) and a signal to write on the drum. Then with the stethoscope, listen for the first and second sounds, closing the key for each. Calculate the time for several observations and take the mean. Calculate the time for diastole and pause. For systole. Expt. XXXIII. Human Pressure Pulse. Frequency:— Palpate the pulse with ball of first, second, and third fingers. With the touch alone you can learn the rate, rhythm, volume, strength, and com- pressibility. (a). Note the frequency per minute when the subject is standing, sitting, lying down and after exercise. A pulse which is obliterated by slight pressure is termed "soft". If considerable pressure is required is "hard". (b). With a sphygmomanometer compare the pulse with the blood pressure in a student in different attitudes. Expt. XXXIV. Carotid Pulse or Cardiogram. Place the button of the cardiograph over the cardiac impulse; or the thistle tube without a membrane joined to a delicate tambour (side branch open until adjusted) over the carotid artery, then close the side branch of the tambour. Obtain tracings, calculate the frequency and compare with the form and frequency of curves obtained when standing, sitting and after exercising. 42 Outlines of Experimental Physiology Expt. XXXV. Radial Pulse. (a). Cover the small thistle tube with a rubber membrane having- a small cork cemented to its centre. Place the cork on the radial artery and record throug-h the tambour a radial pulse. In order to secure a satisfactory curve the deg-ree of pres- sure must be carefully regulated. The pulse curve gives only the variations in blood-pressure and form of the pulse, (does not give absolute blood-pressure — hardness, amplitude or size). These are better obtained by palpating fingers. (b) Sphj'gmograph Tracing. Let the subject stand at the right of the observer' resting the dorsal surface of the left arm upon the observer's knee. The observer standing with his right foot on a chair. Mark with pencil the location of the radial artery and adjust the sphj'gmograph so that its pad is in position and the tension and pressure adjusted for maximum swing of the tracing needle bj' means of the pressure screw. Observe whether there are variations among the members of the class in the location of the radial artery and- whether adipose or muscular tissue hinder the observations of the pulse. (1). Obtain the pulse rate, (2)., the pulse curve when standing, (3)., sitting, (4)., lying, (5)., after exercise, (6)., after inhaling two drops (on no account more) of the nitrite of amyl on a handkerchief. Observe as the face flushes the pulse will be softer. (7). Effect of the vertical position of the arm, (8) , compressing the arteries at the elbow. (9). Inhale some ether. Get the pulse before and after smoking. Expt. XXXVI. Pulse Volume. Connect the Plethysmograph through a tam- bour, side tube open, to write the variations of air pressure on a drum. Obtain a time record under the pulse record of the finger inserted not to impede the circula- tion in the plethysmograph. (a), horizontal and (b) vertical position and {c) effect of forced respiration upon the curve. Expt. XXXVII. Effect of Drugs upon the Heart's Action. (1). Intracardiac Inhibitory Mechanism. Expose a frog's heart. Note the white crescent between the sinus venosus and the right auricle. Arrange an induc- torium for weak tetanizing currents and put the points of the electrode on the cres- cent; stimulate. After one or two beats the heart will stop. (2). Action of Muscarine. Expose the heart of a pithed frog in a heart-holder. Test the action of the vagus, either with Musken's or with ordinary electrodes. Get a curve, then with a clean pipette put a few drops of 0.1 per cent muscarine on the ventricle, (b)., a 0.5 per cent, (c)., again stimulate the vagus. Result? (3). (a). Atropin O.S per cent, a few drops placed on the ventricle with a clean pipette. Get a curve before and after applying the drug. [b). Stimulate the vagus; what is the effect? (c). Stimulate the crescent; effect? Upon what part does atropin act? Expt. XXXVIII. Nicotine. Apply 0.1 per cent solution of nicotine to the ven- tricle, After a few minutes, stimulate the vagus nerve. The heart is not inhibited. (b). Now lift the heart with a glass rod and stimulate the intracardiac inhibitory centres. The heart is inhibited. Nicotine paralysis some inhibitory mechanism between the vague and the intracardiac nerves. It is known that nicotine does not paralyze nerve-trunks, hence it is probable that the cardiac inhibitory fibres do not pass to the cardiac muscle directly, but end in contact with nerve cells which take Outlines of Experimental Physiology 43 up the impulse and transmit it throug-h their processes to the muscular fibres of the heart. (t). Repeat with 0.5 per cent Nicotine. Expt. XXXIX. Reflex inhibition of the heart. (Use a small frog). In a very lightly etherized frog, expose the pericardium by cutting away the chest wall over the heart. Count the number of beats in periods of 20 sec. Con- tinue the heart count while an assistant strikes gentle blows with the handle of a scalpel upon the abdomen at the rate of about 140 per minute. The frequencj- will usually diminish; and in favorable cases, the heart will at length be arrested. Note the effect on respiration. Cut both vagi and repeat the experiment. The reflex inhibition of the heart cannot be secured. It has been shown by Bernstein that the afferent nerves in this experiment are the abdominal branches of the sj'm- pathetic nerve. The stimulation of the central end of the abdominal sympathetic in the rabbit also produces reflex inhibition of the heart. Expt. XL. Digitalin. (The commercial varies in composition, decomposes soon, and is soluble, one part in three of water). (a) Pith a frog, expose the heart, obtain a normal curve, and note the size of the auricles and ventricle. Appl}^ 4 gtt. of tincture of digitalis to the heart. How quickly- does it affect the heart? Which part contracts more vigorouslj-? Is the tonus of the muscle increased? Is the size of the ventricle? What is the change in heart curve? {b). Digitalis acts on the vascular sj'stem, central nervous sj'stem and increas- es the irritability of the heart muscle. Fasten a pithed frog on the cork board and stretch the tongue or web over the cover-glass that is secured over a hole in the cork by means of sealing wax. Focvis the microscope on a certain arteriole and measure its diameter with an e\'e-piece micrometer. Measure the diameters of the heart and make drawings of the same. Now inject into the dorsal lymph spaces, 3 gtt. of Tr. of Digitalis and measure the arteriole at intervals of 10 minutes. Keep moist with normal saline solution. (Digitaline, 3gtt. of 0.5 per cent maj' be used instead of tincture ) («). What change occurs in the diameter of the arteriole? (b). What effect would you expect this to have on arterial pressure? {c). Would its action on the arteriole help to account for its effect on arterial pressure? Expt. XLI. Morphine is eight times stronger than opium. Ascertain (when the frog is quiet against the side of a glass dish,) the rate of re- spiration, and, if possible, also of the heart action, and the size of the pupil. Opium acts on the central nervous S3-stem. Inject 4 drops of a 0.5 per cent solu- tion under the skin of a frog. Note the effect on the movements — clumsj' and awk- ward. Place in an awkward position; note the results. Compare the reflexes with those of a normal frog. (Reflexes are decreased for perhaps an hour and then much increased). Study the effect on respiration. Note the heart action (slow and weak when a large dose is given) and change in the pupil. Expt. XLII. Pith the frog, note the size of a special bloodvessel in the mesen- tery placed under the microscope and micrometer scale. Place a drop of 1 per cent Suprarenal Extract over the vessel. Note the effect. 44 Outlines of Experimental Physiology (b). Get a heart curve. Adda few drops of a 1 per cent Suprarenal Extract. Ag-ain obtain a curve. Note the effect on the heart's action. (c). Thoroug-hly wash off the Suprarenal Extract from the heart. Obtain a cardiac curve. Put two drops of 33 per cent alcohol on the heart. Note the first and later effects. (Alcohol evaporates). If the heart ceases to beat stimulate with the electrical current. The effects vary with the dose. Expt. XLIII. Effect of Physostig-ma. Pith a frog or a curarized frog may be used. Physostigma acts on the central nervous system, heart respiration, muscles and e3'e. Obtain a muscarin curve both before and after phj'sostigma. Expose the heart; apply a drop of phj^sosligma; obtain a curve. The drug slows the heart but increases the irritability of heart muscle, so that vagus stimulation has little effect. Muscarine slowing is abolished because the muscle is more irritable, not because the nerve endings are affected. Expt. XLIV. Action of Aconite. Obtain a heart curve from a normal frog. Inject two drops of tincture of aconite. Obtain curves at intervals of from 5 to 30 minutes. (i>). Take a sphygmographic tracing from a student. Note the pulse. Ad- minister by mouth 0.2 c.c. tincture of aconite and 0.06 c.c. every 10 minutes until the action on the pulse is noticeable. Count the pulse at short intervals. How is the heart rate affected ? What subjective sensations are produced ? Expt. XLV. Transfusion of Salt Solutions through the Turtle's or Frog's Heart. (Walden, Am. Jour. Phys Vol. III. Howell, Amer. Jour. Phys.) Ligate or break the turtle's neck or pith the frog, fasten it back down in the holder, expose the heart and proximal vessels, ligate all vessels except the post cava and left aorta. Into the aorta, tie a canula with a rubber tube attached, pushed partly into the ventricle for an outflow tube. Connect a canula with the post cava or tie it into the right auricle for an inflow tube; join to this aT tube connected with the bottle or burette of solutions. Fasten the tip of the ventricle by a silk thread to a recording lever or use the lever with the heart holder for curves. Remove the clot from the heart. (a). Irrigate with 0.7 per cent NaCl everj^ ten min.utes until the heart slows. (b). Irrigate with 100 c.c. 0.7 per cent NaCl plus a few drops of 1 per cent CaClg everj^ five minutes. Continue to add until 6 c.c. CaCl2 have been added. Obtain curves to show the effect of the.CaCls- (c}. After half an hour or so, if the heart beats feebly add 100 c.c. NaCl 0.7 per cent; 2.3 c.c. CaClg, 1 per cent; 1.5 c.c. KCl, 1 per cent. Effect ? (d). After about half an hour, if the heart beats feebly, irrigate with milk diluted with nine volumes of 0.7 per cent NaCl. (e). If the heart beats feebly after an hour or less, revive with 100 c.c. NaCl, 0.7 per cent; 2.3 c.c. CaCls, 1 per cent; 1.5 c.c. KCl, 1 per cent; 0.6 c.c. Na2C03, 1 per cent. What is the effect? What are your conclusions regarding proteid and inorganic salts on the heart action? {Milk contains certain salts). Expt XLVI. Action of Inorganic Salts upon the Heart. Sever a ring about three mm. wide, parallel with the auricular-ventricul 'fur- row, from the ventricle of the turtle or frog. Put on two ligatures with loops close Outlines of Experimental Physiology 45 tog-ether and divide the ring- between them. Fasten one loop on the strip to the bent end of a small canula in a g^lass tube made especially for the purpose; and the other end, with its loop to an inverted counterpoise lever arrang'ed to record on a slowly moving- drum. Immerse in a 0.7 per cent NaCl solution in the g-1 ass tube provided with a rubber outflow tube. Have a dish ready for the solution. Protect the tube with a cap of oiled paper or rubber tissue. After a latent period, the contractions soon reach a maximum and then die away. Sodium cannot maintain continued activitj'. It diminishes the tonus. Gradually add, drop by drop, a solution of 1 per cent CaCls". This is isotonic with 0.7 per cent NaCl. Is a chang-e noted? Con- tractions may cease in NaCl but Ca added will leng-then the period during which the muscle will contract. Calcium increases the tonus. (a). Put the muscle in NaCl 0.7 per cent again, if it still contracts. Add gradually KCl 0.9 per cent until a change is evident. (b). Potassium chloride 0.9 per cent is approximately isotonic with 0.7 per cent NaCl. Potassium causes all contractions to cease. (c). Modified Ringer Solution. 100 c. c. NaCl, 0.7 per cent; 3.5 c. c. CaClg, 1 per cent; 2.8 c. c KCl, 0.9 per cent). Long continued contractions of the tortoise strips will be secured. Rhythmical contractions may be due to chemico-phj'sical stimulus of inorganic salts in the blood being present in definite proportions. Most observers are agreed that the inert action of these salts is essential. The sinus may be the more sensitive to the chemical stimulus. (ci). Influence of Temperature on Frequency of Contractions. Get a normal curve With a pipette, put on NaCl at 30 °C. Fill the spoon of the holder. Replace with NaCl at 5 ^C. Note the difi'erence in the curves. This can be tried either on the heart directly or on the strips of the ventricle. (Greene, Am. Jour. Phj's.). Expt XLVII. Have ready the foUovviug. M/8 NaCl; M4 Cane sugar; 90 c. c. M/8 NaCl plus 10 c. c. M/8 CaClo. (a). Put the ventricular strip in M/8 NaCl. Try sodium citrate, also. {b). When the strip is beating in (a), put it into M/8 cane sugar. (c). Return the strip to M/ NaCl. Do the beats return or change? (d). When NaCl acts toxic, put the strip into the NaCl plus CaClo solution. Effect? Expt. XLVIII. Lingle's Experiment (a). Non-conductors. M 4^ Cane sugar is equal to M/8 NaCl (No beats). Prove that the strip is not killed by putting it into an electrolyte. (b). In NaCl M/8, after one hour, it beat for 1-3 hours. (c). Number of Ions. 40c.c. M /4 cane sugar plus 10 c c. M/s NaCl gives beats. {d). NaBf yi/i gives stronger beats than 'M.^ NaCl. (effect of too many ions). After a NaCl standstill for H hour, put it into M/^ cane sugar. It beats again. (f). CaClgM/, no beats. Remove the excess of Ca bj-^ putting it in M/^ NaCl. The beats are resumed. iS"). 48 c. c. M/s NaCl plus 2 c. c. M4 CaClj. It beats but '^7 c. c. M/8 LiCl plus 3 c. c M -8 CaCl2, does not beat. 48 c. c. M/s NaCl plus 0.003 KCl, loss of tone. {k). KCl M/8, no beat. , Lingle, Am. Jour. Phys. ( 46 Outlines of Experimental Physiology Expt. XLIX. Action of Strychnine. Pith a frog". (The brain inhibits spasms). Ligate the thig-h, except the sciatic nerve, at its junction with the body. Be sure to protect from drying-. Turn the frog- over and make a long-itudinal incision to one side of the median line, about one inch long, on the unligatured side. Press- ing- aside the viscera, pick up the sacral plexus of nerves going to the uninjured leg. The sacral plexus may be readily recognized lying on either side of the median line. Pass a thread around the nerves loosely, so as to quickly find them when wanted. Inject into the dorsal lymph sac one or two drops of 0.8 per cent strychnine. (a). What part of the frog is reached by the poison? What part is protected from it? Illustrate by a diagram. (6). If strychnine acted upon the central nervous system, would the legs be equally convulsed? If it acted on the cord? If it acted on the motor nerves? On the muscles directly? Are both legs convulsed? Are the sensory nerves affected? (c). To what parts of the reflex arc have you limited the action of the strychnine? B. Using as a guide the thread formerly passed around the sacral plexus pick it up, tie, and sever it between the two ligatures on the uninjured side. (a). Does the strychnine reach the motor nerves and muscles of the uninjured leg? (b). If strychnine were a convulsant through its action on either the motor nerves or the muscles, or both, would the injured leg still participate in the convul- sions? (c) Demonstrate that the muscles, sciatic, and sacral plexus below the point at which it was severed, are still intact, by stimulating the distal portion of the sciatic. (d). To what elements of the reflex arc have 3'^ou limited the possible action of the strychnine? C. Expose the heart of a small frog and ligate the aortae at the base. With an aneurism needle pass a fine thread around the aortae, taking care not to injure the auricles, and ligate. With a scalpel, cut through the occipito alantoid mem- brane and bend the head forward. Remove the posterior wall of the upper end of the spinal canal by inserting the smaller blade of strong scissors into the spinal canal and cutting; taking care not to injure the spinal cord. Allow a drop of the solution of strychnine to fall directly upon the cord, or with a fine hypodermic need- le, inject two drops (not more) into the arachnoid space. (a). What effect has ligation of the aortae upon the circulation? (b). Would stoppage of the circulation prevent the drug from reaching the peri- pheral terminations or trunks of the sensory nerves? Motor nerves? Muscles? (c). Where, then, must the strychnine act to produce the observed symptoms? (d). Would cessation of the circulation delay the action of the strychnine on the cord by slowing the rate of absorption by the latter? D. After observing the results in Expt. C destroy, first, the upper, then the lower portions of the cord, by passing a wire down the spinal canal. (c). Do the results agree with those of the previous experiments? Note. Destruction of the upper part of the cord may take place during the pre- paration of the animal. If so, the upper limbs will not take part in the convulsions. Expt. L. Independent Irritability of Muscle and the Influence of Curare. Prepare the frog as for the strychnine experiment, with the important difference that the sciatic plexus is not exposed in this experiment. Inject a few drops of a 1 Outlines of Experimental Physiology 47 per cent curare, into a Ij-mph sac. Test the reflex, soon the leg from which the poison is excluded still respond to the stimulus of its own foot as well as to strong stimulus of the other What paths therefore, are still functional? Wh^n all reflexes except in the ligatured limb have ceased, (a), stimulate the sciatic be- low the ligatured area. There will be no contraction. (If. Stimulate above the ligatured area and also the sciatic of the unligatured leg supplied with poison. ic). Stimulate the muscle directly of both legs. What structure has curare paral3'zed? Can muscle contract without the agencj' of nerves? Have you proved that curare does not affect the cord, the nerve trunks, afferent nerve fibres, or heart action? Why should curare not be emplo3'ed as £in anaesthic? Expt. LI. Production and Inhibition of Muscular Twitchings. Normal frog. Notice posture, coordination, respiration, (1, rate, 2. mode) and reflexes. Inject into the Ij-mph sac of an unpithed frog 2,'^ c c. of M/8 sodium citrate. Notice carefulh- all changes which take place. Do any parts of the body show tremors or tetanus? If so, where and to what extent? When the tremors become well established, inject 1}4 c. c. M/^CaCls- Note ef" feet. Compare in every point your results with those of other. Note the heart ac- tion, respiration, and reflexes after '2 hour. Make j-our observations carefully'. (a) How does the destruction of the upper part of the cord effect the convul- sions? (b). What is the effect on the entire cord? Does reflex still j>ersist? Does stimulation of the muscle or sciatic, directly, give a response? Expt. LII. Influence of Veratrine. Prepare a frog as for curare experiment. Inject 5 drops of 0.1 per cent. Vera- trine into a dorsal Ij-mph sac. Notice the behavior of the muscles during and after a jump, heart action, respiration and reflexes. Stimulate the ligatured, then the other leg and muscle directly. Sever the sacral plexus. Has the duration of the contraction of its muscle been altered? To what element in the reflex arc have j'ou limited its action? (b). Remove the lower jaw to the hyoid. Drop 0.1 per cent Veratrine on either the hv'oglossus, gastrocnemius or sartorius fastened to a clamp and muscle lever. Record the direct stimulation of a few contractions on a slowly moving drum, and compare the curves with normal ones obtained from the same kind of muscle. Expt. LIII. Arterial blood pressure, A preliminary operation on the Carotid, Superior laryngeal. Phrenic, Depress- or, Sympathetic. -Sciatic, and Femoral. These dissections are performed on dead animals as a preparation for the work. Be sure to take notes on the effect of the anaesthetic. Anaesthetize a cat or rabbit with either. (For a dog, use a hypodermic injec- tion of 2 per cent Morph. Hydrochlor; for large dog, about 8c. c. or 1 c c. per kilo of body weight, or use chloratone, C4 H^ O CI3, dose 0.2 gram per kilo. The dog will anaesthetize for hours. Given in warm aqueous solutions per mouth. Pulse slightly lowered. Little effect on blood pressure. Put the animal in a holder, moisten and clip hair from the part to be operated upon. Make a longitudinal 48 Outlines of Experimental Physiology median incision one and one-half inches long-, just posterior to the larnyx. The skin, muscles and fascia are. thus cut and the sterno-hyoid and sterno-mastoid are exposed The thin narrow sterno-thyroid lies below the sterno-hyoid, inserted to the th3'roid cartilages. The thyroid g-lands are latero-posterior to the larynx. Loosen the skin from the muscle with the handle of the scalpel and hook apart with weights. Note the large external jugular just under the skin at the external edge of the sterno-mastoid extending across it. Separate the two muscles and, in the dog, you will find the carotid; vagus, and internal jugular, surrounded by the cervical facia or common sheath In the cat and rabbit the vagus and sympathetic run a separate course. The vagus is external to the carotid and internal to the jugular. Isolate the carotid and vagus on both sides, the crural nerve, exteral jugular or femoral vein, and the superior laryngeal of one side. (a). Inserting the Canula. Select a proper" sized T canula fitted with rubber- tubing, the latter clamped; the right-angled end is connected to a manometer. Iso- late an inch of the carotid and pass two ligatures around it. Tie the ligature that is farthest from the heart and clamp the carotid about an inch below it with bull- dog forceps. Now make a V shaped incision in the part of the carotid that is isolated. Use sharp scissors and cut through only Yj, of the circumference of the vessel. By means of a seeker, insert the canula, trying it with the second ligature. When ready for the blood, remove the clamps from the carotid and tubing. Don't forget to have the beaker ready and graduated for hemorrhage. (b). The superior laryngeal branch of the vagus arises far forward beyond the anterior end of the larynx. At its origin from the vagus is an enlargement of the nerve known as the ganglion of the trunk. Trace the superior laryngeal, to the larynx which it enters. It anastomoses with the inferior laryngeal by a branch passing beneath the wing of the thyroid cartilage. Somewhat posterior to the origin of the superior laryngeal, the sympathetic trunk separate from the vagus until it ends in the superior cervical ganglion. [c). The phrenic branches from the 4th, 5th, and 6th cervical nerves, unite near the first rib to form this trunk. It passes mesial to the sternal artery and dorsal to the brachial artery into the thorax. The right rests on the lateral aspect of the pre and post cava on its way to the diaphragm. {d). The depressor in the rabbit is formed by the union of a branch from the vagus and superior laryngeal, extending mesial to the sternal artery and dorso- mesial to the carotid, (See Stirling, p. 302). The depressor is the smallest of the three nerves. [e). The femoral artery lies between the femoral vein and the anterior crural nerve. The vessels are just under the skin, ventral side of the proximal part of the thigh, parallel with the femur. (Stewart p. 177). .(/■). The sciatic nerve lies on the dorsal side of the prbximal part of the thigh. Make an incision on the external surface, median line, and separate the skin with hooks. Part the muscles with the handle of the scalpel. The sciatic lies deep between the muscles. The vastus externus may be cut if thought best, B. Blood Pressure Tracing. In connection with this experiment, if two animals are used, one may be used for determination of the circulation time or for transfusion with warm saline solu- tion. Put a dog under Morphia (10c, c. 2 per cent sol.). Setup an induction ma- chine arranged for an interrupted current. Fill both arms of the manometer with I Outlines of Experimental Physiology 49 mercury to the height of about 10 cm Attach a rubber tube to the proximal arm of the manometer and fill with a 25 per cent solution of Mag-nesium sulphate or, better, a saturated solution of Na2C03. Be sure that all the air is out of the tube and this end of the manometer. Blow into the rubber tube so as to cause a difference in the two limbs of about 10 cm of mercurj'. Without releasing the pressure, clamp the tube. Attach the tube and clamp to the free curved end of the Guthrie manometer, a T to the other end, connected with a bottle of salt solution. Arrange the writing point of the manometer float so that it will write on a smoked drum. Keep the needle in contact with the drum without undue friction Below the pressure tracing, tace a time tracing, having the marker beating seconds. Fasten the animal on-the holder, back down. Give either. Insert the tracheal canula (for the purpose of artificial respiration). Avoid cutting the blood vessels. Insert a glass canula, three wa3', one armed with a rubber tube, into the central end of the carotid. Leaving the forceps on the artery, fill the canula and tube with Mg-SO^ or Xao CO 3 saturated solution. Now connect with the manometer, being very careful that all connections are well filled with the solution of MgS04. Before taking off the bulldog forceps be sure to get the line of zero pressure. Take off the forceps and allow the drum to revolve at slow speed. The writing point of the manometer will trace a curve ,'get record of height and pressure), showing an elevation for each heart-beat and longer waves due to the movements of respiration. Which part is due to inspiration? Is the pressure higher in inspiration or expiration? Order of work. 1. Notes on the anaesthetic. 2. Zero pressure in Hg. and abscissa line on the drum. 3. Blood pressure curve. (a) Expose the crural or sciatic in one leg. Double ligature and divide. Stimulate the central end; the blood pressure piobablj' rises and the heart may be accelerated. Stimulate the peripheral end of the nerve. There is little change in the blood pressure and none in the heart. (b) Now expose and ligate the vago-sympathetic nerve in the neck. Ligature double and cut between the ligatures. Stimulate first the peripheral, then the central end and note the effect on the blood-pressure curve. Signal during stimu- lation. (c) Expose and divide the other vago-sj-mpathetic while a tracing is being taken. Again stimulate the central end, Effect? (d) Again stimulate the peripheral end of the vagus of both at the same time, while a tracing is being taken and see how long it is possible to keep the heart from beating. (May cause death of dog). (c) Effect of Intravenous Injection of Nicotine of 'i c. c. of 1 per cent solution Inject carefully to prevent the entrance of air into the femoral vein, clamped with bull-dog forceps in a dog. What is the effect on the heart, blood pressure and respiration' D. Effect of transfusion on the blood pressure. (a) While a tracing is being taken, inject slowlj- about 100 c. c. normal saline solution heated to 40° C) , through a canula in the femoral vein, by means of a funnel supported by a stand at such a height that the solution runs in easily. A stop cock inserted between the funnel and canula should be closed bef«re the funnel 50 Outlines of Experimental Physiology is empty, so as to ob%iate risk of getting air into the vein. Continue to inject por- tions of 100 c. c. until a distinct change in pressure has occurred. Keep a record of the amount injected and when the first change in pressure was seen, and mark the time of each injection on the curve. After 3o minutes, again measure the height of mercurj^ in the manometer. (Kef. Brodie p. 177-18U.) E. Effect of Haemorrhage on blood pressure. Note the scale of the manometer. While a tracing is being taken, draw off 10 c. c. of blood from the femoral artery and notice whether any effect is produced on the tracing. Mark the tracing the moment the removal of blood begins and ends. Now run off 10 c. c. of blood and note the eft'ect on the blood pressure. If none occurs run off 10 c. c. at a time until the pressure falls, noting the amount of blood removed and the change in pressure. F. Circulation Time. Methylene blue. A 0.2 per cent methylene blue in 0.6 per cent XaCl solution is warmed to 40 ° C. and placed in a burette, sloped to make an angle with the hori- zontal, 5 in. above the level of the canula. Expose the external jugular and place two ligatures under it — compress the cardiac end with bull-dog forceps and tie the head end. Insert a three way canula and tie it in place with the second ligature. Fill the canula and rubber tube with a saline solution by means of a long pointed pipette. Be sure to exclude the air. Expose the carotid of the opposite side. Place under it a piece of sheet rubber and between the rubber and the artery a piece of white glazed paper. Connect the canula (free from air) with the burette. Concentrate the light on the carotid; gee the time record .started; and the moment the methj-iene blue flows into the jugular, close the key to the signal (Write above the time). Allow 10 c.c. or more to flow in, and again signal when the blue ap- pears in the carotid. Take as many observations as possible and calculate the mean circulation time of both the greater and lesser circulations. G. To Expose the Heart. (a). After the introduction of the respiratory canula, make a median incision over the sternum from the anterior to the posterior tip. Strip the skin back to the junction of the costal cartilages and ribs. Place strips of absorbent cotton wet with a half saturated solution of tannic acid along the cut surfaces. With strong scissors, cut quickly through the costal cartilages of both sides, parallel with the sternum, clamping the mammary arteries. Study the relation of the heart to the pericardium and other structures in the thorax. Note changes in the form, color, and tension as felt by the hand during the cardiac cycle. As long as artificial respiration is continued, it will hardly be possible to kill the dog. (b). If asphyxia curves are desired, close the trachea, tie the animal firmly in place and obtain the characteristic Traube-Herring curves. (c). If asphyxia curves are not desired, open the right auricle. The animal will then quickly bleed to death without pain or convulsions. {d). Autopsy — Note the lungs, kidney and bladder. Outlines of Experimental Physiology 51 H. Electrical Methud. A canula connected with a burette containing a 1.5 per cent NaCl solution is tied into a jugular vein. For the lesser circulation time, a carotid is then isolated and laid on bent insulated platinum electrodes. To further secure insulation, a piece of india-rubber is slipped between the artery and the tissues. The artery, by means of electrodes, is connected as one of the resistances in a wheatstone bridge (induction coil for interrupted current — one cell in the primary and a telephone instead of a galvanometer according to Kohlrausch's method of measuring resistances of electrolj-tes). The bridge is balanced by adjusting the resistance until the sound heard in the telephone is at its minimum intensity-. One c.c. of saline solution is run into the jugular. Reaching the artery it causes a diminution of electrical resis- tance. This disturbs the balance of the bridge and the sound in the telephone becomes louder. The time from the beginning of the injection to the alteration in the sound is the circulation time between the jugular and carotid, read off b}" the time-marker on the drum with a signal, gives the circulation time. Expt. LIV. A. Effect of Suprarenal on Respiration, Heart Action and Blood Pressure. Have all apparatus, drugs, graduated tubes and vessels, electrodes, etc., read 3'. Do not cut the drum paper for the pressure curve. Put the time record on each drum. Set off at a slow rate and have electro-magnetic signal in each circuit to record the stimulations and time of giving drugs. Use shielded electrodes and a weak current. For 1 per cent adrenal extract, take 2 gr. capsule to 12 c.c. of 0.7 per cent XaCl, 1 gt. acetic acid. Boil, filter, and make up to 12 c.c. Make up also, a 2 per cent adrenalin solution. Fasten the dog with a chain and inject 6-10 c.c. of 2 per cent morphine. Have sawdust ready. Again note the changes produced by morphine. Fasten the dog to the board, giving a little ether, if necessary Xote changes in the eye, re r,iiration and heart. OPERATION. For blood and respiratory pressure. (a) Clip the hair from the neck and jaw of one side. Make a 2-3 inch median incision, beginning below the larynx. Expose both carotids. Tie the head end, put the bull-dog forceps on the heart end, and ligature in the three way canula for connection with the manometer, through tubing filled with a saturated solution of Nag CO 3. Expose both vagi and place silk finders under them. Next connect the trachea through tubes on one side with the respiratory bottle and the other with the ether bottle, if necessary, or not until after normal curves are secured. Put a finder under the superior laryngeal. Expose the femoral vein, con- nect the canula and keep the bull-dog forceps on the vein or rubber tubing above the canula to exclude the air except just when injecting and then be sure to expel the air from the canula with a long pipette or syringe. Clamp off the vein before all the fluid from the pipette enters. Place a silk finder under the crural nerve. ORDER OF WORK. Get normal curves of respiration, blood pressure and heart action. (Connect the respiratorj' bottle with the tambour and T from the trachea. Be sure all is air- 52 Outlines of Experimental Physiology tight and connections close tog-ether). An abscissa line is obtained for both on a slowly moving drum. Have the time record and electric signal ready for use. Re- move the bull-dog and get the blood pressure and a short curve on the one drum and at the same time obtain a normal respiratory curve on another slovirly moving drum, using electrical signal and time marker. Stop the drums. The dog in the meantime, is breathing fresh air. C. Stimulate. Cut the crural nerve while the dog is breathing from the bottle. Note the signal and get curves. Stimulate the peripheral end of the crural nerve. Effect? D. Inject about 4 c. c. adrenalin, 1 per cent (be careful not to inject air) and clamp off. Start all the drums a few seconds before injecting into the femoral vein and at the moment of injecting, mark on all the drums with electric signal, the time of injection. Obtain curves. Obtain curves showing the influence of drugs on blood pressure, respiration and heart; then stimulate the crural nerve again and note the effect. E. Effect of stimulating the vagus. Ligate twice, cut between the ligatures stimulate the central end then the peripheral end of the other vagus, then stimulate again the superior laryngeal. Note the time and get the curves on all the drums. H. Inject 3 c. c. 2 per cent adrenalin and get curves. I. Get asphyxia tracing showing Traube-Herring curves. Be sure to tie the animal firmly; or, if the animal is in good condition and time permits, obtain first the circulation time and the effect of transfusion or hemorrhage as described in the experiment on blood pressure; then obtain asphyxia tracings by clamping the trachea. B. Effect of Suprarenal Extract on Secretion, Respiration, Blood pressure and Heart Action. Have all apparatus, drugs, graduated tubes and vessels, elec- trodes, etc. , ready. Put the time record on each drum, set off at a slow rate and have an electro-magnetic signal in each circuit to record the stimulation and the time of injecting drugs. Use shielded electrodes and a weak current. For 1 per cent adrenalin extract, 2 gr. capsule to 12 c. c. of 0.7 per cent NaCl plus Igt. Acetic acid, boil filter and make up to 12 c. c. Prepare also 0.2 per cent adrenalin solution. Note the rate of respiration, the temperature, and the pupil. (2). Fasten the dog with a chain aud inject 6-10 c. c. of 2 per cent Morphine- Have sawdust ready. Again note the change produced by morphine. Fasten the dog to the board, giving a little ether, if necessary. Note the changes in the eye, respiration and heart. OPERATION. As for blood and respiratory pressure, (a). Clip the hair from the neck and jaw of one side. Make a 2-6 inch median incision, beginning below the larnyx. Expose the carotid on the side opposite to the one selected for the operation on glands or for the transfusion experiment: also both carotids. Tie the head down, put bull-dog forceps on the heart end and ligature in the canula for connection with the manometer through tubing filled with a saturated solution of NagCos. After having exposed the vagus on both sides, placing black silk find- ers under them and ligatures under the superior laryngeal, connect the trachea Outlines of Experimental Physiology 53 through the tube, on one side with the respiratory bottle and on the other side with the ether bottle and bellows, if necessary, but not until the normal curves are secured. {fi). Expose the femoral vein, insert the canula and keep the bull-dog- forceps on the vein or rubber tubing above the canula, to exclude the air, except just when injecting from the burette or S3'ringe; and then be sure to expel the air from the canula with a long pipette. Clamp off the vein before all the fluid from the pipette enters it. (3). Effect on Secretion. Note. —Study carefully the submaxillary region on the dead animal {a). The anterior limit of the median cut made to expose the trachea, ma}' be carried through the skin to the angle of the jaw, avoiding the external jugular and noting the following directions, or the following plan may be used. (6). Make an incision parallel and along the inferior median border of the lower jaw about 1cm. distant from the border, only through the skin. The cut will be carried from about the middle to near the angle of the lower jaw. Carefully divide the platysma myoides and expose the external jugular with its branches. Ligate doubly such branches as are in the way and cut them, except those that carrj- blood from the glands. It may be only the branches extending anteriorly above the jaw border that need be ligated. Feel for the facial artery at the border of the jaw and masseter, between the digastric and masseter. Ligate it Loosen the digastric from the jaw border to its insertion. There ligate twice and cut between the liga- tures. Lift the digastric and notice just beneath, the broad .mj'lohj'oid with its nerve lying on it. Carefully lift the mj'lohj-oid and the lingual nerve and the ducts will then come to view. The mylohyoid may be cut parallel to and near the jaw- border, avoiding the facial artery and ducts. Ligate the branch ' of the facial that supplies the digastric. The lingual nerve is then seen emerging from under the border of the jaw, about }< the distance from the angle of the jaw to the angle of the mouth, a little nearer the masseter or jaw angle, and extends anteriorly and trans- versely toward the median line, across both ducts, then forward to the tongue par- allel to the larger hypoglossal. Ligate and cut the lingual nerve far from the border of the jaw, lift the lingual nerve b}- the ligature and trace it back to the jaw- where its chorda t3-mpani branch is seen running back along the duct. Ligate the lingual central to the chorda, so that the chorda can be placed on electrodes. Insert an angular canula into the larger duct (the one nearer the ramus of the jaw), after placing a small bull-dog forcep on the duct. To reach the sympathetic, divide the hypoglossal nerve just where it crosses the carotid and raise the central end. Close to the inside of the carotid lies the vagus and when it is raised, the sympathetic is seen underneath. The sympathetic from this point goes separately to the superior cervical ganglion — from this ganglion, fibres accompany the carotid to the gland. The sjmpathetic fibres may be ligatured and later cut for stimulation with a weak induction current, or the sj-mpathetic branch may be traced bj' means of the cervical vagus to the cervical ganglion. ORDER OF WORK. A. Note. Get normal curves of respiration (one end of T from the trachea goes to the respiratory bottle, the other to ether), blood pressure and heart-action. 54 Outlines of Experimental Physiology (1). Abscissa line for zero pressure is obtained on both slow moving- drums. Have the time record and electric sig-nal ready for use. Remove bull-dog- and get the blood pressure and a short curve. (2). at the same time obtain a normal respiratory curve on the other slow- moving drums, using the electric signal and time marker. Stop the drums, the dog in the meantime breathing fresh air. (3). Have a 5. c. c. graduate placed at the mouth of the canula inserted in Wharton's duct, shielded electrodes on the chorda and on the sympathetic. Stim- ulate for an exact leugth of time about (3 sec.) with definite strength, weak induc- tion current. Note every IS sec. the height of the secretion. Then stimulate the sympathetic exactly as the chorda was in time and strength. Note the number of drops secreted every IS sec, (4). Note the pupils and temperature. C. Action of Atropin. Inject into the femoral 2. c. c of 0.1 per cent atropin. Just before injection, ob- tain curves of the blood pressure, respiration, and heart and note with the signal, the time of injection. Obtain curves, showing the influence of the drug and stimu- late the chorda and then the sympathetic as before — same time and strength of current — and note for each IS sec, the amount of secretion in drops. D. Effect of Section of Vagi. Ligate twice and cut between the ligatures on one vagus. Connect the head end of one with the shielded electrode. Just before ligating and cutting, obtain a short distance curve for pressure, respiration and heart. Stop the drum until ready to cut, then cut and note the time of cutting with the signal. E. Effect of Stimulation of one Vagus. Stimulate the head end of the vagus for a few seconds noting the time with signal and obtain curves of pressure, respiration, and heart. Repeat, stimulating the heart end. Ligate and cut the other vagus. Simulate first, the head and then the heart end of the vagus, F. Effect of 4 c. c. of 2 per cent Adrenal Extract. Just before injecting 4 c c adrenal extract of 2 per cent. Obtain a curve. Note the time of injection and the effect on pressure, respiration, heart and secretion, stimulation as before. G. Effect of 3 c c. Adrenal Extract. Just before injecting 3 c. c. of 2 per cent adrenal extract, get short length of curves and note the time of injection and the effect on blood pressure, respiration heart and secretion. H. Get asphyxia tracing showing Traube-Herring curves. Expt. LV. The Nervous Regulation of the Respiratory Movements. (See Stewart, p. 188.) Set up the apparatus after the following directions and see tliat it is air-tight. A recording tambour is connected to a large bottle through a double perforated cork. One hole is used for regulating the pressure by means of a clamp. From a hole in the lower edge of the bottle, make connections with a T tube to an ether bottle and Outlines of Experimental Physiology 55 from there to a canula and thence to the trachea. Have also an inductorium and electrodes arranged for an interrupted current, and a signal. Anaesthetize a cat. Insert a canula into the trachea and connect with the bottle. Set off the drum at a slow speed and take a tracing-. (2). Clamp off the ether bottle and remove the clamp from the large bottle for air. Dissect out the vagi in the lower part of the neck, pass the ligature under them but do not tie. Close the tube in the large bottle and while a tracing is being taken, tie the crural. Then stimulate its central end with weak shocks, marking the time of stimulation on the drum. Repeat the stimulation with strong shocks' and take a tracing. Stimulate the peripheral end; note the effect. (3). Cut the vagus; stimulate the central, then the peripheral end. (4). Apply a strong solution of potassium chloride with a camel's hair brush to the central end of the vagus, while a tracing is being taken, and observe the effect — slowing- or expiratory stand still. (5). Open the regulating tube. Isolate the superior laryngeal branch of the vagus, which will be found coursing inwards to the larynx at the level of the thy- roid cartilage. Ligature the nerve and divide it between the larj-nx and the liga- ture. Close the tube and take a tracing; then cut it. Stimulate first with weak then with strong currents, the central end of the superior laryngeal. Effect ? (6). Open the reg-ulating tube while a tracing is being taken; cut and stimulate the central and heart end of the other vagus. Effect ? (7). Isolate both carotid arteries for as great a distance as possible. Take two pieces of lead tubing about nine inches long and bend up about two inches at each end, nearly to a right angle. Place one end of each of the tubes in contact length- wise with each carotid, securing contact with loose ligatures. Support the tube in clamps, so that the arteries are not pressed upon. Connect two adjacent ends of the tubes b}' a short rubber tube. Connect one of the remaining ends to a funnel, supported on a stand, and the other to a rubber tube hanging over the edge of the table above a jar, Slip two or three folds of paper between the tube and the vagus nerve. Heat two or three litres cff water to 60 oC. Now take a tracing while the hot water is passing through the tube to the jar. Mark on the tracing the point at which the circulation of the hot water was begun, and go on passing it until an ef- fect was produced. Then stop the drum and circulate water at ordinary- tempera- ture till the breeithing is again normal. Then, while a tracing is being taken, pass ice cold water through the tubes — note the effect. (8t. Effect of hemorrhage. Insert a canula into the carotid artery. While a tracing is being taken, allow the blood to flow. Dyspnoea and exaggeration of the respiratory movements will be seen when a considerable quantity of blood has been lost. Mark and varnish the tracings. Tie the animal T tubes firmly or they will be pulled out. Note the first effect and the amount of blood lost when the effect set in. In the whole of this experiment the clamp on the regulating tube is to be open except when the lever is actually writing on the drum, in order that the period dur- ing which the animal must breath into the confined space of the bottle may be di- minished as much as possible. The clamp on the tube from the ether bottle is to be closed except when more ether is needed. Expt. LVI. Determination of the Lung Capacity, by Means of the Spirometer. Always clean the mouth piece with corrosive sublimate before using. 56 Outlines of Experimental Physiology (1.) Tidal Air. Partially fill the reservoir of the spirometer with air and then breathe normallj' several times throug-h the mouth-piece and have the helper record the upper and lower limits of each respiration. The average of these esti- mates should give the normal tidal air. Do not watch the scale. (2). Complimental Air — Have the spirometer nearl}' full of air. Take a few normal breaths through the mouth piece, having the assistant make a record of the reading of the instrument at the close of each expiration. Then take as deep an inspiration as possible and note the reading of the instrument. (1). Supplemental Air. Have a small amount of air in the reservoir of the spirometer. Breathe regularly a few times, making records of the upper and lower limits. Then exhale as strongly as possible and then make note of the scale reading. (4). Vital Capacity. Have the spirometer set at zero. Inhale as deeply as possible and then exhale through the mouth piece all the air you can. This will be the greatest possible amount of air which can be forced out of the lungs when filled to their greatest extent. (5). Determine the average rate of respiration at different times during the day, during exercise, fatigue, and rest. Expt. LVII. Respirator^' Pressure. Fasten the tube of a pneumometer with a little cotton wool in one nostril; breathe through the other with closed mouth and observe the amount by which the mercury is altered in ordinary inspiration and expiration. (b). Repeat the observation with forced breathing once a second, also evei-y two seconds, pinching the tube at the height of inspiration and expiration. Read off the maximum inspiratory and exspiratory pressure. (c). Repeat (o) with the tube covered with rubber; held between the teeth and with the nostrils open. (d). Repeat {b) with the tube in the mouth and the nostrils closed. Expt. LVIII. Chest measurements with calipers. (a). Dorso-ventral at the heighth of the sixth rib (junction with sternum). (b). Doro-ventral at the height of the ninth rib (c). Lateral at the height of the sixth rib. {d). Lateral at the height of the ninth rib. Obtain both phases of quiet and forced breathing. Compare carefully these measurements with those obtained in the following related experiments LIX and LX and fill out the anthropometric record. Expt. LIX. The Stethograph records the movements of the chest wall, the curves indicating the relative time between inspiration and expiration and with the curves obtained with a thoracometer, show the difference in diameter or width at difi'erent levels of the thorax in inspiration and expiration, and these should agree with the caliper and tape measurements. In making observations with the stetho- graph, the subject should sit with his back or side to the table. The observer may readily adjust the stethograph to record any lateral dorso-ventral diameter of the thorax. For all observations upon the respiratory changes in the body, the subject should keep the parts of the body symmetrically disposed. Outlines of Experimental Physiology 57 Observations. (1.) How much may be learned of man's respiratory movements bj' simple inspection? Make a careful enumeration and record. (2.) Adjust the stethograph and make a record of the lateral diameter of the thorax at the ninth rib. Does the stethograph show more than can be learned from inspection? If so, what? Note the diflerence between inspiratory and expiratory curve and time. (3.) Take a stethogram of the lateral diameter at the ninth rib. How does it differ from 2 ? Account for the difference. (4.) Take a stethograph of the dorso-ventral and lateral diameter of the thorax over the lower end of the gladiolus. Compare with the records of forced inspiration and expiration at this region. (5.) Take a lateral ninth rib stethograph- while the subject reads a paragraph; sighs; coughs; and laughs. Account for the peculiarities. (6 ) Take a lateral ninth rib stethograph after the subject has taken vigorous exercise. What changes are to be noticed? (7.) After a series of stethographs have been taken for others, compare. Determine the essential features; give cause of these. (8.) Seek the causes of the differences which exist between stethographs of different individuals. May they be accounted for bj' stature, condition, occupation or habit Expt. LX. The Thoracometer curves bear an accurate ratio to the movements of the chest. Remove from the stethograph the wooden rod which bears the receiving tambour and slip thereon the lever, computing its magnification for use in determining the variations in thoracic diameters. So adjust the lever that the slightest movement of the button will be instantly responded to by the lever. (1.) Carefully measure the arms of the lever to determine how much the tracing point of the lever will move for every millimetre that the button moves. (2.) When the button is pressed outward in inspiration, in what direction does the lever move? Take tracings of the changes in the dorso-ventral diameter at the level of the nipples. Determine by measuring the tracing how much the dorso-ventral expan- sion is. What is the average expansion during forced respiration? Make a similar series of observations on the lateral diameter in the plane of the nipples. (5.)^ Repeat observations on the lateral and dorso-ventral diameter at the ninth rib. ANTHROPOMETRIC DATA (Thoracic). Name Home Flat or Hills Age .... Height .Weight Previous Occupation Habit. Inactive, Active. .Tennis, Bicjxling, etc, ; Condition, . .Fat, Lean, Medium, Muscular, Flabby Lung Capacity. Respiratory Pressure from Pne'umonometer Ordinary Inspiration Ordinar}' Expiration Forced Inspiration Forced Expiration . 58 Outlines of Experimental Physiology Girth of Chest in Nipple Plane, (1.) In Repose Ord Inspiration Ord. Expiration Expansion.. Girth of Chest at Xinth Rib. (1 ) In Repose Forced Ins Forced Exp Expansion. .. Diameter of Chest at Xipple Plane. Dorso-Vcntral. (1.) In Repose Ord. Insp Ord. Exp Expansion (2. ) Forced Ins Forced Expir Expan Lateral. (1.) In Repose Ord. Insp Ord. Exp Expansion (2. ) Forced Insp Forced Exp Expansion Diameter of Chest at Plane of Ninth Rib. Dorso-vcntral. (1), In Repose Ord. Ins Ord. Exp Expan (2). Forced Ins Forced Exp Expan Lateral. (1). In Repose ....... .Ord. Ins Ord- Exp Expan (2). Forced Ins Forced Exp Expan Date Examiner Exp. LXI. The Stethog-oniometer. The purpose of this instrument is to record the outline of any horizontal section of the thorax, though it could be used as well for tracing- the periphery of the abdo- men, of the head, or of a limb. To use the stethogoniometer for the purpose here intended, let the subject sit beside a table upon a stool adjustable for height. So adjust the stool as to bring the circumference of the thorax to be observed, even with the upper surface of the table. Fix the point c of the instrument to the table, Let the observer locate, with pen or pencil, upon the side of the subject distal from the table, a point which shall serve as a starting point. When the point b of the instrument rests upon this point of the subject". s thorax, the instrument should be well extended and in equilibrium Fix a sheet of paper to the table under the recording pencil at a. To take a graphic record of the contour of the thorax, proceed as follows: (a). Let the observer place the tracing point b upon the .starting point in the distal side of the thoracic perimeter. Let the subject also sit with hi.s back to the table and take his record. (b). Sweep the tracing point quickl}^ around one-half the perimeter to a point approximately opposite to the starting point. (c). Rotate the curved arm of the instrument upon its axis through 180 ^ . {d). Sweep the tracing point around the other half of the perimeter to the starting point. (e). The movements of the tracing point, b, in the horizontal plane have been faithfully recorded upon the sheet of paper by the recording pencil at a. It is Outlines of Experimental Physiology 59 hardlj' necessar}' to remind the student that the subject must remain motionless duriny the observation. Take a thoracic j^erimeter with the chest in repose. Measure different diamet- ers of the tracing- and multiply by 5 to reduce to actual measurements. Take a tracing- at the end of forced expiration; at the end of forced inspiration. Compare diameters. Make a series of these tracings for different individuals. Compare. Formulate conclusions. Expt LXII. Cardio Pneumatogram. Breathe quietly or suspend respirjition for a short period during- which a trac- ing of the pulse transmitted through the air of the respir^itory passag-es through the tube in the mouth or nose to the tambour which records the movements on the drum. Let some member count the pulse of the experimenter at the same time and compare wi^h your record. Expt. LrXIII. Determination of Carbon Dioxide and Oxygen in Inspired and Expired Air. in). Estimation of Carbon Dioxide, Fill a burette with water and close the pinch cock on the rubber tube. Immerse the wide end of the burette in a large ves- sel of water and fill it with carbon dioxide by putting- into it the tube from the car- bon dioxide reservoir to the graduated point. Hold the burette in a vertical position, its mouth being- still immersed, make the level of the water the same inside and out and read off" the meniscus. Then introduce a piece of stick sodium hydrate, close the burette with the finger or a cork, lift it out of the water and by a sort of see-saw movement, shake the sodium hydrate repeatedlj^ from end to end. Again immerse the burette and read oft" the menisus. Most of the gas will be absorbed. Repeat the shaking. If the reading is just the same, the absorption is complete. {d). Estimation of Inspired Air. Fill the burette with the air of the laboratory. Open the pinch cock and immerse the wide end of the burette till the water reaches the graduation. Then close the cock and read off the meniscus. Introduce a piece of sodium hj'drate and jiroceed as in (a). Notice that there is no appreciable absorp- tion. This method is not suitable for the small Jimount of carbon dioxide present in ordinary air. Now introduce under the water, some pyrogallic acid. This can be done conveniently by wrapping some of the crystals in thin paper, so as to form a kind of cigarette which is pushed up into the burette. A little more sodium hydrate may be added if the first introduced is entirely absorbed. Shake as described in («), until no more absorption is noticed. Then read oft" the meniscus again, always making the level the same inside and outside of the burette. The difterence in the two readings gives the amount of oxj'gen present. "What remains in the burette is nitrogen (and a little argon). Its amounl is, of course, equal to the reading of the burette plus the capacity of the ungraduated part in the narrow end of the burette, which must be determined by a separate measurement {c). Analj'sis of Expired Air. Fill the spirometer with water, breathe into it several times in your ordinary way, but be careful not to inspire any air from the spirometer; then fill the burette with the air from the spirometer. Or simply expire several times through the bur- ette, seeing that none of the inspired comes from it. Determine as in Ux) and (f>), the percentage amount of carbon dioxide, oxygen and nitrogen. 60 Outlines of Experimental Physiology {d). Repeat (c) with air expired after the lungs have been thoroug-hly ventilated by taking a number of deep breaths in succession and determine whether there is anj- difference in the percentage amounts. For calculating gasses where the gas is not each time as it should be placed, so that water inside and outside is on a level. If not on a level, use the following calculation. VP = vp. V = volume of gas under atmospheric pressure. P = atmospheric pressure under water = 950 cm. (76 X 12.5). v = volume of gas under lowered pressure. p = atmospheric pressure minus the height of water in the tube. Expt. LXIV. Mechanics of Respiration. Artificial Scheme. Follow the directions attached to it. Expt. LXV. Chemistry of Respiration. Estimation of oxygen carbon dioxide and water. Apparatus. Two aspirator bottles with a box; wooden tray containing a jar for a guinea pig and six bottles; — Nos. 1 and 4 filled with soda lime to absorb CO 2 : Nos. 2, 3, and 5 filled with pumice stone soaked in H2SO4 to absorb moisture: No. 6, a MuUer valve to prevent the air being forced back through the series of bottles by a wrong coupling of the aspirator tubes. (a). Weigh bottles 3, 4, and 5 (4 and 5 together). Place the guinea pig in the jar and weigh. During one hour draw air through bottles 1 to 6 by placing an as. pirator bottle on its box and allow-ing the water to flow from this bottle to the one remaining on the desk. The rubber connecting tube must be changed when the as- pirator bottles are changed. After one hour, w^eigh bottle 3 and bottle 4 and 5. Tabulate the results as follows. Weight of jar and guinea pig at the beginning grams. Weight of jar and guindea pig at the end grams. Loss grams. Weight of bottle 2 (H2SO4) at the beginning, grams. Weight of bottle 2 (H2SO4) at the end, grams. Gain (water absorbed) Weight of bottles 4 and 5 at the beginning, grams. Weight of bottles 4 and 5 at the end, grams. Gain (CO^ absorbed) Total water and CO 2 absorbed Loss in weight of jar and guinea pig Difference (oxygen absorbed) Respiratory quotient Expt. LXVI. Electro-Physiological Apparatus. Battery or cell. I. Single fluid cells, (a). Place small Zn and Cu plates in separate vessels of 10 per cent sulphuric acid. Note the action. Outlines of Experimental Physiology 61 $ (b). Place Zn and Cu plates in the same vessel (unconnected by wire) of 10 per cent sulphuric acid. Note the action when near and far apart and write the chemi- cal reactions. (c). Connect the plates by wire for an instant. Note the action — reaction. Di- rection of current? Place each wire separately, then together on the tong^ue; result? (d). Amalgamate the zink plate (see footnote). Pure zink in H2SO4 gives off a few bubbles verv slowU'. Now repeat {c) and note the differences between (<:) and (d). Place the plates far apart and then close together while the wires are on the tongue. Note the differ- ences. Illustrate and label all the parts of a single fluid cell. Name the ions set free. If H is electro-positive, a poor conductor, offering great resistance to the flow of the current — what effect may it have upon the direction of the current? (e). Place the electrodes on a piece of filter paper containing a little starch and Kl. Close the circuit. A dark blue spot indicates the anode or X pole. (Iodine is set free at the anode). (/"). From experiment (e) which plate has the positive pole? Give the reaction. II. Double Fluid Cells, (a). Daniell cell, — illustrate, naming the parts and solutions, reactions, anions, and kations, determine with KI and starch which is the positive pole. (b). What advantage does it posses over the single fluid cell in regard to polarization and constancy of currents? Use of porous cup. (c). Which pole gives acid and which alkaline reaction, as proved with litmus paper? III. Leclanche or Dry Cell. Illustrate and name the parts as seen in the separated cell. Write the reaction. Why ought they not be used for long periods of time? And what effect has rest? Test the poles with KI and starch, with litmus. Foot Note. {a). Chemically pure zinc does not need amalgamation, commercial zinc contains iron, arsenic, etc,, as impurities, the contact of unamalgamated zinc and these dissimilar metals with an electrolj'te, causes a difference of potential and paras- itic currents run from the zinc to the foreign metals. These currents are prevented by covering the impurities with zinc amalgam, the electro-motive properties of which toward sulphuric acid are those of pure zinc. As the zinc in the amalgam dissolves out, the mercurj' unites with fresh zinc. Zinc is best amalgamated by cleaning with 10 per cent sulphuric acid and then rubbing on mercury with a brush. (b). If the current passes through a solution, decomposition takes place, elec- trol3'sis, the ions are set free and may adhere to the plates, setting up currents in the opposite direction and weaken the original current. The poles are touched to a solution of KI and starch. Decomposition is effected, in that Iodine is separated and goes to the plus pole, unites with starch to form a blue spot. (c). The zinc is arbitrarily taken as the positive plate, the copper as the nega- tive. The pole attached to the negative plate is the positive pole or anode; that at- tached to the positive plate, is the negative pole or cathode. (d). The latent chemico-phj'sical energy of the cell is transformed into electri- cal energy which becomes manifest in the contact spark, movement of the galvanic needle or lifting of the armature of a magnet. If the copper plate of a cell were weighed before and after using the cell, it would be found that it had increased in 62 Outlines of Experimental Physiology « weig-ht. The amount of electrolysis is an index of the amount of current afPorded by a cell; e. g-., if the negative pole were attached to a cup containing- Ag-NOs and the positive pole to pure silver immersed in the AgNOs, it would be found that one ampere of current will uniformly deposit 0.001118 grams silver upon the cup in one second. This is known as the unit of current or ampere. A dry cell with little ex- ternal resistance can produce a current of about % ampere. The unit of resistance is the ohm. It is the resistance offered to a current by a column of mercury 1 sq. mm. cross section and 106.8 cm. long, or of pure silver wire 1 mm. by 1 metre. The electromotive force is measured in volts. The relation of the units of measurements- are expressed in Ohm's formula, — C=E/R. For our work, the cells are arranged in series; that is, the zinc of one is attach- ed to the copper of the other. Expt. LXVII. Keys or Switches. A. Simple Contact Key. Illustrate, showing- the direction of the current by arrows. B. Du Bois Reymond Key. [a]. As a simple contact key. {b). As a short circuiting key; i. e., so that the current goes back to the cell. Prove with starch and KI, using two cells. C. The Commutator, (a). Set up for a simple key. (b). As a double key; e. g., for two n. m. p. so as to send the current "to either of two pairs of wires. {c). Set up so as to change the direction of a current. Illustrate diagrammat ically, showing the direction of the current with arrows. Expt. LXVIII. The Galvanometer. In order to study the difference of potential, or direction and strength of a cur- rent, a galvanometer or electrometer is employed . In the galvanometer, the points of different potential are connected by a coil of wire near which is suspended a mag- net. When the circuit is completed, the electrical energy acts on the suspended magnet by induction and deflects it to an extent proportionate to the current. A mirror is attached to the magnet by means of which a beam of light is reflected to- ward the scale. The mirror and magnet are suspended by very delicate fibres; hence, when not in use, must always be protected by a support which is moved in place by the lower screw. Adjust the eye piece so that the cross hair is in the focal plane and the scale reading distinctly seen. . [a). Connect the terminal posts through a simple key and a resistance of about 2000 ohms, to the poles of a small battery made of Cu and Zn strips dipping into dilute H3SO4. Note the direction and extent of the deflection of the mirror on the scale. (b). Reverse the poles of the battery. Result? {c). Increase the resistance. Result. Expt. LXIX. The Voltmeter. Connect a dry cell through a simple key, so that the positive pole (the one joined to the carbon plate) is connected with the plus binding post. Close the key and note the voltage of the cell as indicated by the pointer. Repeat this with another cell, then join the two cells in series and ascertain the combined voltage. Does it agree with the sum of the two? Outlines of Experimental Physiology 63 Expt r^XX The Milainmeter indicates the streng^th of the current or amperes directlj'. The scale of the one employed in this laboratory is calibrated in one half thousands of an ampere. You determined the voltage of the dry cell; and to ascer- tain its amperage, connect the positive pole of the cell through a simple key to the positive binding post of the milammeter and read off the current from the scale; add two or more cells and note the scale reading. Be sure never to connect more than twelve cells in the circuit with the milammeter and never join the negative pole of the cell with the positive binding post of the mil- ammeter. Expt LXXI. The resistance of the cell (internal resistance) and the external resistance in the circuit are determined by means of the Wheatstone Bridge. Expt. LXXII. Differences of Electrical Potential, especially very small dif- ferences, are also determined b^' means of the capillary electrometer. The direc- tions for its use and an explanation of it, are posted with the apparatus. Expt. LXXIII. Graduating the Strength of the Current. From the formula C = E/R, it is evident that the current ma3' be altered, eith- er by var5'ing the E M. F , or by altering the resistance. If two poles of a cell or other points of difference of potential be joined by a well- drawn wire, the potential through the wire will uniformly fall from the anode to the cathode. The greater the resistance in the wire, the more uniform will be the fall in potential. By means of the rheostat we are able to send more or less of the current of a voltaic cell through a nerve or muscle by changing the resistance of the current. This is done by putting a long thin wire into the circuit, the current being inversely pro- portional to the resistance. The greater the resistance, therefore, the smaller the current. Since the strength of the galvanic current depends upon the character and number of the cells employed and the total resistance in the circuit, the strength of the current can be easily varied b3^ altering the resistance, since C = E/R. There are a number of forms for this purpose. One convenient form is the rheostat which is a box containing coils of known resistances, connected with brass plates. The current enters bj' the binding posts, passes from block to block through thick plugs of verj^ low resistance. If the plugs are pulled out, the current travels coils of wire of known resistances. Another method of altering the strength of current is to employ some form of shunt to split the current so that only part passes the nervei i. e , an arrangement b}' which a greater or less proportion of current can be sent through the galvanometer; e. g , if the plugs' resistances are labeled and the resis- tance of the galvanometer known, then 1-9 plug inserted indicating the ratio be- tween the resistance of the galvanometer and itself and 1-10 of the current is sent through the galvanometer. A current takes the path of least resistance and if two paths are open to it, more or less can be sent through one of them by decreasing or increasing the resis- tance in the other. The simple rheostat of 10 meters or more fine wires stretched on a board and combined with slider and binding posts so that more or less of the wire can be put into the circuit bj' changing the slider or attachments of the posts is the modified form of the one used in the labor atorj*. 64 Outlines of Experimental Physiology Divided circuits. If a circuit divides into two branches at A, uniting tog-ether again at B, the current w^ill also be divided. The relative strength of current in the tvpo branches will be proportional to their conductivities, i. e., inversely propor- tional to their resistances. If. r be a wire of 2 ohms and r' of 3 ohms, current r will be to current r' as 3 : 2; or 3-5 of the current will flow through r and 2-5 through r'. The joint resistance of the two currents will be less than that of one branch because the current now has the-choice of both and the conductivity will be the sum of the two and the joint resistance will be R. It follows that yR = /r + /r' = ^ ^^'^' or ^^^ >^ + /s rr' and R= — = -S- which is less than 2. or r+r' ® the joint resistance of the divided conductors will be the product of both resistances divided by their sum. (1). Rheostat in Series. Connect a cell through a simple key with a galvanometer or milammeter and introduce the rheostat. Connect the positive pole of the cell to the plus or zero post of the rheostat and from the post marked 3 to the other post of the galvanometer, in- troduce a wire. Tabulate results. If necessary, shunt the galvanometer by con- necting its binding posts through about 4ohms resistance wire. Then introduce the rheostat in the galvanic circuit with gradually increasing resistances. Re- sult and Conclusions? (II). Rheostat in Parallel. Divide the current. When the resistance in the galvanometer is very high, then the fraction of the potential of the cell, passing through the galvanometer will be directly proportional to the fraction of the resistance in the rheostat. Divide the current so that X) /^) Hf and 1 respectively, of the current passes through the galvanometer. Connect each pole of the battery through a key with the end poles of the rheostat and from the o pole and the other pole of the simple rheostat, chosen as the one that gives the desired fraction of the whole resistance of the i-heostat, con- nect wires to the galvanometer. This will give practically the fraction of the cur- rent desired. Tabulate results and prove your conclusions by determining the voltage with the voltmeter and record your readings for comparison. A milammeter may be used instead of a galvanometer, since the resistance is only 210 ohms, the readings are not perfectly correct. From the arrangement of our apparatus, you notice that from the cell through the rheostat back to the cell makes a complete circuit. Having reached the metal- lic slider (S), the circuit has two paths presented. .. Outlines of Experimental Physiology 65 First, from S to B. vSccond, from S throuf,'^h G back to B. The total current is divided into two parts; C which passes along- the wire from S to B and C, the derived circuit which passes through the galvanometer. Suppose the resistance to the last named current is R' and that to the direct current is R, the relative strength of these two currents is expressed in the following proportion; C':C::R:R'. But the resistance of the german silver wire may be conveniently divided into 100 equal (100 r). If the slider be placed at any position along the wire, say at X centimeters from the end, then the formula would be C':C::xr:R'. C = — C Suppose that R=l ohm (r=0.01 ohms); R'=2 ohms and x=0, i. e., the slider R' * xr tobeatB. Then C'=:^ C =0. This shows that no current will then pass through the galvanometer. (1), What is the relative strength of the two currents when x=10? Whem x= 50? (2). What is the relation of C ' to C when x = 99? When x = 100? From this course of reasoning, it is evident that with the simple rheostat we can vary a derived current from zero to a maximum Just what the value of the derived currents will be, will depend upon the voltage of the cell and the total resistance and its distribution. Expt. LXXIV. Induction Currents. A most useful method of electrical stimulation of living tissues is by the induced current, and a clear idea of the phenomenon of induction must be obtained. (1). Iron filings and a magnet. Note the lines of force. (2). Faraday's Experiment. — Remove the secondary (larger) coil of the induct- orium from its slideway and connect its terminal with the capillaj-3' electrometer or galvanometer. Raise the brass bridge between the binding posts, that nearly or practically all the electricity produced in this coil will pass over the bridge, instead of bj' the relativelj- long thin wires leading to the electrometer, galvanometer or milammeter. Tr3' a magnet near the galvanometer without the coil. Effect. Bring the meniscus into the field. If the galvanometer is used, put the coil far from the galvanometer so that the magnet shall not affect the magnetism of the gal- vanometer. Thrust the north pole of the magnetized rod within the coil at a definite speed. The needle will move, indicating that an electric current has been induced in the secondar}- coil. The meniscus will return to its former position. Evidently the induced current is of momentary' duration. Withdraw the mag- net quiclilyt The meniscus will move in the opposite direction. Insert the south pole. The induced current now has the direction opposite to that of the current inlu^e.l by the insertioa of the north pole. Withdraw the magnet quickly. The induced current has the direction opposite to that of the current induced by the withdrawal of the north pole. Thomson, 210-211, Induced Currents. 106-109, Magnetic Field. Expt. LXXV. Lines of Force. The space about a magnet in which the mag- netic forc3s act is called the "field" of the magnet. If very fine iron filings are dusted through a muslin cloth on'io a thin card perforated near the centre by a copper wire or other conductor, and a strong current is passed through the wire, the filings will arrange themselves in concentric circles around the wire, particu- 66 Outlines of Experimental Physiology larly if the card be gently tapped. The position of these lines of force shows the direction of themag-netic force and their number is an index of its intensity. Expt. LXXVI. To produce electric induction, the lines of magnetic force must be cut in the circuit. Hold the magnet at right angles to the axis of the coil; and, keeping it in this position, rapidly advance it towards the coil. The galvanometer or electrometer will show no current, because the number of the lines of magnetic force which pass through the field of the conductor has not been altered, LXXVII. Electric-magnetic Induction. An electro-magnet may be used in place of the bar magnet to produce induction. Connect a dry cell through a simple key with the galvanometer. Get the direction of the deflection with a cell to note its direction in the galvanometer. Then connect the posts 1 and 2 of the primar3^ coil. Open the key. When the current passes through the primary coil, the core of iron in the coil will be magnetized as is shown bj" its attracting the head of the Wagner hammer. (2). Connect the posts of the secondary coil to the galvanometer. Approach the primary to the secondary as in the experiment with the magnet. Get the direction of the deflection. With- draw the primarj- coil. The galvanometer shows the presence of induced currents as before. These currents are momentary. The first induction current is inverse; i. e., it runs round the second arj'^ coil in the direction opposite to that taken by the battery current in the primary' coil- The second induced current is in the same di- rection as the primary current. Place the coils at right angles to each other. Ap- proach one toward the other. No current will be induced. When does the induced current have the same direction as the battery current? At make or break? (3). Make and Break Induction. Open and close the key in the primary cir- cuit; thus making and breaking the primary current. Is the effect the same as if the primary were suddenly brought up to a secondary coil from an infinite distance and removed again? Is the make induction current in the opposite, the break in the same direction as the primary' current? Turn the secondary coil on its pivot until the axis is at right angles to the primar}' coil. Make and break the primary current. No induction will take place, provided the angle between the coils is precisely at Q'J '-' . Expt. LXXVIII. The Inductorium. Examine the construction of the induc- tor ium. The primary coil consists of a few turns of thick wire. More turns would increase resistance and self-induction and the counter induction set up in each turn of the primary wire by the passage of the primary current through the neighboring turns without increasing the induction effect in the secondary coil. The iron core adds to the number of lines of magnetic induction which pas? through the coils. It has been already shown that the lines of magnetic induction produced by the pas- sage of an electric current through a wire are closed circles. If the centre of the coil were filled with air, most of the circles would remain closed about their own wire, for air is not readily permeable to magnetism. But when the iron core is placed within the coil, the greater part of the magnetic induction follows the iron (because it is more permeable) from end to end of the core, returning outside, through the air. The number of effective lines is increased, if bundles of iron wire are used instead of a solid core, because no induced current is then possible through Outlines of Experimental Physiology 67 the mass of iron, as would be the case in a solid core Such a current would slow the speed of mag-netization and demag-netization. Expt LXXIX. The secondary coil is made of many turns of fine wire, be- cause the object of the inductorium is to transform the low E. M. F. of the cell into the high E. M. F. of the induced current. In the induction coil, as in other trans- formers, the electro-motive force in the primary circuit is to those produced in the secondary circuit approximately as the number of turns of wire in the primary is to the number in the secondary circuit. If the induced current is to be passed through conductors of low resistiince, the high internal resistance of the secondary coil, due to its great length of wire will be of importance Place a dry cell with a simple key in the primary circuit of the inductorium (posts 1 and 2). Con- nect the secondary coil with the galvanometer. Note the excursion of the needle with a break, iilso a make induction current. Replace the secondary coil with one of fewer windings. Let the distance between the primary and secondary coil be the same as before. The excursion of the needle with a break induction current will be increased, or at least not proportionately diminished. If, on the other hand, the induced current is to be passed through nerve, muscle, or skin, the resistance of the secondary coil will practically be nothing in comparison with the enormous resistance of animal tissue. Expt. LXXX. Repeat the preceding experiment, introducing . into the secon- dary circuit a high external resistance; i. e., nerve. The secondar3' coil with many turns of fine wire now causes a greater deflection of the galvanometer needle than the coils with fewer turns. Note: — the more turns used, the more induction currents or E. M. F. available. Expt. LXXXI. Interrupter. Instead of making and brejiking the primary circuit by hand an automatic interrupter is provided. The primary circuit passes through a screw, the point of which conveys the current through a flat spring upon which is mounted an iron disk, opposite and near to the core of wire in the primary' coil. When the current enters the primary coil, the core is magnetized and draws upon the iron disk The spring to which the disk is attached, is thereby drawn away from the screw point through which the current is passing. Thus the current is broken and ceases to flow through the primary coil, the core is no longer magnet- ized, and releases the iron disk; the spring again makes contact with the screw point, the current is re-established, only to be at once again broken. Thus a rapid series of make and break induction currents is secured. (d). Draw a diagram of the primary circuit indicating the connections of the inductorium. Expt. LXXXII. (a) Test the effect of a galvanic current, 1 cell, simple key and electrodes place latter on the tongue, at make and break. (b). Extra currents at opening and closing of the primary current. Remove the secondary coil from the inductorium. Connect pasts 1 and 2 of the primary coil with a dry cell, using a key. Fasten the ends of the electrode wires to the same posts. Close the primary circuit Place the electrode points against the tongue. Open the key. Is a shock felt? It was caused by a self-induced current developed in the primary coil. Is it stronger at make or break? Illustrate, showing the di- rection of the current. 68 Outlines of Experimental Physiology Expt. LXXXIII. Galvani's Experiment. Decapitate a frog-, fasten the hook of the copper holder 'carefully throug-h the vertebral column and twist the holder quickly so that one of the frog's legs comes in contact for an instant with the insulated rod. The other leg in the meantime, is held off with a glass rod. Are the contractions confined to one leg or both? Explain. Expt. LXXXIV. Lay the frog on the glass plate covered with moistened filter paper. Study the sciatic plexus, uotiug the number of fibers that compose it and their origin. (a). Make a V-shaped wire composed of zinc and copper Touch the sciatic with one end of the wire and the muscle with the other, first one, then the other, then together. (b). Are there indications of fatigue of the muscles? (c). Note whether the current varies with zinc on the muscle or copper on the muscle. (d). Sartorius — alternately and together, wires on the eight nerve and sartorius. (). The nerve lying on the muscle whose nerve is directly stimulated, is ex- cited by what current, that of the battery or by the action current of the muscle? (c). What portion of C is traversed by the electric current? (cf). Does the electrical current traverse the muscle C between the points sepa- rated b}' the nerve-lifter? Preparation B is called a rheoscopic frog. If the contractions are caused by differences of potential or electricity, they should be detected with the galvanometer or electrometer. Expt. cm. Electrical Phenomena in Muscle. A. Demarcation Current in Muscle. Place small platinum electrodes connected through a short circuiting key to an inductorium arranged for tetanus, in the posts further from the muscle and a pair of N. p. E. connected through a key to a galvanometer or electrometer. Test the N. P. E. by placing the brushes in contact. If any movement in the galvanometer oc- curs, the N. P. E are polarized and must be set up again. Fasten the femur of the N. M. P. in the clamp and the injured tendon end pinned to a piece of cork stuck in the clamp rod of a moist chamber. (n). Lay one N. P. E. on the injured and the other on the uninjured part of the muscle, open the key and note the direction and extent of movement of mercury or the spot of light on the scale. (b). Determine which is the positive portion of the muscle, judging from the galvanometer or electrometer movements, B. Action Current in Muscle. Without disturbing the position of the muscle, lay the nerve on the platinum electrodes, open the electrometer key and wait until the mercury comes to rest, then 78 Outlines of Experimental Physiology stimulate with a weak tetanus, the N. M. P. by opening! the short circuiting- key. Note the direction and extent of the movement of the galvanometer or capillary elec- trometer and the variation of current as indicated by the galvanometer. Which di- rection does the current take in the muscle? Which is greater, the current of injury (demarcation) or current of action? [C]. Demarcation and Action Current in the Nerve. Repeat [A] with the nerve alone, laying the injured cut end on one and the lon- . gitudinal side of the neiwe on the electrode. (a). Determine the direction and (b) extent of the deflection in the galvanometer, and [c] the direction of the current in the nerve. [D]. Repeat [B]. Is the action current greater than the demarcation current in the muscle or in the nerve? Expt. CIV. Contraction of Human Muscles. Arrange the inductorium for single shocks with one cell and key in the primary circuit. Determine the cathode pole of the secondary coil when the primary current is broken through the galvanometer. To this pole, connect the small electrode; to the other, the lai"ge one. Cover both with a cotton cloth wet with saline solution. The smaller place over the nerve, the larger on an indifferent region, as the arm sto- mach or neck. With the smaller electrode make out on anterior and posterior side the motor points [where the nerve enters the muscle] of the arm muscles, — anterior sur- face, Lumbricales, Digiti, Pollicis, Flexor Pollicis Longus, Ulnaris, Radii, Pro- nator radii teres Posterior Surface. Interosseous, Abductor minimi digiti. Extensor pollicis longus add brevis. Extensor communis digitorum, Extensor radii longus. Make, then break the current. Does contraction follow at make or break at anode or cathode? Illustrate the points. Polar Stimulation of Human Nerves. Connect terminal zinc and carbon of 8 cells in series through a pole changer with cross wires, to a small and a large elec- trode covered with cotton moistened with NaCL. Place the small electrode over the ulnar nerve between the internal condyle and olecranon a little above the furrow. Make and break the current, add cells up to 12, never more than 16 until contraction occurs. (a). Cathode over the nerve, first contraction at make. With this strength of current, break contraction will be absent. {b). Turn with the pole-changer, bringing the anode over the nerves. A strength of current will be reached with which make will cause contraction, but not break- (c). A slightly greater current will bring out the anodal break contraction. (Some- times anodal break precedes the make). (d). Further increase gives catliodal break contraction and may cause anodal make tetanus. Tabulate the results. Test the strength with the milammeter. [Dose, 2-10 milliamperes] . For this purpose, the milammeter is employed. Remember the positive pole, the one from the carbon plate is joined to the positive post of the inilammeter and the small elect, rode to the negative pole of the cell. Place the electrodes on the neck and arm and when the body is in circuit, read off the strength of the current directly from the scale. [/]. Place the cells in the circuit with the Voltmeter, determine the voltage directly and {^) from these data, calculate the resistance offered by the body. Outlines of Experimental Physiology 79 Expt. CV. Anatomj' of the Frog-'s Brain. References, — Wiedersheim, Ecker, and Schenk. [1]. Make a median cut throug-h the skin beginning at the level of the occipi- tal artery and in the skin. Note the cutaneous artery imd vena magna. Carefully lift and remove the roof of the skull. Make a careful drawing [enlarged] and note the relations of different regions of the brain with landmarks of the body; e. g. , eye, nares, tympanum. Note the olfactory lobes, cerebrum, thalamencephalon, pineal body, cerebellum, and medulla. Note the optic, olfactor3'. and other nerves as far as the eleventh, given oft' from the medulla. Lift the optic lobes and see the pituitarj' body. Expt. CVI. Excision of the Cerebral Hemispheres in the Frog. Method. The following articles are needed for the operation. Scissors, scal- pel, forceps, trephine, silk, cotton corrosive, 1 per cent adrenelin, NaCl, frogboard, ether, and a bell jar. Sterilize the instruments in boiling water. When everything is in readiness, put Ic, c. of ether on a small piece of cotton on a frog under the bell jar. Test the aft'ect of the anesthesia by the reflex of the eyes. How does the anesthesia affect the respiration? A. When anesthesized make a median incision over the skull yi inch long fasten back the skin with pin hooks, after having washed the skin in corrosive, and having fastened the animal to the frog-board. Expose and remove the cerebrum, be sure not to get corrosive in the wound and to remove all nerve tissue in front and not to injure the thalamencephalon and neighboring parts. Use NaCl solution on the wound and adrenelin to stop the bleeding. Suture the skin and wash in salt solution. Date and note all observations regarding the position and irritability during and after the shock. Compare with the normal frog, — ej'es, nares, respiration, when placed on its back or on an inclined plane, in water, obstruct its path, stimulate its croak centre, by stimulating its side back of the front legs. Will it catch flies when put in its pan? After several hours, days or weeks, note any furthur changes. What is the conclusion regarding the frog's cerebrum? Co-ordination? B. Removal of right Cerebral Hemisphere. With the same precautions as observed in the preceding operation, remove the right cerebral hemisphere and sew the skin together. Note and date all the important observations immediately and for hours and days afterwards. Keep the frogs in clean moist moss. C. Remove the thalamencephalon. Compare with A. D. Pith a frog; i. e, remove the whole brain. State the important differences between A, C, D. Suggestions. The parts to be removed must be clearly seen before thej' are cut. Notice the overlapping of the cerebral lobes. Expt. CVII. Reflex Action of the Spinal Cord. Simple Reflex. Expose the brain and remove onl\' the part anterior to the optic lobes. [1]. How long before the shock eft"ect passes off? Test by pinching and posi- tion. Note; position of bodj-, head, legs, respiration, eye reflex, croak reflex, turn- ing-over reflex. Compare with a normal animal. 80 Outlines of Experimental Physiology [2] . Suspend the frog- by its upper jaw in a clamp. Does this stimulate? Why not? [a]. Pinch one foot, first gently, then increase the streng-th. Result? [6]. Hold the toe and pinch the leg. Result? Why? Afferent and efferent path? [c] . Stimulate the leg with a weak induction shock of such a strength that sin- gle stimuli cause no reflex contraction. Stimulation of subminimal stimuli will produce reflex. [3]. Purposive Character of Reflex. Dip 4 m. m. squares of filter paper in 20 per cent acetic acid, others in 40 per cent. Place one upon the flank. Result? Wash the skin and after a short period of rest, apply another, holding the leg of that side. Result? Wash the skin and study the effect bv altering the position and strength of the stimulus. [4]. Reflex Time. Prepare 4 beakers containing fresh water and H2SO4 of 0.1 percent, 0.3 percent, and 0.5 per cent. Arrange a signal with key, one cell and time marker to record on a slow moving drum. Move one leg aside with a glass rod and let the other dip into the solutions, beginning with the weakest. Record with the signal the instant it is withdrawn. Wash the leg thoroughly and allow a period of'Test to intervene between each test. Tabulate the results, noting the relative time and strength of the stimuli. (5] . Inhibition of Reflex through Peripheral Afferent Nerves. Get the reflex time with 0,1 per cent H 2 SO'4. Expose the sciatic nerve about one inch, ligate, cut, and place the central stump of the nerve on the electrodes and stimulate with the weakest induction current. Dip the other leg in the acid again, recording'the 'instant of immersion and withdrawal while the nerve is being stimu- lated. Wash off the acid thoroughly. Has the latent period of reflex been prolonged? Why? [6] . Through Central Afferent Paths, the Optic Lobes. Expose the optic lobes. Determine the reflex time with 0.1 per cent or 0.3 per cent H3 SO 4. Wash off the acid and after a time put NaCl crystals on the cut sur- face of the optic lobes. When the salt has had an effect, determine the reflex time as before. Has the time of reflex been increased? Why ? [7]. Place calcium chloride crystals on the lobe and determine the reflex time. Result? Conclusions? [8]. Place the frog in water, wash off the salt and remove the optic lobes. Again determine the reflex time. Has the time been shortened? Why? Expt. CVIII. Reflexes in Man. Reflexes play an important part in the maintenance of the human body and a disturbance of certain ones are of value in the diagnosis of diseases of the central nervous system, since the reflex^depends upon the integrity of the corresponding reflex arc. [1]. From the Skin. Rub the plantar surface of the foot with a hard object. The foot will be retracted. [2] . From the eye, cornea, and pupil. [3]. Throat Reflex. Stimulation of the posterior wall causes swallowing. Outlines of Experimental Physiology 81 [4]. Tendon Reflex. Cross the leg- and tap the tendon below the patella with the edg-e of the hand. The quadriceps extensor muscle will be suddenly stretched and responds with a quick contraction which throws the foot forward in a kicking motion. [5] . Ankle Jerk. Bend the foot at right angle to the leg and strike the Tendo Achillis. The gastrocnemius contracts. Expt. CIX. Reaction Time. »[A]. Arrange a fast speed drum, time record 100 per second. Directly over the latter, place the point of the signal. The signal is connected through a cell and two keys to posts 1 and 2 [shocks] of the inductorium and the stimulating electrodes from the secondary coil to the tongue. Let the subject close his eyes and hold one key closed until he feels the stimulus. The observer starts the drum and tuning fork and stimulates by closing the second key in the primary circuit. Determine the interval between the stimulation and response. This inter- val is the reaction time plus the error of observation; e. g , the latent period of the signal. Record briefly the links in the chain between the stimulus and response and errors of observation. B. Reaction Time with Choice Connert the secondary posts with the pole changer without cross bars; from one set of posts join wires to platinum electrodes, from the other, to neck and hand [brass] electrodes. Tell the subject to signal only when the tongue [or hand] is stimulated. Physiological Optics. Expt. ex. [a]. Reflection from Concave Mirrors. Place the 2 mm. diaphragm in front of the condenser in the lantern. Place the concave mirror (segment of a 5 cm radius sphere) in the optical box at right angles to the pencil of parallel rays. The ra3's are reflected 2.5 cm. from the mirror. (Smoke will make them visible). The point to which the parallel rays are converged is the principal focus of the mirror. The distance between the mirror and the principal focus is half the radius of curvature? Principal focus? {b) Replace the diaphragm by an L apertured one; remove the mirror from the box and place it in the axis of the beam of light. At the principal focus hold a small screen with a handle. Describe the image seen on the screen. Is it erect, smaller, real, or virtual? (c). When the distance between the mirror and object is less than the radius of curvature but greater than the focal distance, state whether the image is real, inverted and the size of the object. Are real images from concave mirrors always erect or inverted? (d). Place the mirror at less than the focal distance from the luminous point. Where is the unreal or virtual image? (^) Hold a tiny object nearer than the principal focal distance to the mirror. Results? (/) Hold a candle in front of concave watch crystals of different curvatures. Where are the images seen? size? position as compared with the object? Relation of size as compared with the radius of curvature. 82 Outlines of Experimental Physiology (^). Determine bj' construction the length of the image of an arrow 2 cm. long- placed 10 cm from the middle point of a concave watch cr3'stal5cm. radius of curvature. Expt. CXI. Reflection from Convex Mirrors. [a]. Repeat Expt. c, replacing the concave with the convex mirror. State the [a] size, [b] position, [c] form of the image as compared with the object. [6]. Determine by construction the length of an image of an arrow 2 cm. long placed 10 cm. from the middle point of a convex mirror of 5 cm. radius. [c]. Hold a candle in front of convex watch crystals of difi'erent degrees of cur- vature. Describe the change in size, position, and whether erect, and other changes as compared with the object and the curvature of the mirror. Expt. CXII. Refraction by Convex Lenses. [a]. Principal Focus. Place the biconvex lens 5 cm. from the window of the box, parallel rays are brought to a focus 10 cm. from the lens. The 2mm. diaphragm is in the lantern. Place the black wooden screen at this point. A real image of the diaphragm aperature is seen. Where is the principal focus of the biconvex lens?- [b], E.stimation of the Principal Focal Distance. Remove the tube of projection lenses from the lantern. Place in front of the con- densing lens the L diaphragm. Place the convex lens in the axis of the pencil of rays at a place greater than the focal distance. On the other side of the lens, place a screen where it will give a clear, enlarged image of the L. Note the distance of the lens from the image [?]. Is it erect or inverted? Note the distance of the object from the lens [o]. An easy method of determining the focal distance of a lens de- pends upon the relation of the distance o the conjugate foci to the general focal distance. This relation may be expressed thus; — The sum of the reciprocals of the conjugate foci equals the reciprocal of the focal distance. The distance of the object [o] represents one and that of the image [z] , the other of these conjugate focal distances; so one maj' say, the reciprocals of the distance of the object from the lens l/o equals the reciprocal of the general focal distance T7 oi 1 /F, Study the general formula, l/o plus 1 /i = l/F. r = o + i Determine o and i f or several lenses and substitute their values in the equation. "When the object is twice the focal distance what is the distance of the image? 2. When the distance of the object is greater than 2F how does the distance of the image compare with 2F? 3. When the object is at a very great distance [ow=qo] at what distance will the image be formed? The principal focal distance of a double convex lens is approximately equal to the radius of the curvature. Expt. CXIII. Conjugate Foci. Place the 2 mm. diaphragm in the lantern, the tube removed, the convex lens against the window of the box, and the black screen twice the focal distance ©f the lens. Get a clear image on the screen, Points from which rays diverge and con- verge again after passing a lens are conjugate foci. Measure the distance of the luminous aperture from the lens. It will be twice the focal distance. When the point of diverging rays is twice the focal distance from the lens, the point of conver- gence is equally distant from the lens. Move the lantern further from the lens. The conjugate focus will approach the lens. As one conjugate focus recedes, the other approaches the lens. Outlines of Experimental Physiology 83 Virtual ImcOge. (a;. With the tube removed and the 2 mm. diaphrafrm in the lantern, the convex lens at a distance from the luminous point less than the princi- pal focal distance, one sees the diverging- rays passing through the lens continue to diverge; but prolonged backwards would unite in a virtual imag^ on the same side as the luminous object. The virtual image is further fr6m the lens than the object, is never inverted, and always enlarged. [b] . Look through the convex lens at printed words placed between the lens and its principal focus. The image is virtual and enlarged. Expt CXIV. Construction of Images Obtained with Convex Lenses. Draw a horizontal line through the optical centre of the lens of 10" aperture, (a lens of such curvature that parallel rays will be refracted through the principal focus only when the aperture of the lens does not exceed approximately IQO) and 5 cm. radius. The radius of curvature being 5cm. , the principal focus will lie ap- proximately Scm. from the optical centre. Draw through the optical centre a line 10 mm. long, connect its ends with the principal focus. The angle included will be about 10", At anj' distance greater than 5 cm., draw a 10 mm. arrow bisected by the principal axis. Draw incident rays from the ends of the arrow parallel to the principal focus. From each end of the arrow draw a line through the optical centre of the lens. Theintersectionofthe.se lines mark the limits of the imao-e. Note that it is real and inverted. If the object is twice the focal distance from the lens, the image will be the size of the object; if at less than twice the focal distance the image will be larger than the object; if at more than twice the focal distance smaller than the object; if between the principal focus and the lens the image will no longer be real but virtual and larger than the object. Expt. CXV. Refraction by Concave Lenses. Place a 2mm. diaphragm in front of the condenser. Let the rays fall upon the concave lens. The parallel rays will diverge. Look through the concave lens at the printed words. The image is virtual, upright, and smaller than the object. It is nearer the leas than the object and always within the principal focus. Expt. CXVI. Preparatory Experiment For Purkinji Sanson Images. Place a large convex lens on a table; hold a watch crystal in the left hand in front of the lens and a few inches from it. Movj a lighted candle at the side of this arrangement and observe the three images. Substitute a convex lens of shorter focus, observe the change in the reflected images. Explain the phenomena, using drawings Purkinji Sanson Experiment. Hold a lighted candle to one side of the observed eye in front and on a level with it. Look obliquely at it. Ask the person to look at a distant object (far accommodation) and look into his eye frcm the opposite side of the candle, notice the relation in size and position of the three images. At the mar- gin of the pupil and superficially, an erect image is reflected from the anterior sur- face of the cornea In the middle of the pupil is a second less bright erect image which appears to lie further back. It is reflected from the anterior surface of the lens. The third image lies toward the opposite margin of the pupil. It is the smallest and inverted and is furthest back. It is reflected from the posterior surface of the lens. 84 Outlines of Experimental Physiology Expt. CXVII. Refraction by Segments of Cylinders. Place in the optical box the 2mm. diaphrag-m in front of the condenser. Throw a pencil of parallel rays into the box. Place the cylindrical lens in the axis of the pencil in such a position that the curvature shall be from side to side; i. e , in the horizontal meridian. The image of the circular aperature in the diaphragm will be a vertical line with blurred convex ends. (b). Turn the cylinder so that the curvature shall be in the vertical meridian. "What is the image? Illustrate. {c). Astigmatic Lens. Place the diaphragm with the horizontal slit in the lantern, throw parallel rays into the box. Place the cylinder in the axis of the pen- cil with its curvature vertical. The horizontal line is a fusion of illumined points. From each point rays diverge in all directions. Those passing from any point in vertical planes through the cylinder, converging in the vertical meridian, will be focused by the convex surfaces in the corresponding point in the image. The over- lapping of such points will form a horizontal line with clear upper and lower edges. The rays passing from any point in the illuminated line in horizontal planes through the cylinder with vertical curvature, will be refracted by plane glass surfaces and will not come to a point but will form a faint horizontal line. The overlapping of the imcLge of the bright points which unite the rays passing in vertical planes and the faint horizontal lines formed by the rays passing in horizontal planes will form upon a screen a horizontal line with blurred ends. (d). Place the vertical slit in front of the condenser. Draw a diagram illus- trating the formation of the image. (e). Turn the cylinder so that the curvature will lie in the horizontal meridian. Into what images are the horizontal rays united? (/). Astigmatism. Draw on a card two lines of equal thickness intersecting' each other at right angles. Fix it vertically at the limit of accommodation and look at it. Probably either the vertical or the horizontal line is seen more distinctly. Test each eye separately. The line most distinct corresponds to the meridian of least curvature of the cornea. Expt. CXIII. Myopia. Let parallel rays pass through the convex lens (condenser) of about 10 cm. focal distance, and the focusing lens placed against the window of the optical box. Find the principal focus and then move the screen 2.5 cm. further away from the lens. The image is blurred. The screen will intersect the rays diverging from the focal point. Hold the weak concave lens (1-2) in front of the window. The lens has a focal distance of two diopters or j4 metre. If the image is clear, the myopia in this case is-"2D. Note the numbering of the lens. Lenses are numbered according to their refractive power. The unit is a lens with a focal distance of one metre (one diopter, ID). A lens of two metres focus is )i the refractive power or 0.5D. Lenses ordinarily employed in ophthalmic practice extend from 0.12D to 22 D. The principal focal distance of any lens in the dioptric system may be found by dividing one metre (100 cm,) by the number of dioptrics. For example, 4D=^100X =25cm. Convex lenses are positive, concave negative and they are. marked -j-or— . When two or more lenses are placed together, the dioptric power of the system thus formed equals the algebraic sum of the dioptric powers of the lens in the system. Outlines of p]xperimental Physiology 85 Expt. CXIX. Hypermetropia. Have the condensing lens in the lantern. Find the focal distance. Place a white screen 2 ocm. nearer the lens than the principal focus. The image will be blurred. The screen will intersect the rays before they have converged to the focal point. Hold the weak convex lens marked -\-2 in front of the window. The image will be clear. The hypermetropia, is therefore, in this case, ^2D, Expt. CXX. Chromatic Aberration. (a). Make a pin hole in a black card. Behind the hole place the cobalt glass. Look at a white flame through this arrangement. Cobalt glass allows violet and red rays to pass Accommodate for violet by approaching the light about 30 inches. What does the flame s how. (b). Accommodate for the red by receding. The center of the flame is what color? What is the halo? What is the diff"erence in distance for the red and blue focal images. (c). Each spectral ray due to the difference in wave length and rate of vibra- tion, on entering a refracting medium, pursues its own path and its own principal focus. In other words, the images for the several spectral colors do not coincide precisely. Violet crosses nearest the lens, then blue, green, yellow, orange and red. The peripheral part of a lens refracts rays parallel to the principal axis more strongly than the axial portion. Hence chromatic aberration increases with the aperture of the lens. (d). Put the ground glass plate and the diaphragm with the 2 mm. aperture in front of the condenser. Let the raj^s from the illuminated spot of ground glass pass through the lOD lens placed about 15 cm. in front of the ground glass, i. e., a distance somewhat greater than the focal distance of the lens (10 cm.). Place a white screen about 15 cm. in front of the lens. The image of the white spot upon the ground glass will be a disc with a violet center and a red margin. It is best seen in a darkened room. (e). Remove the white screen further from the lens. At a distance of about 30 cm., the center of the image and its border will change; in what respect? The im- age in this experiment is blurred because the rays which pass through the peripher- al portion of the lens cross the principal axis sooner than the rays which pass through the axial portion. If the screen be placed at the focus of the more axial rays, this focal point of the image will be surrounded by dispersion circles made of the rays which have been refracted from the periphery through foci nearer the lens and which are now diverging from their foci. If the screen be placed at the princi- pal focus for the peripheral rays, this focal point will be surrounded bj' dispersion circles made by rays that have not yet converged to the principal axis. The aberration is avoided by a diaphram. {/) Place a strip of red and one of blue 3 mm. apart on a black background. Look intently, first at one, then at the other. Which appears nearer? Why? Expt. CXXI. Place the 2mm. diaphragm in front of the condenser. Throw parallel raj's into the box Set in the box near the window, a cj'linder bottle filled with water (refracting cylinder). Show into what kind of a focal image the circular pencil of parallel rays are brought. Note the outer rays of the pencil pass through the outer part of the cylinder and are therefore more strongly refracted than those nearer the optical axis. Each refracted ray 86 Outlines of Experimental Physiology intersects the refracted rays nearer than itself to the principal focus. These inter- sections form two curved surfaces extending from the principal focus (in this case not a point) toward the cylinder. Place a wire on the side of the bottle. This throws a shadow on the opposite side showing the crossing of the rays. {b). The curvature of the caustic surface will be more noticeable if the 2mm. diaphragm is placed over the aperature in the box and light from the lantern is concentrated on it and then diverging raj's from this pass through the cylinder. Expt. CXXII. Accommodation, Have a person look at a distant object. The parallel rays of light proceeding from it, are focused on the retina. Observe his eye from the side and somewhat from behind Half of the pupil projects beyond the margin of the cornea. Have him look now at an object about six inches from the eye, without moving the eye-ball. The rays of the near object are divergent, yet are also focused on the retina with a consciousness of a distinct effort. The power of voluntarily' bringing divergent rays to a focus on the retina is termed accommoda- tion. Note that as he looks at the near object that the whole pupil and a part of the iris next the observer are projected forward owing to the increased curvature of the anterior surface of the lens. This requires careful observation. Expt. CXXIII. Scheiner's Experiment Prick two smooth holes in a card at a distance of 1mm. from each other; i. e., less than the diameter of the pupil Fix two pins in a cork. Place the pins in line with the holes on a strip of wood, one about 8 in. the other 2U in. from the card. The latter maj' be turned horizontally', turning the card so that the holes lie hori- zontally or verticall}' according to whether the vertical or horizontal needle is to appear. (1.) Sit with the back to the window, close or bandage one eye and with the other look through the holes placed horizontally at the near pin which will be seen distinctly; the far pin will appear double, both images being somewhat dim. Move the eye away and the two images separate. Move the eye closer and they ap- proach. (2). With the other card while accommodating for the near pin, close the right hand hole, the image disappears; close the left hand hole and the left image disappears. (3). Accommodate for the far pin the near pin appears double. Close the right hand hole and the left hand image disappears. Close the left hand hole and the right hand image disappears. Move the eye gradually away and the images -ap proach each other, while on moving the eye toward them, they separate. (4). Explain the phenomena, drawing figures which show just what must take place in the eye for all experiments. (a). When the eye is accommodated for the far point, why the images approach each other when the needle is moved away from the eye. {b). Why the images separate when moved toward the eye. (c). When the eye is accommodated for a point nearer than the needle (near point), why the images separate when the needle is moved away from the eye {d). Why the images approach each other when the needle is moved toward the eye. (e). Why closing one of the holes does not effect the image. Outlines of Experimental Physiology 87 (b). Why stopping- one of the holes when the eye is focused at a greater dis- tance than that of the needle, causes the image of the opposite side of the field to disappear. (ff). If tlie eye is focused for a shorter distance, the image of the same side as the blocked hole disappears. Expt. CXXIV. Determination of Near and Far Points. (a). Cut two corks so that they slide easily along a ruler. Mount a needle ver- tically in one cork and a card with two holes in a horizontal line in the other cork. Place the needle about 25 cm. from the card. Close one ej'e. Look through the two holes as in Scheiner's experiment; and when one distinct image of the needle is seen, gradually bring the needle toward the card. Observe that it becomes double at a certain distance from the eye. Why? This is the near point of accommodation. Make several trials and measurements of the distances from the needle to the eye when the doubling appears. Record the average. (b). Determine in a similar manner the near point with a horizontal needle and card with the holes vertical. The average measurements of (a) and (b) may differ. Why? {c). Stand six metres in front of a card of Snellen's test-types. If you see the numbers VI clearly, the acuteness of vision is normal and the far point (punctum remotum) is infinite. If you read I at one meter, II at two, but cannot read VI at six meters, bring the test card toward the eye until VI are seen clearly. This is the far point. Expt. CXXV. Diffusion. (a). Close one eye. Hold a pin upright before the other eye and gradually bring it nearer. When the pin is from 12 to i5 cm. from the ej'e, a change in dis. tinctness will be observed. Of what character? Why? {b). Prick a smooth hole in a card. Arrange the pin at the proper distance to obtain the previous diffusion effect and introduce the card 'between the pin and the ej'e and look through the hole in the card. Distinctness? Apparent size of the pin? Explain and make constructions. Expt. CXXVI. Identical Points. {a). Having both e3-es open, hold a pencil a foot from them and look at a dis- tant object The pencil appears double. Close one eye. Which image disappears? Why? Close the left. Which image disappears? Why? Reverse the experiment by accommodation for the near object. The far object will appear double. Close one eye. Which image disappears? This experiment is easiest done by having one object 20 to 30 cms. from the eye and the other 45 to 60 cms. awaj'. (b). Look at a near object and then press one eye ball out of place. The object appears double. Why? Expt. CXXVIT. Macula Lutea or Yellow Spot. Close the eyes for a minute, open them and while holding a bottle of chrome- alum solution between one eye and a white cloud, look through the solution. An elliptical spot, rosy in color, will be seen in the otherwise greenish field of vision. The size of the spot depends on the distance to which it is projected. The pigment in the yellow spot absorbs the blue-green rays, hence the remaining rays, red and greenish blue which pass through the chrome-alum, give a rose color. 88 Outlines of Experimental Physiology Expt. CXXVIII. Purkinji's Figures (of the Retinal Blood-vessels). In a dark room, hold an electric lig"ht at the level of the nose at the malar pro- cess and stand in front of a monochromatic wall. While looking steadily with one eye toward the wall, accommodate the eye for a distant object, hold the light close to the side of that eye well out of the field of vision, downwards and laterally from the eye and move the light up and down. It is better to direct the eye outwards, keep- ing it accommodated for a distant object. Ere long, dark, somewhat red-brown branching lines, shadows of the retinal blood-vessels, will be seen on a black back- ground, due to the shadows cast by the retinal vessels on the percipient parts of the retina. Therefore, the parts of the retina stimulated by light must lie behind the retinal blood-vessels. If the light be moved in a vertical plane, the shadows move upward or downward with the light, but apparently opposite. If the light be moved horizontally the shadows move in an opposite direction, but apparently in the same direction Modified Method for the Purkinji's Figures. Concentrate a beam of sunlight by a lens on the sclerotic at a point as far as possible from the corneal margin, passing the rays through a parallel-sided glass bottle filled with alum solution to sift out the low heat rays. The eye is turned toward a black ground. The field of vision takes on a bronzed appearance and the retinal vessels stand out on a dark net-work which appears to move in the same direction as the spot of light on the sclerotic, The area of the yellow spot is devoid of shadows. Illustrate. Expt. CXXIX. The Eye as a Camera Obscura. From the eye of an ox, remove the posterior part of the sclerotic and choroid from a spot one centimeter in diameter near the temporal side. Cover the exposed retina with a watch glass. Turn the cornea toward an incandescent light. Move a pencil in front of the light in different directions and note the movement of the shadow upon the retina. What kind of an image is cast upon the retina, erect or inverted? Expt. CXXX. Perimetry. Object:— To test the limits of indirect vision. In direct vision the image of an object falls upon the fovea centralis. In indirect vision, the image is formed on the peripheral part of the retina. Method. Turn the semicircle horizontally and place the chart on the brass disc, right side up, with the chart surface toward the reader. Fasten concentri- cally by the two screws. Charts are labeled right and left for the use of the right or left eye respectively. Ask the subject to close the left eye if the reading is to be taken of the right eye and to look steadily at the revolving point of the semicircle marked by a white disc, while the chin is upon the rest, so arranged that the open eye is directly in front of and on a level with the white disc. With the semi-circle held steadily in the horizontal position, take the handle which has a white disc at one end and pass it along the inside of the semicircle, from the extremity of the right arm toward the centre until it comes into the area of indirect vision. Take the reading at this point and mark at the same reading on the small scale on the chart. Turn the semi-circle through any desired meridian and as above, get the readings and record, thus plotting the area in which a white object can be distinguished with that eye. Outlines of Experimental Physiology 89 (2). Repeat the experiment for the other eye. (3). Repeat (1) and (2) for green. (4). Repeat for blue. (5). Repeat for red, using- in each case the respective colored pencils for re- cording. Do not let the subject see the color. Bring it from without inward. (6). Conclusion regarding the field of vision for a white object to the horizontal meridian and the outer, temporal, and nasal side and for which color is the field more extensive? (7). The perimeter is also useful for mapping out defects such as blind spots. As the observer moves the white disc along the arc, the subject may tell you that at certain points it disappears but comes again into view as you continue to move it further along. Then bv- shifting the arc into other meridians, these spaces ma^' be determined. Expt. CXXXI. (a). State what happens in a fellow student's eye when he looks at far or near objects. Pupil? (b). State the effect on the right pupil if light falls into the left eye only. Screen the right eye. {c). The Blind Spot. Make a cross and a circle three or four inches apart on a large card. Closing the left eye, hold the card vertically about 10 inches from the right ej'e and so as to bring the cross to the left side of the circle. Look steadily at the cross with the right eye when both cross and circle will be seen. Gradually bring the card toward the eye, keeping the axis of vision fixed on the cross. At a certain distance, the circle will disappear; i. e. , when the image falls on the entrance of the optic nerve. On bringing the card nearer, the circle reappears, the cross, of course, being visible all the time. Expt. CXXXII. Mapping out the Blind Spot. (a). Close the left eye and look steadily at a cross on a sheet of paper held about 10 inches from the right eye. Place the blackened point of a glass tube or point of pencil near the cross and gradually move it to the right until the black becomes in- visible, keeping the head steady and the axis of vision fixed. Mark this spot. Carry the point further outward until it becomes visible again. Mark the outer and inner limits of the blind spot. Begin again moving the pencil, first in an upward, then in a downward direction, in each case marking where the pencil becomes invisible. If this be done in several diameters, an outline of the blind spot is obtained. (6). To Calculate the Size of the Blind Spot. Let f=the distance of the ej-e from the paper. F=the distance of the second nodal point from the retina (about 15 mm.) d=the diameter of the sketch of the spot drawn. D=the corresponding size of the blind spot. d D Then -^^^ Therefore, D=Fd/ f. Expt CXXXIII. Description of the Opthalmoscope. A tilting mirror for reflecting raj's into the subject's eye. A large disc of convex lenses of seven different dioptric powers ranging from 1 D to 7 D, marked in white figures. These are separated from the eight concave lenses of-1 D to -8D diopters, — marked in red. A second disc of two convex lenses 16 D and 0.5 D marked in red figure^. These can all be rotated in fr^^nt of the hol^ in the mirror. 90 Outlines of Experimental Physiology By adding the 0.5D to those of the large disc we can get one or more whole diopters with a half diopter. "With the 16 D in front of the opening and rotating the lenses of the large disc we get as high as 23 D with the convex and -24 D with the concave lenses. Ophthalmoscopic Study with the ArtiHcial Eye and Lantern. (1). Direct Method, Remove from the lantern the tubes holding the projecting lens. Place the ground glass screen before the condenser. See that the inner tube of the artificial eye is drawn out to line C. The eye is then accommodated for distant vision. Set the eye on a level with that of the observer's near the edge of the table. Place the light on the right side of the artificial eye slightly behind it. Hold the ophthalmo- scope in the right hand close to the right eye at a distance of about SOcm. from the artificial eye and through the mirror's aperture. Hold the elbow close to the side and the head vertical, so that the observer's eye and the artificial may have the same visual axis. Keep reflected light on the pupil of the artificial eye. With the pupil illuminated approach the artificial eye until the lens-bearing disc lies in the anterior principal focus (50 mm. in front of this eye and 15. mm. in front of the cornea of the human eye). Artificial eye accommodated for a distant object. The observer's eye is also accommodated for distant vision. The power of volun- tarily relaxing the ciliary muscle is obtained b}' practice. The observer should try to look through and beyond the eye at some distant object or place the -3D be- fore his eye. If the observer be myopic or hypermetropic, his refractory error should be corrected by placing the appropriate lens before the opening of the mirror. As the eye is approached, the details of the fundus will come into view. Find the optic disc. The image of the branch of the central artery and vein is virtual and magni- fied about 16 times and erect. The apparent size of an object held 10 inches from the eye would give a retinal image 1.5 mm. (the size of the optical disc) which can be found. B, apparent size of the optic disc. B:1.5::250:1S. 10 inches=250 mm. B 2 5^0 1.5. B=25 mm. (2). Indirect Method. Arrange the light and artificial eye as directed for the examination by the direct method. Hold the ophthalmoscope 30 cm. from the artificial eye. With the other hand hold a convex lens 20 D, at its own focal length of 50 mm. in front of the cornea. The rays returning from the fundus, pass through this lens, and form an image in the air between the observer and the lens. Examine this image through a mag- nifying glass of-)-5D placed behind the aperture of the mirror. If the observer be myopic, in moderate degree the aerial image will be near his far point and he will need no magnifying or correcting glass; if the myopia be excessive, a weak concave glass should be used. If the observer be hypermetropic, the degree of his hyper- metropia should be added to the focal distance of the magnifying glass. The con- fusing bright reflexes from the surface of the 2D lens may be avoided by holding the lens slightly oblique to the optical axis. Subject's eye and 20D lens form a re- fracting system like the objective of the compound tnicroscope. The 6phthalm6scope lens plays the part of the ocular. The image is real, inverted and magnified, but Outlines of Experimental Physiology 91 appears upright. B, size of the aerial image is to b, size of optic disc (1.5 mm.) as the focal distance of the 20 D lens D, (50 mm.) is to D, the distance from the nodal point to the retina (15 mm.). B:1.5-:50:15. (3). With the aid of the condenser study the fundus of a fellow student's eye with the ophthalmoscope, both with the direct and indirect methods. State your ob- servations. Expt. CXXXIV. Positive After Images. Rest the retina by closing the eye a minute. Suddenly look for two seconds at an electric light covered with a white globe. Then close the eye. An image like the one looked at will be seen. Try in a dark room. Expt. CXXXV. Negative After Image or Successive Contrast. (1), Rest the retina and then stare steadily for half a minute or so at a white square or a white cross on a black ground To insure fixation of the balls, make a small mark in the center of the white paper and fix this steadily. Then suddenly slip a sheet of white paper over the whole. A black square or cross will appear on the white background. (2). After looking at the square or cross, close the eyes. Can you see the nega- tive after image? (3). Stare intentlj' at a bright red square on a black surface for twenty or thir- ty seconds. Suddenly interpose a white surface. Observe the color of the after- image. Try blue, yellow, and other colors. In each case, the negative after image is of the complementary color. (4). Try moving the eyes in their sockets to see whether the after image is due to a condition of the brain or in the eye. Expt. CXXXVI. Testing Color Vision. (a). The palest shade of green (neither yellow nor blue-green) is selected as a test. Select all the other skeins of the same color or one or more confusion colors. (Those which a color-blind person picks out according to defect. These may be grey, light red or light purple). The fact that any confusion color is picked out, shows that he is color blind. (b). Determination of the Kind and Degree of Color Blindness. The second test is a medium purple. The test is continued until all of the pur- ples or certain confusion colors have been selected. A person proving color blind with the first test, but who selects purples, blue and violet, is completely red blind. If he selects green and gray with purple, he is completely green blind. (c). Third test. Select a shade of medium yellowish red. The red blind chooses with reds, greens and browns of darker shades. The green blind chooses with reds, greens and browns of lighter shades, Expt. CXXXVII, Color Vision. Mixing White and Black. (1). Place on the spindle the graduated wheel and then white and black disc, so arranged as to show a part of each. Rotate with sufficient velocity to cause blending. Note the resulting shade. All mixtures of black and white form grays. In other words, gray is white of less intensity. Us- ing black, and white discs and the speed, tester, ascertain the number of revolutions (interruptions) just sufficient to produce blending. 92 Outlines of Experimental Physiology (II). Talbot-Plateau Law, etc (I). Use disc No. 1. Note that the propor- tion of white and black are equal in all the circles but differently distributed. Rotate and observe the greys produced. Are they alike? (2). Mix any two colors; using just enough speed to insure a uniform color. Increase the speed. Does the color produced depend on (1), the number of interrup- tions, (2), the manner of distribution of the components, or (3), the proportion of the components? Expt. CXXXVIII. Complementary colors. See Bradley, pp. 50-53. Place on the wheel the graduated circle, the medium-sized R, C, and B wheels and the small W and N wheels. The problem is to pi'oduce by mixing R, G, and B the same gray as by mixing W and N. In this case the proportions should be about R, 41>^ ; B, 22^2 ; G, 36, in the larger disks, and W 15; N 86; in the smaller. Having approxi- mated the grays as nearly as possible, take out the red and combine the G and B to make the full circle, but in the same relative proportion as were found above; (e. g., as 22 Yz is to 36). Rotation of this combination will give the complementary of R. It is greenish-blue. Test this by the negative after image method. Expt. CXXXIX. The Skin as a Sense Organ. (1). Using a metal point cooled in ice determine the distribution of cold points on various parts ot the skin. Make careful diagrams of the back of the hand, palm and forearm. (2). Using a hot point, determine in the same way the distribution of the wai-m points. Make diagrams as in (1) of the size so that they may be superim- posed. Which points (cold or warm) are most sharply defined? Why? Which are more numerous? Have they any relation to hairs? (3), Place a piece of wood and a piece of iron on ice until they are thoroughly and equally chilled Which feels the colder? Why? Repeat, using heat instead of cold. Test the sensation caused by dipping the finger into water and mercury at the same temperature. (4). Arrange two vessels of water so that the temperature of the water may be changed at will and determine it accurately by means of thermometers. In these, heat or cool two metal points and by applying them to the skin, determine the smallest difference in teitiperature which can be appreciated by the skin of various parts of the body Is the difference greater or less when the points are warmer or colder than the skin? Make tabulations, (5). Is there a latent period in the appreciation of cold and heat? Which is the larger? (6/. Hold one hand in water at 15° C and the other in water at 40 OQ. After several minutes, plunge both hands into water at 25 ° C. Explain the sensation, Expt. CXL. Hearing. (1). Hold a ticking watch between your teeth, close both ears and observe that the ticking is heard more plainly. Why? Unstop ohe ear. In which ear is the sound then the louder? (2). Hold a vibrating tuning fork to one ear until it is no longer heard. Now place it on the incisor teeth until you no longer hear the sound; now close both ears. How about the sound in each case? > Outlines of Experimental Physiology 93 (3). Find where the ticking of a watch just vanishes'as the "watch is moved away; measure the distance from the ear. Also find the point where the tick can just be heard as the watch is brought nearer; compare the two distances (II'. Direction of Sound. Blindfold a person and test his sense of direction of sound. Expt. CXLI. Taste. It is well not to smoke on the day previous to performing these experiments. At least the student should not smoke on the same day that he performs his experi- ments. It is best in the following experiments that two students work together. The experimenter should get his results from the statements made by the student worked upon, who should not be allowed to know what solution he is given to taste. (1). Let the student experimented upon, thoroughl3' rinse his mouth with dis- tilled water. Give about 4 cc. of M/200 hydrochloric acid What is the taste? Give the same amount of M/ 100 sodium chloride. Has it a sour taste? What de- termines the sour taste of HCl? Give a 4 cc. of M/400 Sulphuric acid; of M/400 nitric acid. What is the taste of these? What gives the sour taste to acids? Begin with an M/1000 HCl solution and gradually increase the strength. Where do you get the first taste of the H ions? Is the first taste a sour one? (2). Taste an M/200 sodium hydrate solution. What is the taste? Has an M/200 NaCl solution, a similar taste? Taste an M/200 potassium hj'drate solu- tion. What determines an alkaline taste? Find the weakest solution in which you can recognize the OH ions. (3). What is the taste of an M/25 sodium chloride solution? Has an M/25 sodium acetate solution the same taste? What determines the salty taste of sodium chloride? Try an M/50 and stronger solutions of sodium iodide and sodium brom- ide. Have these a salty taste? Compare with equimolecular solutions of sodium acetate. What is the taste of halogen ions? (4;. Taste. Dry the tongue thoroughly and place on the tip a crystal of sugar; or on the back, one of quinine sulphate. Neither will be tasted until it is dissolved. (5). Apply with pointed non-polarized electrodes a constant current to the tongue. A distinct sensation of taste will be felt. (6). Determine in this way the relative sensitiveness to taste of the various parts of the tongue. Make a diagram. Can different sensations be obtained by stimulating different regions of the tongue? Where is sensation most acute? Re- peat with dilute acetic acid. Also with sulphate of quinine and common salt. (7). After-tastes, (a). Make a solution of sugar which has no sweet taste, wash the mouth out with salt water and then test the sugar solution. (b). Wash the mouth with dilute sulphuric acid. Even distilled water then gives the sensation of sweetness. Expt. CXLII. Study of the Larynx. Draw a side and longitudinal view of the human larynx, illustrating the glosso-epiglottidean fold: lip and cushion of the epiglottis, cartilages of Wrisberg and Santorini; vocal cord; ventricular band; processus vocalis; cricoid cartilage and arytenoid commissure. Experiment on a living person. M Outlines of Experiraental Physiology (a). Place the person uprig^ht iu a chair. A good artificial light with a con- dmser is plaxied near the side of the subject's head a little above the level oi the month. The observer seats himself opposite and close to the patient, places the large mirror on his forehead and either looks through the central hole in it with one eye or raises it so that he can just see under itss lower edge. Seated in front of the subject, the obser%'er directs a beam of light so that the lips of the patient are brightly illuminated. The subject is then directed to incline his head slightly backward to open wide his mouth and to protude his tongue. Place a clean nap- kin over the toague and have the student hold it himself or use the tongue depressor and 0.2 per cent cocaine spray. Move the head mirror until the throat and uvula are brig^htly lighted. Note the tonsils Take the small laryngeal mirror in the right hand and warm it gentlj- over the lamp to prevent the condensation of moisture on its surface. Test its temperature on the cheek, lest you burn the patient. Holding the handle of the mirror as one does a pen, rapidly carry it horL&ontall^' backwards, avoiding contact with any structure in the mouth, until its back rests against the base of the uvula. At the same time direct the light upon the laryngeal mirror when an inverted image of the larynx will be seen more or less perfectly. By mov- ing the laryngeal mirror, not however pressing to much on the uvula or continuing ibe observation for too long a time one may explore the whole of the larynx. Per- haps only the posterior part of the dorsum of the tongue is seen at first; if so, slightly depress the handle of the mirror when the curved fold of the slightly yellowish epiglottis and its cushion with the glosso-epiglottidean folds come into view. In the middle plane are the true vocal cords which are pearly white and shining and best seen when a high note is uttered; and between the chink of the glottis. Above these are the false vocal cords which are red or pink, the ary-epi- glottidean folds, with the cartilages of Wrisberg on each side furthest out, the cartilages of Santorini internal to this, and the arytenoid cartilages near the middle plane. Ask the patient to sing a deep and then a high note, or to inspire feebly or deeply and observe the change in the shape of the glottis. On uttering a deep note, the tracheal rings may be seen. N. B. What is fieen by the observer in the laryngeal mirror on his right cor- responds to the patient's left and vice versa. Also the lower part of the mirror gives an image of the more posterior structures, while the anterior structures are reflected in its upper part. Glosso-epiglottidean fold, three ridges on the pharynx side of the epiglottis, like thefrenumof the tongue, cushion of epiglottis, thickening on larynx side of epiglottis bantorini—Comiculum laryngis. Wri»berg=Cuneiform cartilage. Material;— Cocaine, cotton, light, carbolic, towel, cuspidflar. COLUMBIA UNIVERSITY LIBRARIES This book is due on the date indicated below, or at the expiration of a definite period after the date of borrowing, as provided by the rules of the Library or by special arrange- ment with the Librarian in charge. DATE BORROWED DATE DUE DATE BORROWED DATE DUE ' C28(i14i)m100 QP44 'Hyde fi99 Outlines of « H^^