in tl|? (Hxt^ of Npm f nrRT 
 
 S^ftr^nr^ Htbrary 
 
Digitized by the Internet Archive 
 
 in 2010 with funding from 
 
 Open Knowledge Commons (for the Medical Heritage Library project) 
 
 http://www.archive.org/details/laboratoryguideiOOedmu 
 
Laboratory Guide 
 
 IX 
 
 Experimental Pharmacology 
 
 BY 
 
 CHARLES W. EDMUNDS, A.B., M.D. 
 
 PROFESSOR OF MATERIA MEDICA AND THERAPEUTICS 
 IN THE UNIVERSITY OF MICHIGAN 
 
 AND 
 
 ARTHUR R. CUSHNY, A.M., M.D., LL.D., F.R.S. 
 
 PROFESSOR OP PHARMACOLOGY IN UNIVERSITY COLLEGE, 
 
 LONDON ; AND LATE PROFESSOR OF MATERIA, 
 
 MEDICA AND THERAPEUTICS IN THE 
 
 UNIVERSITY OF MICHIGAN 
 
 George Wahr 
 
 publisher and bookseller 
 
 ann arbor, mich. 
 
CoPYRIGHTj 1918 
 
 GEORGE WAHR 
 Ann Arbor, Mich. 
 
 
 THE ANN ARBOR PRESS 
 ANN ARBOR 
 
PREFACE TO 1918 EDITION. 
 
 In the years which have elapsed since these notes were 
 first put into permanent form the book has been placed in 
 service in different pharmacological laboratories over the 
 country, and in this revision which the progress of the sci- 
 ence makes necessary, the suggestions which have been re- 
 ceived from teachers of the subject have proved most 
 welcome and helpful. It would seem that the wish ex- 
 piressed in the preface to the first edition that these notes 
 might prove useful to others has been realized. 
 
 It need hardly be pointed out that the object of the book 
 is not to furnish an exhaustive treatise on laboratory meth- 
 ods, but rather a simple outline of experiments which can 
 easily be carried out by the average medical student in any 
 laboratory equipped for the purpose. That the arrange- 
 ment is entirely practical and workable is proved by the fact 
 that these directions essentially as here published, have 
 been in use in this laboratory for considerably over twenty 
 years. 
 
 The more important changes which have been made in 
 this revision may be mentioned shortly. The section on 
 pharmaceutical work has been enlarged by the introduction 
 of directions for making a few preparations other than those 
 already included in the earlier edition of the book, and all 
 of these directions have been collected and placed in the 
 early part of the book. This will allow of the omission of 
 this section entirely in schools in which medical students are 
 given a special course in pharmacy. In case no separata 
 course in this subject is given the students, the devotion of 
 
4 LABORATORY GUIDE IN 
 
 a few hours to such work as is given herein wih prove very 
 instructive. 
 
 In the pharmacological work proper a section on biological 
 assays has been' introduced, and in addition directions for 
 work on isolated tissues and organs. Attention might be 
 called also to the experiment on the action of "Digitalis in 
 auricular fibrillation" on account of its importance from the 
 clinical standpoint. The number of drugs studied has not 
 been increased as it has been felt that if the student worked 
 out thoroughly the action of one drug as typical of a group 
 he could work out the other members. Such an arrange- 
 ment tended to simplicity and to economy in time and ani- 
 mals. 
 
 Finally, attention may be called to the fact that in the 
 earlier pharmacological work the use of apparatus is re- 
 duced to a minimum, the effect of this being to train the 
 student's powers of observation. The making of accurate 
 protocols of the experiments in the laboratory should be 
 emphasized as much as the making of good clinical notes 
 in the hospital, and the former may v;ell serve as a train- 
 ing for the latter. 
 
 C. W. Edmunds. 
 Ann Arbor, Mich., 
 
 Jan. 1st, 1918. 
 
PREFACE TO THE FIRST EDITION 
 
 The following course has been gradually developed in the 
 pharmacological laboratory of the University of Michigan, 
 and it has been suggested that if put in a more permanent 
 form than hitherto it might be helpful to other teachers of 
 this subject. 
 
 The practical course has hitherto been given as an intro- 
 duction to the didactic lectures and it has been found advis- 
 able to limit the number of drugs examined to those possess- 
 ing the most typical action. The object has been to train the 
 student entering on the subject in the method of work and to 
 impress on him that the study of drugs is to be approached 
 in the same objective way as other branches of medicine. 
 
 The order in which the experiments are arranged is a 
 purely arbitrary one, necessitated by the conditions of the 
 laboratory, and can doubtless be modified with advantage to 
 suit other classes. 
 
 A few days are devoted to purely pharmaceutical work, 
 but the chief weight is laid on the experimental part of the 
 course. The few hours devoted to pharmacy, however, train 
 the student in observing the characters of the much larger 
 number of drugs met in the lecture course, and have been 
 found to be of considerable value. A beginning may be 
 made in prescribing as occasion offers, but this, as well as the 
 therapeutic applications of the results obtained in the phar- 
 macological experiments, is better left to the teacher than 
 expounded in the laboratory directions. 
 
 Arthur R. Cushny. 
 CHARiviiS W. Edmunds. 
 
 Ann Arbor, March i8th, 1905. 
 
!!(' ! \' 
 
 CONTENTS. 
 
 The Pharmacopoeia 9 
 
 General directions for anaesthetics and operations. ... ii 
 
 Physiological solutions for animal tissues 19 
 
 Chemistry of drugs '. 20 
 
 Percolation 44 
 
 Powders ' 5 ^ 
 
 Capsules 55 
 
 Cachets 59 
 
 Pills 60 
 
 Emulsions 67 
 
 Ointments 72 
 
 Suppositories . 76 
 
 Physiology of frog's nervous system 80 
 
 Soporifics or Hypnotics 88 
 
 Nux vomica 96 
 
 Camphor group 112 
 
 Opium 116 
 
 Curara 127 
 
 Nicotine 132 
 
 Veratrine ■ 135 
 
 Caffeine 136 
 
 Cocaine 143 
 
 Belladonna 147 
 
 Cinchona 156 
 
 Anaesthetics . 160 
 
 Digitalis series 168 
 
 Aconite 183 
 
 Drugs on the frog's heart 187 
 
 Drugs on the turtle's heart 191 
 
8 CONTEXTS. 
 
 Drugs on the blood pressure 200 
 
 Drugs on the mammahan heart 207 
 
 Analysis of tracings 215 
 
 Digitalis in Auricular Fibrillation 220 
 
 Perfusion of blood vessels 227 
 
 Action of drugs on isolated tissues 232 
 
 Diuresis 239 
 
 Drugs on the sympathetic system and on intestinal 
 
 peristalsis 247 
 
 Salivary and pancreatic secretion 255 
 
 Antipyretics 267- 
 
 Biological assay 272 
 
 Pilocarpus 283 
 
 Nitrites 287 
 
 Suprarenal extract 288 
 
 Pituitary extract 291 
 
 Intestinal peristalsis 292 
 
 Vasomotor effects in the intestine 299 
 
 Vasomotor effects in the kidney 304 
 
 Perfusion of isolated frog's heart 311 
 
 Perfusion of isolated mammalian heart 315 
 
 Drugs and reagents required 317 
 
The Pharmacopoeia. 
 
 When a physician prescribes a certain drug from time to 
 time in his practice it is of greatest importance to him to 
 know that his patients are always getting the same prepara- 
 tion, containing the same active principles in uniform 
 strength. These requirements could not be carried out if 
 there were not some standard recognized by the manufac- 
 turers and followed by them in the preparation of the vari- 
 ous medicinal remedies. Most countries possess such a 
 standard in their "Pharmacopoeias," which are generally 
 published by the government. In this country, the govern- 
 ment does not issue the pharmacopoeia, but it recognizes its 
 authority. The book is revised every ten years by a Commit- 
 tee of Revision composed of members appointed or elected 
 by a convention of various medical and pharmaceutical so- 
 cieties and colleges. 
 
 It need hardly be remarked that in such a work only such 
 remedies are included as have become well established. 
 These are designated as "ofhciaF' remedies, while drugs not 
 mentioned are "non-ofhcial." 
 
 The pharmacopoeia gives, first, the Latin title of the drug- 
 followed by the English name, and the official abbreviation, 
 and in the case of chemicals the formula and molecular 
 weight are given. Any synonyms are mentioned and 
 wherever it is necessary, a short, concise definition of the 
 drug is added, telling its nature and its source. The book 
 then gives tests by which the identity of the drug may be 
 recognized, and its purity and strength tested and finally 
 mentions in what ways and in what doses it may be ad- 
 ministered. 
 
lo LABORATORY GUIDE IN 
 
 Official preparations are therefore made according to the 
 directions given in the pharmacopoeias and they should 
 possess uniform strength and properties ; such an ideal, 
 however, is hard to attain, as some of the crude drugs, even 
 when collected according to pharmacopoeia! directions, dif- 
 fer in their content of the active constituents. 
 
. BXPBRIMBNTAL PHARMACOLOGY n 
 
 General Directions. 
 
 Before giving the specific directions for the experimental 
 work a number of general directions are given for the com- 
 moner operations which are carried out on animals some of 
 which are usually necessary in subsequent manipulations. 
 
 The various anaesthetics employed in experimental work 
 are also described. 
 
 Anaesthetics. 
 
 No animal is to be operated on unless it is under complete 
 anaesthesia. Ether and chloroform may be used for this pur- 
 pose, but have the disadvantage of requiring constant atten- 
 tion. They are therefore only used to supplement other 
 anaesthetics which experience has shown can be used to give 
 full anaesthesia in animals without the necessity of their be- 
 ing continually administered. For this purpose the follow- 
 ing combinations of drugs usually give very good results, al- 
 though in rare cases chloroform or ether may have to be 
 given in addition. 
 
 Anccsthctic for Rabbits. 
 
 Paraldehyde, 1.7-2.00 cc. per Kg. of body weight. 
 
 While your assistant holds up the animal by all four legs 
 and head, place a gag in the mouth and pass a stomach tube^ 
 through the opening in the gag, being very careful not to 
 pass it into the lungs. Draw the paraldehyde into a pipette 
 and place the point of the pipette in the opening of the 
 stomach tube and blow the drug gently into the stomach. 
 
 * A soft rubber catheter serves very well as a stomach tube for rabbits and 
 cats, a size of lo or 15 F. beinR most satisfactory. These catheters will pass 
 mcire easily if they are first moistened with water. 
 
12 
 
 LABORATORY GUIDE IN 
 
 and then withdraw the tube. If after waiting 15 minutes 
 the animal is not completely anaesthetized ether may be ad- 
 ministered cautiously or a second small injection of par- 
 aldehyde given. 
 
 In place of paraldehyde urethane in doses of i G. per 
 Kg. of body weight may be used as an anaesthetic. The 
 drug is dissolved in water and given by the stomach tube. 
 
 Anesthetic for Cats. 
 
 Chloretone, 0.3 G. - 0.4 G. per Kg. of body weight. 
 
 Dissolve the chloretone^ in as small a volume of alcohol 
 as possible and add about an equal volume of water. Place 
 the animal in a cat box- and administer the chloretone so- 
 lution by means of the stomach tube. The stomach tube 
 is passed through a hole in the center of a gag which is 
 firmly held between the jaws of the animal by an assistant. 
 
 Anccsfhetic for a Dog. 
 
 Morphine sulphate, o.i G. to 0.25 G. Chloretone, 3 G. 
 to 4 G. 
 
 • One or two hours before the animal is needed inject the 
 morphine solution under the skin by means of a hypodermic 
 syringe. 
 
 ^ It is convenient to keep a stock solution of chloretone in 95% alcohol on 
 hand so that the required amount of drug may be measured out quickly. A 
 useful strength is the one in which one gram of chloretone is contained in 
 each 3 cc. of the finished solution. 
 
 ^ A cat box is of the greatest service as a means of protection to the op- 
 erator whenever this animal is used. It consists of an ordinary wooden box 
 about 30 or 35 cm. long, 18 cm. wide and 15 or 18 cm. deep. It is provided 
 with a sliding cover, which has a V shaped cut in the middle of one end. In 
 the corresponding end of the box a second V cut is made. When the lid 
 is shut these two cuts leave enough room for the animal's neck. The lid is 
 kept closed tightly by means of a nail passed through the opposite end of the 
 cover into the end of the box. 
 
. EXPERIMENTAL PHARMACOLOGY 
 
 13 
 
 Just before the operation, dissolve the chloretone in 
 alcohol and add about an equal amount of water and give 
 it to the dog by means of a stomach tube^ passed through 
 a wooden gag. Another very satisfactory method of giv- 
 ing the chloretone is to dissolve the drug in 5 or 10 cc. of 
 olive oil which may be warmed to hasten solution. When 
 dissolved it can be drawn up into a large veterinary all- 
 metal syringe. While the animal is lying on its side the 
 abdominal wall can be punctured quickly and the oily solu- 
 tion injected into the peritoneal cavity. Anaesthesia is pro- 
 duced in about 10 minutes by this method. 
 
 Operations on Frogs. 
 
 Pit] ling. 
 
 Before exposing the heart of a frog or carrying out any 
 other operation upon the animal its brain is destroyed 
 (pithed) in the following manner: Hold the frog in the 
 left hand with its back upward, tip the head forward with 
 the left index finger, and feel for the prominent angle made 
 at the junction of the skull and spinal column. Just back 
 of this angle make a cut with small scissors through the 
 skin in the median line. Now push the sharpened end of 
 a match forward through the foramen magnum into the 
 skull, rotating it in the cranial cavity to destroy the brain. 
 
 Injecting into a LympJi Sac. 
 
 In the intact animal, drugs to be tested are usually in- 
 jected into the anterior lymph sac. This is carried out in 
 the following manner: Lay the animal back downwards in 
 
 ^ The average stomach tube, such as is used in clinical medicine, is about 
 the right size for a dog. It should be about 75 cm. long. 
 
14 LABORATORY GUIDE IN 
 
 the palm of the left hand. Hold one of its forelegs firmly 
 between the thumb and index finger and the other foreleg 
 between the middle and ring fingers. Draw its hind legs 
 downward and hold them against the palmar surface of 
 the hand by means of the little finger. 
 
 Having the drug in the glass injecting pipette/ which is 
 held in the right hand, force the animal's mouth open with 
 the point. Pass the pipette into the mouth avoiding ihe 
 tongue, which is attached anteriorly and direct the point to • 
 Vs'ard the floor of the mouth which with a little pressure it 
 will pierce, entering the lymph sac. As it is pushed down 
 the sac the point can be seen beneath the skin of the ab- 
 dominal wall. The finger is now removed from the upper 
 end of the pipette and the drug allowed to flow into the sac, 
 cr if necessary blown in. 
 
 In case the anterior lymph sac has been destroyed, for 
 instance, when the heart has been exposed, the point of the 
 pipette may be pushed onward and made to enter either one 
 of the leg lymph sacs or one of the lateral sacs. 
 
 To Expose the Heart. 
 
 Pith the frog and tie it on its back on the frog board. 
 With a pair of forceps raise the skin in the median line of 
 the body just below the point of the sternum and make a 
 nick in it with a pair of scissors. Starting at this point 
 
 iTo make a glass injection pipette take a piece of glass tubing about 7 mm. 
 in diameter and about 20 or 30 cm. long and heat the middle point in the Bun- 
 sen flame, rotating the tube constantly, and when it is red hot remove it from 
 the flame and draw it out to a capillary size; cut it in the middle so as to leave 
 a fine tube about 8 cm. long. Heat the large end of the pipette to smooth it 
 oft so it will not cut the tongue or lips. 
 
EXPERIMENTAL PHARMACOLOGY 
 
 15 
 
 cut up and outward on each side as far as the pectoral girdle 
 and turn this V-shaped piece of skin upward toward the 
 head. Raise the sternum by placing the forceps beneath 
 the point and with scissors cut it through its entire length 
 
 
 
 
 
 1 
 
 ' 
 
 I. 
 
 M. 
 
 ll 
 
 1 1 
 1 
 
 _ 
 A 
 
 \\'^ 
 
 -^1 \ ^p 
 
 %' ll 
 
 F^ 
 
 I "^^ i^lffP^r? 
 
 p^w***^ ' ' 
 
 V^^S 
 
 if ' 1 ' 
 
 
 ;| m 
 
 f''l 
 
 1 
 1 
 
 1 , 
 
 1 i 
 
 I 1 
 
 1 
 
 
 i 
 
 ral 
 
 : 1 
 
 ', 1 
 
 1 
 
 1 
 1 , 1 
 
 1 
 
 r' 
 
 1( ( 
 
 1 
 
 i 
 
 
 \''I 
 
 k ' 1 
 
 ,1 
 
 ■ i 1 
 
 
 . ' - i ' 
 
 I 
 
 1 A 1 • 1 
 
 Fig. Xo. i. 
 
 avoiding injury to the underlying heart. Tighten the strings 
 on the fore legs so as to pull the sides of the sternum well 
 apart. The heart is now seen enclosed in the pericardium, 
 which may be carefully opened and cut away. 
 
i6 LABORATORY GUIDE IN 
 
 Directions for Operations on Mammals. 
 
 The following are the most common operations necessary 
 in experimental pharmacology. 
 
 To Insert a Venous Cannula. 
 
 This is to allow of the intravenous injection of drugs. 
 
 Two sizes of glass venous cannulas (with rubber tubing 
 attached) are shown in Fig. No. 2. The larger is best suited 
 for a dog, while the smaller is used in a rabbit or cat. Vari- 
 ous sizes should be provided. 
 
 Fig. No. 2. 
 
 Operation. When the anaesthesia is complete make an in- 
 cision through the skin over the site of the vessel and deepen 
 the cut through the fascia until the vein is exposed. Avoid- 
 ing injury to the vessel from forceps or tearing, clear about 
 two inches of the vein, removing all the fascia surrounding 
 it so as to leave it entirely free. Pass two threads about a 
 foot long under the vein and place "bull-dog" forceps on it 
 toward the cardiac end of the cleared portion. This allows 
 distention of the vessel by the blood. Tie one of the threads 
 tightly around the upper end of the cleared portion. Hold 
 
EXPERIMENTAL PHARMACOLOGY 
 
 17 
 
 the end of this Hgature in the left hand and place the left 
 index finger under the vessel, thus making it tense. With 
 sharp scissors make a cut in the distended vein and place the 
 tip of the cannula in it, and tie it in place with the loose 
 thread previously placed around the vessel. Fill the cannula 
 and vein with salt solution (0.8 %), taking great care that 
 ,all air is expelled. 
 
 Precaution. When drugs are injected intravenously it is 
 of the utmost importance to see that no air is allowed to 
 f^nter the veins. A small bubble of air is almost invariably 
 fatal to rabbits. Be sure the cannula and syringe have all 
 the air expelled. 
 
 To Insert an Arterial Cannula} 
 
 This is to allow records of arterial blood pressure to be 
 taken. 
 
 Operation. Expose and clear the artery, usually the 
 ■carotid, in the manner described above for the vein. Pass 
 two ligatures under the vessel and tie the thread which is 
 ■distal to the heart. Now clamp the artery with the "bull- 
 dog" forceps, placing them on the "heart end" of the cleared 
 portion. Place the index finger under the vessel as before 
 and make a cut in it and tie a cannula in place with the loose 
 ligature. This cannula is to be filled with sodium sulphate 
 solution to prevent clotting of the blood.- 
 
 1 Arterial cannulas are exactly like venous cannulas. Fig. No. 2. 
 
 2 Various solutions may be used to prevent coagulation of the blood. Some 
 •of the best are as follows: 
 
 Sodium Sulphate, half saturated. This is very satisfactory and it is non- 
 toxic, but it is rather dirty in case some is spilled. 
 
 Sodium citrate, 5-10%. 
 
 Magnesium sulphate, 25%. Very satisfactory but must be used with care 
 as it is quite toxic in case some is allowed to enter the heart. 
 
i8 LABORATORY GUIDE IN 
 
 To Insert a Tracheal Cannula. 
 
 A tracheal cannula is inserted to allow of artificial respira- 
 tion being carried on in case the chest is to be opened or if 
 the anpesthesia is so deep that the respiratory center is 
 seriously depressed or paralyzed. 
 
 Fig. No. 3. Glass tracheal cannula with rubber tubing attached. 
 
 A convenient form of tracheal tube is shown in Fig 3. 
 They can be made of various sizes of glass tubing so as to 
 fit the trachea of any animal. When in use the piece of rub- 
 ber tubing shown in the cut is partially constricted to give 
 tlie proper degree of inflation to the lungs. 
 
 Operation. Make a .median incision in the neck through 
 the skin and fascia beginning just below the thyroid cartil- 
 age. Expose the trachea by separating the muscles with the 
 fingers or blunt hooks and dissect away all structures from 
 around it. Pass a strong thread under it and then make a 
 transverse cut with the scalpel between two rings about two- 
 thirds of the way through. Into this opening pass the 
 tracheal tube and tie it in place with the loose ligature. 
 
EXPERIMENTAL PHARMACOLOGY 19 
 
 Physiological Solutions for Animal Tissues. 
 
 Physiological Salt Sokition for Frogs 0.65% 
 
 For Mammals 0.9 % 
 
 Ringer's Solution for Frogs : 
 
 NaCl 0.6 % 
 
 KCl 0.0075 
 
 CaClo (crystals) 0.026 
 
 NaHCOg o.oi 
 
 Locke's Solution for Mammals : 
 
 NaCl 0.92% 
 
 KCl 0.42 
 
 CaClo (crystals) 0.024 
 
 NaHCOg 0.015 
 
 Dextrose o.i 
 
 Tyrode's Solution: 
 
 NaCl 0.8% 
 
 KCl 0.02 
 
 CaClo (crystals) 0.02 
 
 NaHCOg 0.1 
 
 Na^HPO^ 0.005 
 
 MgClo 0.01 
 
 Glucose 0.1 
 
20 LABORATORY GUIDE IN 
 
 Chemistry of Drugs. 
 
 Alkaloids. 
 
 Among the most important of the organic compounds em- 
 ployed in medicine is the group of vegetable bases known as 
 Alkaloids. They are found in almost all parts of plants, but 
 in greatest abundance in the seeds and roots. They consti- 
 tute in almost all cases the active constituents of the plants 
 in which they are found and some of them are among the 
 most powerful poisons known. 
 
 The alkaloids contain carbon, hydrogen, nitrogen, and 
 usually oxygen, but in a few instances the latter is not pres- 
 ent. 
 
 In their chemical properties they resemble ammonia very 
 closely, having an alkaline reaction and uniting with acids 
 without the elimination of hydrogen to form salts. These 
 salts differ from the alkaloids in their solubility, and upon 
 this fact is based one method of isolating them in a pure 
 form from the plants in which they are found. 
 
 One of the most familiar alkaloids is quinine, which is 
 derived from various species of cinchona bark, and it may 
 be taken as a type of the group, as the reactions given by it 
 are characteristic of most of the other alkaloids. 
 
 Taste quinine and quinine sulphate. 
 
 I. Test the solubility of a few crystals of quinine 
 (alkaloid) and of quinine sulphate (alkaloidal salt) in lo 
 cc. of each of the following: 
 
BXPBRIMENTAL PHARMACOLOGY 21 
 
22 
 
 LABORATORY GUIDE IN 
 
EXPERIMENTAL PHARMACOLOGY 
 
 23 
 
 Solvent. 
 
 Alkaloid solubility. 
 
 Alkaloidal salt solubility. 
 
 Water. 
 Alcohol. 
 Ether. 
 Chloroform. 
 
 II. Add about 100 cc. of water to 0.2 G. quinine. Shake 
 the flask and observe only a small part goes into solution. 
 Test the reaction of the solution with litmus. How do you 
 explain the reaction? 
 
 Add I cc. dilute sulphuric acid and shake until complete 
 solution takes place. The sulphuric acid formed the bisul- 
 phate of the alkaloid. (Note the fluorescence of the solution 
 which is characteristic of quinine.) Use this solution in 
 making the following tests. 
 
 III. To 5 cc. of the solution add slowly potassium car- 
 bonate solution until a distinct alkaline reaction is obtained. 
 What happens? Render the mixture acid with dilute sul- 
 phuric acid. 
 
 Alkaloidal salts are very feeble bases and are thrown out 
 of solution by the addition of the fixed alkalies and their 
 carbonates which unite with the acid of the alkaloidal salt. 
 The precipitate therefore was the freed alkaloid which is 
 insoluble in water and which on the addition of the acid 
 formed the sulphate (or bisulphate) again, which passed 
 into solution. 
 
 IV. To 5 cc. of the solution add potassium hydroxide 
 until the reaction is alkaline. Add distilled water until the 
 test tube is nearly full and then shake the mixture and see if 
 the precipitate dissolves. 
 
24 
 
 LABORATORY GUIDE IN 
 
 The precipitate here, as in No. HI, is the free alkaloid. 
 Ihe alkaloids are spoken of as being insoluble in water, but 
 this is only relatively correct, as they are soluble in a very 
 large excess ; in the case of quinine in t6oo parts of cola 
 water. 
 
 In the first parts of experiments III and IV it was seen 
 that an alkaloidal salt is thrown out of solution by the fixed 
 alkalies and their carbonates. For this reason they should 
 not be prescribed together in the same solution, because the 
 alkaloid would fall to the bottom of the bottle and even if 
 it were shaken each time before a dose was taken the chances 
 are that the last dose in the bottle would contain a much 
 larger amount of alkaloid than was intended, and alarming 
 if not fatal symptoms might result. The same objection 
 holds in regard to prescribing alkaloids and tannic acids to- 
 gether in solution (see Exp. V a). 
 
 This incompatibility of alkaloids and alkalies may be over- 
 come by the presence of a sufficient amount of alcohol. 
 
 V. Put 5 cc. of the solution into each of 5 test tubes and 
 add slowly small quantities of the following reagents ; note 
 the formation of any precipitate and if any occurs test its 
 solubility by adding water and shaking as was done in Exp. 
 IV. Tabulate the results. 
 
 
 Reagent. 
 
 Precipitate? 
 
 Soluble 
 
 in water? 
 
 a* 
 
 Tannic acid. 
 
 
 
 
 b 
 
 Picric acid. 
 
 
 
 
 c 
 
 Iodine in potassium iodide. 
 
 
 
 
 d 
 
 Mercury-potassium iodide. 
 
 
 
 
 e 
 
 Phosphotungstic acid. 
 
 
 
 
 ^ See experiment VI. 
 
MXPERIMBNTAL PHARMACOLOGY 25 
 
2S LABORATORY GUIDE IN 
 
EXPERIMENTAL PHARMACOLOGY 
 
 27 
 
 The precipitate in (a) was the tannate of the alkaloid; in 
 (b) the picrate ; (c) polyiodide (usually); (d) double 
 iodide of the alkaloid and mercury; (e) phosphotungstate of 
 the alkaloid. These five reagents, with a few others, of which 
 the most important are gold chloride and platinum chloride, 
 are known as the "alkaloidal precipitants" and they are 
 largely used for the detection of the different members of 
 this group. 
 
 VI. Filter the precipitate obtained with tannic acid in. 
 Exp. V a, and wash it with a little water and then taste 
 it comparing its taste with that of quinine sulphate. Qui- 
 iime tannate is sometimes known as tasteless quinine. 
 
 VII. To 10 cc. of the quinine solution add potassium 
 hydroxide until the reaction is alkaline. Now add 5 to 10 
 cc. of chloroform and placing the thumb over the mouth of 
 the test tube shake it vigorously for a minute or two or until 
 the precipitate disappears. Set it aside for a short time until 
 
 , the chloroform settles in the bottom of the tube when it is to^ 
 be drawn off from the water by means of a glass pipette and 
 put into a clean, dry, evaporating dish and set aside to allow 
 the chloroform to evaporate. Also draw off some of the 
 water into a clean test tube and test for the presence of 
 alkaloid by adding a few drops of the solution of tannic 
 acid. Explain the results. 
 
 This experiment illustrates one of the methods used in 
 separating an alkaloid from other bodies which are found 
 present with it in the crude drug. It is known as the "Shak- 
 ing out process" and will be more fully described later. (See 
 nux vomica and belladonna.) 
 
 VIII. Boil 10 cc. of the quinine solution for a few min- 
 utes with a cubic centimeter of sulphuric acid ; neutralize 
 
28 LABORATORY GUIDE IN 
 
 the acid with potassium hydrate and apply Fehhng's test for 
 glucose.^ Is there any reduction? 
 
 Absence of reduction shows no glucoside is present. Con- 
 trast next section (Glucosides). 
 
 Examine specimens of other alkaloids and their salts : — 
 
 Strychnine, from the nux vomica bean (Strychnos nux 
 vomica) . 
 
 Morphine, from opium, the dried juice of the unripe cap- 
 sules of the poppy (Papaver somniferum). 
 
 Caffeine, from the berry of cofifee (Coffea Arabica), and 
 from tea leaves (Thea Chinensis). 
 
 Atropine, from deadly nightshade (Atropa Belladonna). 
 
 Berberine,^ from golden seal (Hydrastis Canadensis). 
 
 Coniine,- from the fruit of the poison hemlock (Conium 
 maculatum) . 
 
 Nicotine,- from tobacco (Nicotiana tabacum). 
 
 Notice that the names of alkaloids end in "ine," in L,atin 
 *'ina," as for example, strychnina, morphina, etc. 
 
 Glucosides. 
 
 Next in importance to the alkaloids among the vegetable 
 poisons rank the Glucosides. These are substances which 
 break up when acted on by dilute acids or ferments, and 
 yield as one of their decomposition products a sugar which 
 in many cases is glucose ; in fact, glucosides may be regarded 
 
 ^ Fehling's test for glucose is carried out as follows: Mix in a test tube 
 equal quantities (about i cc.) of two solutions, one, a solution of copper sul- 
 phate, and the other, a solution of potassium hydroxide and Rochelle salts. 
 The resulting mixture is a clear, dark blue solution, which is to be boiled 
 and the solution to be tested added to it. If glucose is present a red precipi- 
 tate of cuprous oxide will be thrown down after the solution has stood lor a 
 short time. The precipitate may be clearly seen in the bottom of the test tube 
 if a reducing substance is present. 
 
 ''■ Berberine is one of the few colored alkaloids. 
 
 ^ Coniine and nicotine are tlfe two most important liquid alkaloids. They con- 
 tain r.o oxygen and are volatile. 
 
■EXPERIMENTAL PHARMACOLOGY 
 
 29 
 
^o LABORATORY GUIDB IN 
 
BXPBRIMBNTAL PHARMACOLOGY 
 
 31 
 
 as salts of sugar. The other products differ in the different 
 glucosides, in some cases being a volatile oil, in others an 
 alkaloid, etc. 
 
 In the following experiments amygdalin will be employed. 
 It is a glucoside which is found in bitter almonds, laurel 
 leaves and in the bark of the Virginian prune or cherry. 
 
 Make 50 cc. of a 2% solution of amygdalin in water. 
 
 I. Apply Fehling's test for glucose (see page 28, foot- 
 note) to 5 cc. of the amygdalin solution. Is a reducing sub- 
 stance present? 
 
 II. Boil 10 cc. of the amygdalin solution with i cc. of 
 sulphuric acid (dilute) for 10 minutes. Neutralize with 
 sodium hydrate and apply Fehling's test. Do you get any 
 reduction? Compare with Exp. I, and explain. 
 
 III. To 5 cc. of the amygdalin solution add some saliva 
 and place it on the water bath at 40° for half an hour. Test 
 for a reducing substance as in i and 2 and explain results. 
 
 IV. Put about 5 cc. of the amygdalin solution into each 
 of 5 test tubes and test each with the alkaloidal precipitants. 
 
 Reagent. 
 
 Tannic acid. 
 
 Picric acid. 
 
 Iodine in potassium iodide. 
 
 Mercury-potassium iodide. 
 
 Fhosphotvingstic acid. 
 
 Precipitate? 
 
 V. Pulverize three bitter almonds in a dry mortar and 
 place the powder in a small beaker and add about 10 cc. 
 of warm, not hot, water to it. Boil three more bitter almonds 
 for two or three minutes in about 10 cc. of water and then 
 pulverize them in a mortar and add warm water as in the 
 
32 
 
 LABORATORY GUIDE IN 
 
 first instance. Set both mixtures aside for a short time and 
 compare the odors from the two beakers. 
 
 The odor found is due to the oil of bitter ahiionds formed 
 by the decomposition of the amygdahn through the action 
 of a ferment, emulsin, also present in the almonds and act- 
 ing in the presence of water. This oil consists of benzalde- 
 hyde and prussic acid and is therefore extremely poisonous. 
 
 What reaction is characteristic of the glucosides? 
 
 Examine specimens of other glucosides : — 
 
 Salicin, found in the willow and poplar. 
 
 Digitalin, found in the fox glove (Digitalis purpurea). 
 
 Digitoxin, found in the fox glove (Digitalis purpurea). 
 
 The names of glucosides end in "in," in Latin "inum," as 
 for example, salicinum. 
 
 Volatile Oils. 
 
 The group of volatile oils contains a large number of 
 preparations which are extensively employed in medicine. 
 They are also known as the Bssential or Bthcrcal oils. They 
 are found widely distributed in nature as the odor of the 
 various plants and flowers is due to them or to their oxidized 
 products. In their chemistry they differ greatly from the 
 group of fatty or fixed oils from which they must be care- 
 fully distinguished, the latter being non-volatile. The volatile 
 oils are not to be regarded as pure substances, like the alka- 
 loids, but rather as mixtures of varying composition. Most 
 of them are colorless when fresh and pure, but they tend 
 to become colored after long exposure to light and air. 
 In odor and taste they differ widely from one another, but 
 in both cases these properties are generally characteristic 
 of the individual oil. The majority of them are lighter than 
 water, there being only a few heavier. 
 
■BXPBRIMBNTAL PHARMACOLOGY 33 
 
3^ LABORATORY GUIDE IN 
 
nXPERIMUNTAL PHARMACOLOGY 
 
 35 
 
 As a typical member of the group the oil of turpentine is 
 to be employed in the following experiments. As a type of 
 the fatty or fixed oils cotton seed oil will be used to illustrate 
 some of the differences between the two series. 
 
 I. Solubility of volatile oils. 
 
 Into each of five test tubes put i cc. of the oil of turpen- 
 tine and then add 5 cc. of the following reagents. Shake 
 and note whether the oil is soluble. 
 
 
 Reagent. 
 
 Solubility? 
 
 a 
 
 Water. 
 
 
 b 
 
 Alcohol. 
 
 
 c 
 
 Ether. 
 
 
 d 
 
 Chloroform. 
 
 
 e 
 
 Cotton see'J oil. 
 
 
 II. Solubility of fixed oils. 
 
 Into each of 5 test tubes place i cc. of cotton seed oil 
 and then add 5 cc. of the following reagents. Shake and 
 note the degree of solubility as in I. 
 
 
 Reagent. 
 
 Solubility? 
 
 a 
 
 Water. 
 
 
 b 
 
 Alcohol. 
 
 
 c 
 
 Ether. 
 
 
 d 
 
 Chloroform. 
 
 
 e 
 
 Oil of turpentine. 
 
 
 What differences in solubility exist between the two 
 
 series : 
 
 The two groups of oils resemble one another in leaving a 
 greasy stain when dropped on paper, but that from the fixed 
 oil is permanent. 
 
36 LABORATORY GUIDE IN 
 
 III. a. Put a drop of oil of turpentine on glazed paper. 
 Note the stain. Hold the paper from six inches to a foot 
 above the flame of the Bunsen burner for a few minutes. 
 Result ? 
 
 b. Repeat, using cotton seed oil, being careful not tp use 
 enough heat to char the paper. How does it differ from the 
 previous experiment? 
 
 IV. Decomposition product, 
 
 a. Heat 5 drops of the oil of turpentine in a dry test tube. 
 Note the odor. 
 
 b. Heat strongly 5 drops of cotton seed oil in a dry test 
 tube. Note the odor. 
 
 In the latter case acrolein is formed from the partial de- 
 composition of the fat and is recognized by its irritating 
 and disagreeable odor resembling burnt fat. 
 
 V. Add I cc. oil of turpentine to 5 cc. water and shake 
 the mixture well for a minute or two. Allow the oil and 
 water to separate, and then draw off the latter with a pipette 
 into a clean test tube. Note its odor and taste, which are due 
 to the turpentine, of which the water has taken up a small 
 amount, forming a preparation recognized in the Pharma- 
 copoeia as an "Aqua." These waters flavored by the various 
 volatile oils are largely used to give a pleasant taste and 
 smell to medicines. 
 
 The "Syrupi" are solutions of sugar and water to which 
 is often added a small quantity of a volatile oil as in the 
 aquae. 
 
 Spiritiis, spirits, are solutions of a volatile oil in alcohol 
 \^Exp. lb.); they are largely used in medicine as flavoring 
 agents. 
 
liXPBRIMBNTAL PHARMACOLOGY 
 
 37 
 
38 
 
 LABORATORY GUIDE IN 
 
■EXPERIMENTAL PHARMACOLOGY 39 
 
 Examine specimens of the different volatile oils named 
 below and compare them with the oil of turpentine as re- 
 gards odor, taste, color, etc. 
 
 Oleum Menthse Piperitse (oil of peppermint). 
 " Gaultherise (wintergreen oil). 
 " Lavandulae Florum (oil of lavender). 
 " Eucalypti (eucalyptus oil). 
 " Caryophylli (oil of cloves). ■ 
 
 Resins. 
 
 Among the less important chemical compounds used in 
 iJiedicine are the Resins. This is a miscellaneous class char- 
 acterized by the smooth, shining fracture. 
 
 Using powdered resin test its solubility as follows : — 
 
 Reagent. Solubility? 
 
 a 
 
 Water. 
 
 
 b 
 
 Alcohol. 1 
 
 c 
 
 Ether. 
 
 d 
 
 Chloroform. 
 
 
 e 
 
 Oil of turpentine. 
 
 
 Add water to b and explain result. 
 Examine the Resin of jalap. 
 
 Resin of copaiba. 
 Olcoresins are solutions of resins in volatile oils. 
 Examine Turpentine. 
 
 Burgundy pitch. 
 
 Copaiba. 
 Balsams are like oleoresins, but contain also benzoic and 
 cinnamic acids. 
 
 Examine Balsam of Peru. 
 
 Balsam of Tolu. 
 
40 LABORATORY GUIDE IN 
 
 Glim resins are mixtures of gums and resins, containing 
 usually in addition some volatile oil. 
 
 Examine Asafoetida. 
 
 Gums are amorphous transparent substances belonging to 
 the group of carbohydrates. Some dissolve in water v^hile 
 others only .swell to a jelly. They are insoluble in alcohol. 
 
 Examine Acacia. 
 
 Tragacanth, 
 
 Saponins. 
 
 In a large number of plants are found certain substances 
 of a glucosidal nature which because of close chemical and 
 pharmacological relationships have been included under one 
 group known as Saponins or in the case of the more toxic 
 ones, Sapotoxins. Chemically many of them may be ar- 
 ranged in a series CnH^n-sOio ^^'^ they all possess the char- 
 acteristic glucosidal reaction, being decomposed by acids 
 and ferments with the formation of a reducing substance. 
 
 They possess the property of forming a soap-like solu- 
 tion with a very persistent foam when even very dilute 
 v/atery solutions are shaken. It is on account of this prop- 
 erty that the plants from which some of them are derived 
 have obtained their popular names such as Soapwort 
 (Saponaria officinalis) ; Soapbark (Quillaja Saponaria), 
 etc. 
 
 They also aid in the emulsification of fats and in the sus- 
 pension of fine powders and act powerfully in dissolving red 
 .blood cells. 
 
 Using a 2% solution of saponin make the following 
 tests : — 
 
UXPBRIMBNTAL PHARMACOLOGY 41 
 
42 LABORATORY GUIDE IN 
 
EXPERIMENTAL PHARMACOLOGY 43 
 
 I. To I cc. in a lest tube add 5 or 10 cc. of water. Shake 
 well and then set aside noting the persistence of the foam. 
 
 II. To 2 cc. castor oil add i cc. of the saponin solution 
 and shake well. Now add about an equal volume of water 
 and shake thoroughly. A uniform white mixture consisting 
 of the free fat droplets held in suspension by the saponin 
 results. Set the emulsion aside for a short time and see if 
 the oil separates. Later add to the emulsion about half 
 as much alcohol and after shaking set the mixture aside and 
 see if any change results. 
 
44 LABORATORY GUIDE IN 
 
 Percolation. 
 
 The U. S. Pharmacopoeia gives the following definition of 
 this process : "The process of percolation consists in sub- 
 jecting a comminuted substance or mixture of substances, 
 contained in a vessel called a percolator, to the solvent ac-. 
 tion of successive portions of a liquid termed the menstruum 
 in such a manner that the liquid, as it traverses the powder 
 in its descent to the receiver, shall extract the soluble con- 
 stituents and pass from the percolator free from insoluble 
 niatter." 
 
 The liquid with the soluble portions of the drug dissolved 
 in it is known as the percolate. 
 
 The drug to be percolated is usually ground to a fine 
 powder in order to break up the vegetable cells, etc., so that 
 the menstruum can penetrate it easily, and the menstruum is 
 allowed to pass through the drug only very slowly, giving it 
 .time to dissolve the soluble constituents. To still further aid 
 this penetration, a common plan is to moisten the powder 
 with a small quantity of the menstruum and to allow it to 
 remain tightly covered (macerate) for twenty-four or forty- 
 eight hours before beginning the percolation. This also 
 gives time for the drug to swell, as is likely to occur in some 
 cases. 
 
 Percolators^ are usually cylindrical, or conical, and vary 
 in size with the amount of drug to be exhausted. The 
 lower orifice is to be closed with a tightly fitting cork, which 
 is best put in from the inside, but this is not necessary 
 
 ^ For all purposes in this course the cylindrical percolator of the following 
 dimensions is large enough: Length, 28.2 cm.; internal diameter at top, 3.7 
 cm.; capacity, 275 cc. It should be provided with an ordinary iron stand and 
 ring, the latter being padded with cotton. 
 
BXPBRIMBNTAL PHARMACOLOGY 45 
 
^6 LABORATORY GUIDE IN 
 
EXPERIMENTAL PHARMACOLOGY 47 
 
 with the small percolators. Through this cork passes a small 
 glass tube (5 mm. in diameter), the inner end of the tube 
 being flush with the top of the cork. To the outer end of the 
 tube is fitted a piece of rubber tubing. One method of regu- 
 lating the flow of the percolate is to have this tubing of suf- 
 ficient length to reach nearly to the top of the percolator and 
 to have the free end fitted with another glass tube ; the latter 
 can be raised or lowered until the percolate escapes at the 
 desired rate. The other method, which probably is not quite 
 as good, is to have the rubber tube about an inch long and to 
 regulate the flow by means of a small screw clamp. 
 
 Having fitted the cork and tubing the percolator is ready 
 to pack if the drug has macerated the required length of 
 time. The packing is done by means of a plunger, which is 
 made by taking a cork with a diameter about half as great as 
 the diameter of the upper opening of the percolator and 
 sticking into it a glass rod which is a little longer than the 
 depth of the percolator. 
 
 Place in the bottom of the percolator a small piece of ab- 
 sorbent cotton and press it loosely in place. Only a small 
 amount of cotton should be employed, as its only use is to 
 keep the powdered drug from passing down and clogging up 
 the exit tube. Now put in the drug, packing it uniformly in 
 place with the plunger. Do not use much force or the drug 
 will be so tightly packed that it will be very hard for the 
 menstruum to pass through. Very little more force will be 
 required than would be given by dropping the plunger from 
 a height of a few inches. Experience together with a knowl- 
 edge of the physical and chemical constitution of the differ- 
 ent drugs can alone teach how much force to employ. When 
 the drug is in place cover it with a piece of filter paper cut 
 
48 LABORATORY GUIDE IN 
 
 so as to roughly fit its upper surface and weight it down 
 with a small glass stopper. Pour in the menstruum to the 
 top of the percolator and cover the latter with a small glass 
 plate. If the lower orifice is open the menstruum will soon 
 penetrate through the drug and begin to drop from the rub- 
 ber tube. In cases in which the formula prescribes that the' 
 drug is to macerate for a certain length of time in the perco- 
 lator, the clamp is closed, or if a long tube has been used the 
 end is raised to a level which will prevent any outflow. At 
 the end of the prescribed time allow the percolate to escape, 
 receiving it in a bottle or flask. Regulate the outflow so that 
 it shall not escape faster than ten or fifteen drops in a min- 
 ute. It is best after once starting percolating to keep a 
 layer of menstruum above the surface of the drug so as to 
 prevent the access of air to its interstices. If the latter hap- 
 pens the drug contracts into irregular masses leaving fur- 
 rows and fissures, and when more menstruum is added it 
 will naturally flow through these channels and will not pene- 
 trate the drug evenly. 
 
 The U. S. P. gives general directions for percolation and 
 also in the cases of the individual drugs specifies the size of 
 the powder and the number of grams to be used together 
 with the amount and character of the menstruum. It also 
 gives directions as to the length of time the drug is to mac- 
 erate before percolation. 
 
 Percolation is one of the most important processes em- 
 ployed in pharmacy, as by it are prepared extracts (page 
 147); fluid extracts (page 96); and tinctures (page 168), 
 and it will be used in the manufacture of these preparations 
 in the following pages. In addition it is one of the steps 
 employed in the isolation of alkaloids. 
 
.EXPERIMENTAL PHARMACOLOGY 49 
 
50 LABORATORY GUIDE IN 
 
EXPERIMENTAL PHARMACOLOGY 51 
 
 Powders. 
 
 A very considerable group of medicinal substances is 
 prescribed in powder form. Some substances act better 
 when given in this manner and certain individuals also 
 find them easier to swallow than either pills or capsules. 
 To be best suited for this form of medication the drug 
 should be fairly insoluble so as to avoid taste and should not 
 be given in too large a dose. 
 
 The manner in which powders are dispensed may be 
 learned best by filling a prescription such as the following 
 in which 100 mg. of calomel are to be divided into eight 
 powders. On account of the weight of the calomel the bulk 
 of each powder has to be increased by another powder 
 which is inactive ; in this case, sugar of milk being used. 
 
 R 
 
 Hydrargyri chloridi mitis o.i 
 Sacchari lactis 1.5 
 
 Misce et divide in pulveres No. VIII. 
 Sig. 
 
 Take one powder every 20 minutes until all are taken. 
 
 Weigh out both ingredients and put the calomel into a 
 small mortar and add the sugar of milk, little by little, mix- 
 ing it all the time with the pestle, using only light pressure 
 to avoid the formation of compact layers. From time to 
 time, scrape the powder together into the middle of the 
 mortar. When the two ingredients are thoroughly mixed, 
 empty the powder onto the pill tile and without compress- 
 ing it at all arrange it in an oblong parallelogram, the longer 
 edge being near the scale on the tile. See that the powder 
 
52 
 
 LABORATORY GUIDE IN 
 
 is of a uniform depth over the whole mass and if it is not, 
 scrape some powder very lightly from the high points down 
 to the thinner parts. The powder can now be divided easily 
 into eight equal portions by using the spatula with reference 
 to the scale on the tile. The powders are now ready to be 
 put in papers which are obtained by the dispenser all ready 
 for use. 
 
 Take eight papers and make a narrow fold in each along 
 one of the longer sides. Put a powder in the center of one 
 cf the papers and bring the uncreased edge of the paper 
 over into the fold previously made and then both of these 
 are brought over to the center, creasing the whole paper 
 along the lines formed. The ends of the paper are now 
 creased by pressing them over the sides of a powder box 
 and one end is tucked within the fold of the other. Fix 
 all the powders in the same way and place all in the box, 
 labeling it properly. 
 
nXPERlMUNTAL PHARMACOLOGY 53 
 
54 
 
 LABORATORY GUIDE IN 
 
MXPBRIMBNTAL PHARMACOLOGY 55 
 
 Capsules. 
 
 Capsules are shells of gelatine into which medicinal sub- 
 stances are put in order to cover up the taste. The sub- 
 stances placed in the capsules may be either in solid or liquid 
 form. Empty capsules made of gelatine are purchased by 
 the pharmacist so that the problem is merely one of filling. 
 The process can be best learned by the experience gained in 
 filling one or two prescriptions. 
 R 
 
 Quininas Sulphatis i.o 
 
 Pone in capsulas No. VIII. 
 Sig. 
 
 Take one capsule every three hours. 
 
 To fill this prescription the quinine sulphate is weighed 
 out and placed upon the pill tile and spread out into an 
 oblong parallelogram with the powder of uniform depth. 
 This mass of powder is now divided lengthwise with the 
 spatula and then crosswise in the middle and then on eacli 
 side of the middle, thus making eight equal piles of quinine. 
 The large end or bottom part of the capsule (size No. 2) 
 is now pressed down on the powder repeatedly until all the 
 powder in one of the piles is packed into it and then the top 
 part is placed on the base, thus closing it. The process is 
 repeated with the other seven capsules until all are filled. 
 All traces of quinine must be removed from the outside of 
 the capsules either by wiping carefully with a clean soft 
 cloth or washing them in alcohol and then drying with the 
 cloth. 
 
 There are, of course, various modifications of this method 
 of filling capsules, made necessary by the different char- 
 
56 LABORATORY GUIDE IN 
 
 acteristics of the individual powders which it may be de- 
 sired to place in the capsule. For example, some powders 
 lack adhesiveness and would run out of the shell each time 
 it was raised. Such powders may be put in by means of 
 the spatula or by the use of some form of filling apparatus 
 --many of which are on the market. 
 
 Oily or alcoholic liquids may also be dispensed in hard 
 capsules as for example in the following prescription : 
 
 R 
 
 Olei Terebinthinge 4. 
 
 Pone in capsulas No. VIII. 
 
 For this purpose select eight empty capsules (size No. o) 
 and remove the tops, placing them mouth downwards upon 
 some moist filter paper. This will soften the gelatine at 
 the point of contact with the moisture. Draw up some of 
 the oil of turpentine into a one or two cubic centimeter 
 graduated pipette and taking the bottom part of the cap- 
 sule between the thumb and first finger of the left hand, 
 allow 0.5 cc. of the oil to flow into the capsule, taking care 
 none gets on the outside. Now replace the top, thus clos- 
 ing the capsule, and rotate it between the thumb and finger 
 to seal it by compressing the soften ring of gelatine which 
 was at the mouth of the top portion. Repeat the process 
 with the other capsules. 
 
 Soft or elastic gelatine capsules are also employed in 
 the dispensing of oily liquids. 
 
BXPBRIMBNTAL PHARMACOLOGY 
 
 57 
 
58 LABORATORY GUIDE IN 
 
BXPBRIMBNTAL PHARMACOLOGY 59 
 
 Cachets. 
 
 A convenient mode of prescribing powders, especially 
 those which have a disagreeable taste and are not to be 
 given in too large doses, is by enclosing them in cachets 
 which are small concave discs made of flour. The powder 
 is put between two of the discs and the edges then sealed 
 forming a tight compartment. 
 
 Fill the following prescription: — 
 
 R 
 
 Quininge 'Sulphatis 0.5 
 
 Pone in cachetas No. III. 
 Sig. 
 
 One cachet every three hours. 
 
 Weigh out the qtiinine sulphate and placing it on a pill 
 tile divide it into three equal portions. Holding one of 
 the paste discs in the left hand, by means of the spatula 
 place one of the powders of quinine in the middle of the 
 disc, taking care none of the quinine gets on the edge 
 of the disc. Moisten the edge of another disc with a very 
 httle water, and then place it against the edge of the disc 
 which holds the quinine. Press the two edges together 
 firmly, thus sealing the cachet. Fill the other cachets in 
 the same manner and remove any quinine which may be 
 on the outside. These cachets may be swallowed very 
 easily if they are first softened by being placed in water 
 for a few moments. ' 
 
6o LABORATORY GUIDE IN 
 
 Pills. 
 
 Pills are spherical or ovoid masses of medicinal sub- 
 stances of a size convenient for swallowing. In weight they 
 should not exceed 0.3 G., the average being about 0.2 G. 
 
 This method of administering solid medicinal substances 
 is perhaps more common than any other, partly on account 
 of convenience arid also because substances which have a 
 disagreeable taste may be given most agreeably in this way. 
 Drugs which are corrosive or are very deliquescent or 
 which have to be given in very large doses should not be 
 given in pills. Those which are best suited for administra- 
 tion in this form are powders and extracts. 
 
 The first step in pill making is to form the pill mass, 
 which should be of doughy consistency but not sticky. The 
 mass consists of two parts ; the active ingredients which 
 are to be incorporated in the pill and some substance (the 
 excipient) which will give the proper degree of consistency 
 and adhesiveness. The excipient will vary with the char- 
 acter of the active ingredients. Some of the powdered ex- 
 tracts only require water to give them the proper consis- 
 tency, while dilute alcohol may be needed in other extracts. 
 Some fluid preparations may be made into pills by using an 
 inert powder and on the other hand a non-adhesive powder 
 ma)'' be made into a pill mass by the use of soft extracts 
 or by the use of such an excipient as a combination of 
 glycerin and tragacanth (glycerite of tragacanth). The 
 choice of an excipient is very important, as it largely de- 
 termines the usefulness of pills if they are kept for some 
 time. P'or example, a pill made with acacia as excipient 
 
EXPERIMENTAL PHARMACOLOGY 6i 
 
62 LABORATORY GUIDE IN 
 
EXPERIMENTAL PHARMACOLOGY 63 
 
 may become so hard that it will pass through the body un- 
 changed, while with other excipients the pill may dry out 
 and crumble to pieces after they have been made for a 
 short time. The formation of a pill mass may be learned 
 best by filling the following prescription, which calls for 
 ten pills to be made from a gram of extract of gentian. 
 
 R Extracti Gentianse, i. 
 Divide in pilulas, No. X. 
 
 Counterpoise two slips of paper on the scale pans and 
 weigh out. the correct amount of extract, which is- then 
 transferred to a mortar. (If some of the extract sticks to 
 the piece of paper moisten the opposite side of the paper 
 with water and wait a few moments, after which the re- 
 mains of the extract may be easily scraped off.) 
 
 Make the extract into a pill mass of the character de- 
 scribed above. If it is too hard add a little water or dilute 
 alcohol; if too soft add a small quantity of a powder, such 
 as starch or the powdered extract of liquorice. Triturate 
 the mass thoroughly in the mortar (not in the hands!) so 
 as to get the two substances uniformly distributed through 
 each other. When the mass is completed, work it up with 
 the hands into a short cylinder and transfer it to the pill 
 slab, over which has been scattered a small quantity of a 
 dusting powder, such as lycopodium. Roll out the mass 
 with the wooden roller into an even cylinder of the right 
 length to correspond with the scale on the slab (in this case 
 roll out to 10), taking care that the ends of the cylindrical 
 mass are ''squared up." With the cylinder over the scale, 
 cut it with the spatula into the required number of pieces, 
 taking care that the spatula is always held at right angles to 
 
64 LABORATORY GUIDE IN 
 
 the mass. The Httle sections of the pill mass are now to be 
 moulded with the thumb and fingers into spherical shape 
 and then placed in the pill box with a small amount of ly co- 
 podium and shaken about so that they may be completely 
 covered by powder which will prevent them sticking to- 
 gether. Discard the excess of powder. 
 
 To mask the taste of the pills they are usually coated 
 with some substance such as gelatin, sugar, chocolate, etc. 
 
 Examine the pills after a day or two and see if they have 
 retained their shape or if they have flattened out. Weigh 
 five pills and see how nearly they correspond in weight. 
 
EXPERIMENTAL PHARMACOLOGY 
 
 65 
 
66 LABORATORY GUIDE IN 
 
■ MXPBRIMBNTAL PHARMACOLOGY 67 
 
 Emulsions. 
 
 Emulsions are aqueous preparations in which oils, resins 
 or insoluble powders are suspended by means of gummy 
 substances (emulsifiers). 
 
 The object of emulsification is to render substances in- 
 soluble in water freely miscible in that fluid, so that any 
 disagreeable taste may be more easily disguised and the 
 absorption of the oil rendered more easy. In nature, some 
 emulsions exist already formed, as for example, milk, the 
 juice of plants, the yolk of eggs, etc. 
 
 There are a large number of substances which can be 
 used as emulsifiers, the most important being acacia. Con- 
 densed milk and yolk of egg are superior to acacia for this 
 purpose, but possess the disadvantage of becoming stale. 
 
 In some substances, as for instance in gum resins, the 
 gum needed to form an emulsion is found present with the 
 resin. In the seeds of some plants albuminous substances 
 are found and these act as emulsifiers of the oils present 
 in the seed, rendering the addition of gums unnecessary. 
 
 The various types of emulsions are prepared according 
 to the following directions : 
 
 I. Seed emulsion prepared from bitter almonds. 
 
 Take four or five bitter almonds and remove their skins. 
 This may be done in two ways. They may be dropped for 
 a few moments in very hot water and then in cold, when 
 the skins can be slipped off. The danger with this method 
 is that the boiling water may destroy the ferment, emulsin, 
 which aids in the emulsification. To avoid this, the seeds 
 
68 LABORATORY GUIDE IN 
 
 may be soaked in lukewarm water for half an hour, or until 
 their skins are loosened. 
 
 Break up the seeds and put them in a clean mortar and 
 add a small quantity of water (one part to ten of seeds) 
 and with the pestle rub the seeds up into a thick, creamy 
 paste which shall be free from lumps. More water can be 
 added now, gradually, until a milky solution is formed, 
 which may be strained if necessary. 
 
 II. Emulsion of a gum resin. 
 
 Prepare lOO cc. of the emulsion. of asafoetida according 
 to the U. S. P. 
 
 III. Emulsion of a fixed oil. 
 
 Among the various methods employed in making emul- 
 sions, that known as the Continental is probably the most 
 satisfactory, and is to be used in the following experiment. 
 
 Prepare 60 cc. of a 50% emulsion of castor oil.. 
 
 In this method a primary emulsion or nucleus is first 
 formed with certain proportions of the ingredients and this 
 riucleus can then be diluted with water without the fear 
 that separation will occur. 
 
 The proportion by weight is as follows : 
 
 Oil ' 4 
 
 Water 2 
 
 Gum (powdered acacia) i 
 
 Using this formula, calculate the amount of each ingredi- 
 ent needed and weigh them out carefully in clean vessels. 
 Pour the oil into a clean, dry mortar and add the acacia, and 
 with the pestle stir the two together rapidly until a uniform 
 mixture is obtained. (This must be done quickly, as other- 
 
UXPBRIMBNTAL PHARMACOLOGY 69 
 
yo LABORATORY GUIDE IN 
 
■BXPBRIMUNTAL PHARMACOLOGY 71 
 
 wise the oil and acacia remain in contact too long and the 
 latter tends to become hard and insoluble. The object is 
 merely to get each particle of gum incased in oil.) To the 
 oil-acacia mixture add all the water (as determined by the 
 above formula) and triturate the mixture rapidly until it 
 becomes a thick, white, creamy emulsion. In the trituration, 
 which should take from 3 to 5 minutes, no pressure is 
 needed, but a rapid motion is very important. The pestle 
 should be held loosely between the thumb and first two or 
 three fingers and the motion given not only from the shoul- 
 der and elbow, but also from the wrist and fingers. It is 
 best to move the pestle always in one direction, but it is not 
 essential to do so. 
 
 When the nucleus is complete it can be poured into a 
 bottle and water added to make up the required bulk. It 
 should then be well shaken. 
 
 Such an emulsion may separate after some hours into two 
 layers without showing any separated oil. Shaking will re- 
 store the uniform appearance. Such separation is entirely 
 analogous to that which occurs in milk when the cream 
 rises to the surface. 
 
 Emulsions can be flavored by various agencies, such as 
 the volatile oils, but, as a general rule, some of the less 
 powerful flavors will disguise the taste of the oil better, as 
 for instance, liquorice extract, coffee, vanilla, or chocolate. 
 They may be sweetened by small quantities of sugar or 
 saccharin. 
 
 Acids, alcohol, or glycerin, if added in any except the 
 smallest quantities, cause the emulsion to separate (break). 
 
72 LABORATORY GUIDE IN 
 
 Ointments. 
 
 Ointments are soft fatty preparations which are in- 
 tended for appHcation to the skin. The purpose of an 
 ointment may be merely to act as a protective to the skin 
 or to render it softer and more elastic, in either case the 
 action being merely a local one. Then, too, active sub- 
 stances are sometimes incorporated into ointments in order 
 that they may be absorbed and exert a general or systemic 
 action upon the body. 
 
 In order that ointments may be used for these different 
 purposes, it is evident that substances possessing different 
 properties must be used in their formation. In one in- 
 stance it is not desirable for the ointment to be absorbed, 
 whereas in another case lack of penetrating ability might 
 be disadvantageous. To give these different properties to 
 ointments, there are three substances used most commonly 
 in their formation; namely, vaseline, lard (or lard con- 
 taining benzoin to keep it from becoming rancid), and 
 hydrous wool fat or lanolin. Vaseline is the most un- 
 changeable of the bases, being unaffected by air, moisture 
 or chemicals. However, it is not absorbed well, nor does 
 it give up readily the medicinal agents incorporated in it, 
 so that it is best suited for ointments designed for pro- 
 tective purposes only. Benzoinated lard is very valuable 
 as a base as it will take up about fifteen per cent of water 
 without losing its ointment-like consistency and in addi- 
 tion it readily yields up for absorption any medicinal sub- 
 stances which have been incorporated in it. 
 
 Wool fat is also largely used as it will take up a very 
 considerable amount of water — even an equal weight — and 
 
T^XPBRIMUNTAL PHARMACOLOGY 'j2> 
 
^4 LABORATORY GUIDE IN 
 
UXPBRIMBNTAL PHARMACOLOGY 75 
 
 in addition it is easily absorbed. Its chief disadvantages are 
 that it is not so easily worked as other fats and that it is 
 quite sticky. These imdesirable features may be lessened by 
 incorporating other fats with it in different proportions. 
 
 Prepare thirty grams of belladonna ointment (Unguentum 
 Belladonna) according to the directions given in the U. S. 
 Pharmacopoeia. 
 
 In preparing this ointment note especially the physical 
 characteristics of the lard and the wool fat. 
 
 It is hardly necessary. to say that the finished ointment 
 should be perfectly homogeneous and free from any gritty 
 particles. 
 
 Examine a specimen of vaseline (Petrolatum). Look 
 over also the composition of other ointments than the bella- 
 donna ointment as they are described in the Pharmacopoeia. 
 
76 LABORATORY GUIDB IN 
 
 Suppositories. 
 
 Suppositories are solid medicated substances usually of 
 a conical form and designed for introduction into the vari- 
 ous passages of the body where they will become liquid 
 and exercise a local action or else be absorbed and have 
 a general or systemic effect. They consist essentially, 
 therefore, of some drug and a base with which the medicinal 
 substance is incorporated and which is solid at ordinary 
 temperatures, but which liquifies ' slightly below the body 
 temperature. 
 
 The base most commonly employed is cacao or cocoa but- 
 ter (Oleum Theobromatis) which is obtained from the 
 seeds of the plant Theobroma cacao, from which chocolate 
 is obtained. The melting point of this substance lies be- 
 tween 86° and 95° F. Other bases which are used for spe- 
 cial purposes are glycerin-gelatin and sodium stearate. 
 
 The size and shape of suppositories will depend upon the 
 base employed in their manufacture, and the use to which 
 they are to be put. If they are to be made from the oil 
 of theobroma, the cone shaped rectal suppositories should 
 weigh about two grammes ; the pencial shaped suppository 
 for the urethra about the same amount, and the globular 
 suppositories for the vagina, four grammes. If on the 
 other hand, they are made with glycerin-gelatine as a base, 
 the weights above given must be doubled. 
 
 There are three ways of making suppositories using the 
 oil of theobroma as a base. 
 
 In the fusion process, the oil is gently melted and the 
 medicinal substance incorporated with it while it is still 
 
BXPBRIMENTAL PHARMACOLOGY 77 
 
78 LABORATORY GUIDE IN 
 
EXPERIMENTAL PHARMACOLOGY 79 
 
 warm and as the mass is cooling it is poured into well cooled 
 moulds and allowed to harden. 
 
 In the cold compression method, the mixed drug and 
 base are placed in a special apparatus and under pressure 
 moulded into the required shape. Complete directions for 
 these methods are to be found in the U. S. Pharmacopoeia. 
 
 The third method, and the one usually employed when 
 only a few are to be made is carried out in the following 
 manner, which may be illustrated by filling a prescription 
 calling for three suppositories. 
 
 R 
 
 Acidi Tannici 0.3 
 
 Olei Theobromatis 6. 
 
 Fiant suppositoria No. iii. 
 
 Sig. 
 
 Use as directed. 
 
 These three suppositories are to be made by hand as 
 follows. Put the tannic acid in a mortar and add about 
 an equal weight of the oil, which has been grated. Mix 
 the two thoroughly and then add the remainder of the 
 grated oil, working the mass in the mortar until it be- 
 comes homogeneous and plastic. If necessary to make the 
 mass cohesive, about three drops of oil of sweet almond or 
 of glycerin may be added. Transfer the mass to the pill 
 tile and form it into a cylinder of convenient length and 
 cut it into three equal parts. Form each piece now into a 
 conical suppository by means of the fingers and spatula. 
 They should all be of the same size and shape. Dust them 
 with a little lycopodium or starch to keep them from stick- 
 ing together in the box. 
 
8q laboratory guide in 
 
 Physiology of the Nervous System of the Frog. 
 
 In order to study the action of a drug upon the nervous 
 system of a frog it need hardly be pointed out that it is of 
 prime importance to understand the physiology of the differ- 
 ent parts of that system. To illustrate, a certain drug will 
 depress the cerebrum, while a second drug will stimulate the 
 spinal cord and a third may paralyze the peripheral nerves ; 
 now, only by knowing the normal functions of these di- 
 visions of the nervous system can we recognize and interpret 
 the symptoms arising from an increase or diminution in their 
 activity, and thereby locate the point of action of the drug 
 employed. 
 
 First, examine the normal frog, observing its spontaneous 
 movements and the method of leaping and what degree of 
 irritation is necessary to make it move. Note also its co- 
 ordination of the muscles in swimming; its recovery of bal- 
 ance when slowly turned on a sloping board, its recovery 
 v/hen placed on its back, its reflexes when the toe is pinched, 
 the croaking when its back and sides are stroked, the rate of 
 respiration and heart beat. (The latter can usually be 
 counted when the animal is turned on its back and placed in 
 a good light, a slight shadowy movement of the skin being 
 seen in the cardiac region with each pulsation of the organ.) 
 
 Having studied the intact animal, separate the cerebral 
 hemispheres from the optic lobes by thrusting the point of 
 a scalpel through the roof of the skull on a line joining the 
 posterior borders of the eyelids, gently rocking the scalpel 
 from side to side so as to completely sever the hemispheres 
 from their inferior connections. As will be seen from the 
 
-BXPERIMENTAL PHARMACOLOGY 8i 
 
82 LABORATORY GUIDE IN 
 
EXPBRIMENTAL PHARMACOLOGY 
 
 83 
 
 accompanying figure, ;:uch a cut 
 will also destroy the optic thalami 
 and some symptoms will result 
 from such destruction, but for all 
 purposes here, the differences in 
 results obtained are not important. 
 After allowing the animal a few 
 minutes to recover from the effects 
 of the shock of the operation 
 (which must be done in all cases) 
 compare its condition with that of 
 the normal animal. 
 
 Make full notes of all changes 
 seen. 
 
 Next remove the optic lobes 
 from the same animal by cutting 
 as before with the point of a 
 scalpel on a line joining the cen- 
 ters of the tympanic membranes. 
 These membranes are seen on the sides of the head posterior 
 to the eyes as two fiat circular surfaces about 3mm. in 
 diameter. Wait a short time as before and then study the 
 effects of the operation. Hang up the animal by means of 
 a wire hook passed through the lower jaw and observe the 
 reflexes on pinching the toes or on dipping the toe in very 
 dilute acid (made by adding dilute sulphuric acid to water). 
 Wash off the acid with water. 
 
 Using the same frog thrust a fine wire down the spinal 
 canal so as to destroy the cord. Observe any change in the 
 reflexes. What is the condition of the heart and respiration ? 
 
 Fig. No. 4. Frog's Brain, a. 
 Cerebral hemispheres; b. Optic 
 thalami; c. Optic lobes. d. 
 Cerebellum, e. Medulla. (Stein- 
 er.) 
 
84 LABORATORY GUIDE IN 
 
 Examine the activity of the peripheral nerves in the fol- 
 lowing manner ; hold the frog back upwards in the hand and 
 apply the electrodes from the induction coil (tetanizing cur- 
 rent) to the lumbar spine. Observe how long the muscles 
 will stay contracted before fatigue sets in. 
 
 Dissect out the sciatic nerve by an incision along the pos- 
 terior surface of the thigh and stimulate it with the electric 
 current as before. 
 
 Lay a few crystals of sodium chloride on the nerve and 
 moisten them with a drop of water. Observe any change 
 in the muscles. Explain. 
 
 Expose the gastrocnemius muscle and stimulate it directly 
 with the electric current and also with salt crystals. 
 
 Describe the functions of the different parts of the nerv- 
 ous system in the frog as learned from the symptoms arising 
 from their removal. 
 
 Action of Acid Puchsin. 
 
 In connection with the study of the physiology of the 
 nervous system of the frog the action of acid fuchsin is 
 very interesting and instructive. This drug when injected 
 into frogs in proper dosage apparently produces no effects 
 for from 12 to 24 hours. If, however, it is given in the 
 same-sized dose to an animal suffering from fatigue or to 
 one in which the cerebral hemispheres have been injured the 
 symptoms will appear in about fifteen minutes. 
 
 Experiment i. Using a 1.5% solution of acid fuchsin 
 inject into the anterior lymph sac (see page 13) of a frog 
 0.3 mg. acid fushsin per gram body weight of animal. Re- 
 place the frog under the bell jar and study it from time to 
 time, observing general activity, reflexes, etc. After 10 
 
■UXPBRIMBNTAL PHARMACOLOGY 85 
 
86 LABORATORY GUIDE IN 
 
■EXPERIMENTAL PHARMACOLOGY 87 
 
 or 15 minutes sever the cerebral hemispheres from the rest 
 of the brain, according to the method described earher, 
 and replace the frog on the table for observation. If con- 
 vulsions appear study them carefully, making adequate notes 
 and then try to locate the point of action of the poison by 
 cutting off the optic lobes, destroying the medulla and cord 
 noting changes that occur after each operation. 
 
 Where does the drug act to produce the convulsions and 
 v/hat light does the experiment throw upon the function 
 of the cerebrum? 
 
88 LABORATORY GUIDE IN 
 
 Soporifics or Hypnotics. 
 
 Soporifics or Hypnotics are drugs which are used to pro- 
 duce sleep. They comprise a very large and important 
 group of substances which are extensively employed in medi- 
 cine. In their action they are closely related to chloroform 
 and ether, but they are not used to produce anaesthesia in 
 man owing to the fact that it is impossible to regulate suf- 
 ficiently accurately the depth of the anaesthesia induced by 
 them. 
 
 Chloral (chloral hydrate), the most important member of 
 the series, was introduced into medicine by Liebriech in 
 1S69, and as a pure hypnotic drug is probably the most re- 
 liable in use today. The other members of the group which 
 are of greatest importance are Sulphonal (Sulphonmeth- 
 ane), Paraldehyde and Veronal. 
 
 Examine each drug as to appearance, odor and taste. 
 . I. Examine the solubility of the following soporifics : 
 
 Chloral 
 
 Sulphonal. 
 
 Paraldehyde. 
 
 Veronal. 
 
 Cold water. 
 Hot water. 
 Alcohol. 
 Ether. 
 
 Wrap up a few crystals of chloral in paper, set aside and 
 examine them after a few days. 
 
 In what ways would you think these drugs could best be 
 prescribed in practice? 
 
 II. Put a few crystals of chloral in a test tube and add 
 a small amount of potassium hydrate solution and heat 
 gently. Note the odor of the fumes. What is formed? 
 
liXPERIMBNTAL PHARMACOLOGY 89 
 
go LABORATORY GUIDE IN 
 
nXPERIMBNTAL PHARMACOLOGY 91 
 
 CCl3-CH0-H,0 + KOH = H-COOK + ? 
 Chloral hydrate. Potassium formate. 
 
 III. Select three frogs of about equal size and mark 
 each frog in some way so that it can be recognized. (This 
 is probably done most easily by making a drawing showing 
 the outlines of the marking on the back.) Examine the 
 frogs, making a record of the heart rate and respiration in 
 each. 
 
 Inject into the anterior lymph sac (page 13) of one frog 
 5 mg. of chloral dissolved in water. After studying this 
 animal for about 10 minutes inject into the second frog 2vT 
 mg. of chloral. In like manner after observing this animial 
 for a time and making notes inject into the third frog 50 
 mg. of chloral. These amounts may be obtained most ac- 
 curately by making 5 or 10 cc. of a 5% solution and then 
 diluting small quantities (i cc.) so as to get the required 
 doses. 
 
 Make a record of the time when each injection is made. 
 After injection replace the animals under the bell jar and 
 observe them frequently (especially those with the larger 
 doses) as to spontaneous movements, co-ordination, reflexes, 
 respiration, heart beat, etc. 
 
 Keep a careful record of the condition of each frog at 
 each examination. 
 
 This can be done most systematically, not only in this experiment, but also 
 in those to follow, by keeping a record of the time when each examination is 
 made and the conditions found. By doing this it also gives some idea of the 
 length of time it takes the drugs to act when given in diiferent sized doses. 
 Such a record would start something as follows: 
 Feb. 10, '15 — Frog. 2^ G. 
 
 2:30 — Heart 56, respiration 30 per minute. 
 
 2:32 — Injected 20 mg. chloral in water into anterior lymph sac. 
 2:3s — Frog quiet, apparently normal; reflexes, normal; heart, 54; respir- 
 ation, 28. 
 2:40 — Frog jumps if urged strongly, does not regain sitting position 
 easily when it alights; has difficulty in turning over when 
 placed on back. Reflexes lessened; heart, 40; respiration, 15, etc. 
 
92 LABORATORY GUIDB IN 
 
 Such a record should give an accurate picture of the con- 
 dition of the animal at each stage of the drug's action. 
 
 If complete paralysis occurs in any animal determine 
 whether the action is on the central nervous system or is 
 peripheral. 
 
 This is done by stimulating over the lumbar spine with the electric current 
 and if the muscles of the leg contract the paralysis must be central; if they 
 do not contract see whether the trouble is in the nerve or in the muscles by 
 stimulating the latter directly with the current. 
 
 Also in one of the animals thus paralyzed, expose the 
 heart (page 14) and examine and record its condition. 
 
 The frog given 50 mg. may be used for this purpose. The 
 others may be replaced at the end of the day's work under 
 the bell jar and moistened and examined on the following, 
 day. Some will doubtless recover. Notes should be kept 
 of their progress. 
 
 With the knowledge gained of the physiology of the 
 nervous system study the records of the three frogs and 
 from the symptoms recorded determine the points of action 
 of the drug in the varying doses. 
 
 IV. Inject into the anterior lymph sac of a frog 0.5 cc. 
 of a 10% solution of paraldehyde in water. Compare the 
 symptoms shown by this animal with those manifested by 
 the chloral frogs in Exp. III. Pay particular attention to the 
 condition of the heart, which can probably be sufficiently 
 well studied (as to its rate) through the chest wall without 
 exposing it. Keep the animal, as it will probably recover. 
 
 The difference in action of the two drugs upon the heart 
 is explained by the difference in the constitution of the two 
 molecules. The chloral molecule contains chlorine and these 
 chlorine compounds all have a decided action on the heart 
 muscle which is absent in the case of paraldehyde, whose 
 
EXPERIMBNTAL PHARMACOLOGY 93. 
 
94 LABORATORY GUIDE IN 
 
UXPBRIMBNTAL PHARMACOLOGY 95 
 
 molecule, CgH^aOa, contains no chlorine. Compare the 
 action of chloroform and ether on the heart (page 187). 
 Other members of this group are, Trional. 
 
 Tetronal. 
 
 Urethane. 
 
 Chloretone. 
 
 Chloralose, etc. 
 
 Therapeutics} 
 
 The chloral group of drugs depresses the central nervous 
 system. For this purpose it would be used in cases of in- 
 somnia and of nervous excitement. Chloral and its allies are 
 very valuable also in delirium and in various forms of con- 
 vulsions, as for instance, those due to poisoning with certain 
 drugs or those due to specific diseases (tetanus). In certain 
 forms of insanity (mania) and in some nervous diseases 
 with increased activity of the brain and spinal cord (chorea 
 or St. Vitus's dance) the quieting effects of these drugs are 
 largely made use of. 
 
 On account of its action on the heart chloral must be em- 
 ployed with care in persons who are already affected with 
 certain forms of heart disease and its effect on this organ 
 must also be watched with care in case of its prolonged use, 
 or better, some non-chlorine containing soporific may be 
 used in its stead. 
 
 ^ At the conclusion of the study of a drug group a short disctission of 
 the therapeutic uses of the drugs contained therein will be given. Such dis- 
 cussions are not intended to give compete resume of all the therapeutic uses 
 of the drugs studied, but only those will be mentioned as would be indicated 
 by the results obtained in the foregoing experiments. The student will thus be 
 shown how the knowledge gained by the science of Pharmacology is trans- 
 ferred to and made use of in the Therapeutic art; or, in other words, in 
 rational medicine. 
 
96 LABORATORY GUIDE IN 
 
 Nux Vomica. 
 
 Nux Vomica, the seeds of Strychnos nux vomica, an East 
 Indian tree, contains two alkaloids upon which the activity 
 of the drug- depends. By far the more important of the two 
 is Strychnine which is present in from .9 to 2. per cent; the 
 second alkaloid, Brucine, is present in slightly smaller quan- 
 tities. In action the two alkaloids resemble each other very 
 closely excepting that strychnine is about forty times as 
 strong as brucine. For this reason the activity of nux vom- 
 ica may be considered as dependent upon the amount of 
 strychnine present and in the following experiments it alone 
 will be considered. 
 
 Examine the nux vomica bean and taste a little of the 
 powdered drug. 
 
 I. Place a small amount of the powdered drug in a 
 beaker and pour upon it 10 cc. of boiling water and let it 
 stand until cold. Set this water extract (infusion) aside 
 and use it in experiments IV and V given below. Place a 
 drop of the infusion on the tongue. 
 
 II. Prepare a fluid extract of nux vomica in the follow- 
 ing manner : • . 
 
 Nux vomica. No. 60 powder — 50 grams. 
 
 Alcohol and water in the proportion of 2 : i. 
 
 Moisten the powder with 25 cc. of the diluted alcohol and 
 let it macerate in a well covered vessel in a warm place for 
 48 hours. Pack it in a percolator (see percolation, page 44) 
 and gradually pour on diluted alcohol until the drug is ex- 
 hausted. Reserve the first 35 cc. of the percolate and evap- 
 orate the remainder over a water bath to a soft extract. Dis- 
 
■ EXPERIMENTAL PHARMA CO LOGY. gy 
 
98 
 
 LABORATORY GUIDE IN 
 
- EXPERIMENTAL PHARMACOLOGY 99 
 
 solve this extract in the 35 cc. saved and make the total bulk 
 up to 50 cc. with diluted alcohol. 
 Define a fluid extract. 
 
 III. Isolate the strychnine in the fluid extract in fol- 
 lowing manner : — ■ 
 
 Dilute the fluid extract with about twice its own volume 
 of distilled water and pour it into a separating funnel and 
 render it alkaline with ammonia. Add about 25 cc. of a 
 mixture of chloroform and ether in the proportion of 2 : i 
 and shake gently for three minutes and set the funnel aside 
 to allow the chloroformic solution to separate. When sep- 
 aration has taken place, draw the chloroform and ether off 
 into a beaker and repeat the agitation of the watery extract, 
 using a fresh supply of chloroform and ether. Draw this 
 off as before into the breaker with the first solution. The 
 watery extract can now be thrown away. Pour the chloro- 
 form and ether into the separating funnel, which has been 
 v/ashed, and add about 25 cc. of water and enough dilute 
 HoSO^ to make the solution distinctly acid, shake gently 
 for a few minutes and then separate the water and chloro- 
 form as before, placing the acidulated water in a flask. Re- 
 peat the agitation of the chloroform and ether with fresh 
 acidulated water, adding the latter when separation has 
 taken place, to the first lot in the flask. (The waste chloro- 
 form is to be poured into a large bottle provided for it and 
 later redistilled.) As the acidulated watery solution is prob- 
 ably colored it should be shaken in the flask with charcoal 
 and filtered. Render the filtrate distinctly alkaline with 
 ammonia and shake it out twice as before in the separating 
 funnel, with small quantities of the mixture of chloroform 
 and ether. When this has been done, pour this chloroformic 
 
loo LABORATORY GUIDE IN 
 
 solution into a glass evaporating dish, add to it a drop of 
 concentrated hydrochloric acid and stir it well with a glass 
 rod or put the mixture in a small flask and shake it well. 
 The solution becomes milky from the formation of strych- 
 nine hydrochloride, which is thrown out of solution and 
 may be separated from the chloroform by allowing the lat- 
 ter to evaporate spontaneously or by warming it gently over 
 a water bath. 
 
 Caution. — In this process two things are of prime im- 
 portance in determining the success of the manipulations : — 
 First, to agitate sufficiently the chloroform and ammonia or 
 sulphuric acid solutions to allow thorough intermingling of 
 the solutions. Three to five minutes should be devoted to 
 each shaking. Second, make sure that the correct reaction 
 is present. Test with litmus paper each time and after 
 shaking for a minute or two, test again. (See Alkaloids, 
 Exp. No. II.) The reaction must be distinct each time. 
 
 Explain why each step in the manipulation was carried 
 out. (Compare Alkaloids, Exp. Nos. I, II, VII.) 
 
 The crystallized strychnine hydrochloride is to be used in 
 tests given below. (Exp. Nos. X, XL) 
 
 IV. To a few cubic centimeters of the infusion made in 
 Exp. I add a few drops of ferric chloride solution. The 
 dark color of the mixture is due to the tannic acid present 
 in the nux vomica bean uniting with the iron to form ferric 
 tannate. (To a dilute solution of tannic acid add ferric 
 chloride.) 
 
 V. Inject 0.5 cc. of the infusion into the lymph sac of a 
 frog, and replacing the animal under a bell jar, observe the 
 effect. Keep full notes. If any convulsions come on, after 
 
- EXPERIMENTAL PHARMACOLOGY. loi 
 
J02 LABORATORY GUIDE IN 
 
EXPERIMENTAL PHARMACOLOGY 
 
 103 
 
 studying them carefully, try to find out the point of action 
 of the drug by dividing off in turn the cerebrum, optic 
 lobes, and finally destroying the cord, watching the effect 
 of each operation on the symptoms. The strychnine con- 
 vulsion is "tonic" as distinguished from the "clonic" type, 
 ^v-hich is caused by some drugs and occurs in some diseases. 
 (Compare Camphor ( page 112.) 
 
 Make a drawing of a frog in a strychnine (tonic) con- 
 vulsion. 
 
 VI. Inject into the lymph sac of a second frog ^ cc. of 
 a 1/10% solution of strychnine sulphate diluted with physio- 
 logical salt solution to make a convenient amount. In this 
 animal, as in the previous experiment, study the early effects 
 of the poison before convulsions come on. Look for changes 
 in irritability, in reflexes, etc. When convulsions come on, 
 paint the skin of the animal with a 2% solution of cocaine 
 observing any changes which occur. If the convulsions 
 cease wash the animal off with water and put it to one side 
 observing it again at the end of 10 or 15 minutes. Explain. 
 
 If paralysis should occur in any of the frogs used in these 
 experiments see if it is due to central or peripheral action, 
 as was done in chloral, Exp. III. 
 
 VII. Inject into a small frog 1/20 mg. of strychnine. 
 When convulsions come on place the animal under a bell jar 
 with some absorbent cotton upon which has been poured 
 some chloroform. C)bserve whether any change takes place 
 in the convulsions. If they become lessened, remove the 
 chloroform from the bell jar and allow the animal to recover 
 from its effects. Do the convulsions return? When they do, 
 pith (page 13) the brain of the animal and hang it up by the 
 jaw on the hook for reflexes. Now test the animal's reflexes 
 
I04 
 
 LABORATORY GUIDU IN 
 
 by pinching the toe and by dipping the foot in acid. How do- 
 they differ from those in a normal animal? 
 
 Is any therapeutic hint contained in the first part of this 
 experiment? In experiments Nos. V, VI and VII, it was 
 noticed that the same effects were induced whether the 
 poison was employed as the infusion, or as the pure alkaloid. 
 
 VIII.^ The object of this experiment is to apply strych- 
 nine to the cervical portion of the cord while leaving the 
 lumbar cord unaffected. Instead of pithing a frog as usual, 
 remove the brain entirely by cutting off the head with a pair 
 of scissors. Put the lower blade in the mouth and the upper 
 blade over the top of the head, incline the scissors back- 
 ward slightly and make a quick cut. Expose the heart and 
 remove it from the body in order to stop the circulation. 
 The upper part of the cord will be exposed by the cut first 
 made and a drop of a moderately strong strychnine solu- 
 tion (o.i%) is to be applied to it. After about five minutes 
 test the reflexes by pinching the fore and hind legs. If the 
 experiment is successful a normal reflex should be ob- 
 tained by pinching the toes of the hind foot, while a typical 
 strychnine convulsion involving the entire animal should 
 be brought on by irritation' of the fore limbs. 
 
 How would such results help to locate the point of strych- 
 nine activity? 
 
 (See Pharmacology and Therapeutics, Cushny. Refer 
 to chapter on Strychnine.) 
 
 IX. With a hypodermic syringe inject subcutaneously 
 into a rabbit i mg. of strychnine sulphate. Watch the effect 
 
 ^ This experiment is not always successful when carried out by students and 
 may be omitted if thought best. However, if successful, it is of so much in- 
 terest and importance in locating the point of action of the drug as to merit 
 a place in these notes. 
 
EXPERIMENTAL PHARMACOLOGY. 105 
 
jo6 LABORATORY GUIDE IN 
 
- EXPERIMENTAL PHARMACOLOGY 107 
 
 on reflexes, etc. If convulsions come on control them with 
 a few drops of chloroform on cotton held close to the ani- 
 mal's nose. 
 
 Compare the convulsions in this animal with those seen in 
 the frog. Note the efficiency of chloroform in controlling 
 the convulsions of strychnine. 
 
 Chemical Tests for Strychnine. 
 
 Xa. Put one or two crystals of the strychnine hydro- 
 chloride, prepared in Exp. Ill, on a porcelain cover and add 
 to it two drops of concentrated sulphuric acid, mixing the 
 two with a clean dry glass stirring rod. Put beside the 
 strychnine-sulphuric acid mixture on the cover a minute 
 crystal of potassium bichromate and with the glass rod 
 (which has been washed and dried) draw the bichromate 
 crystal through the strychnine mixture. Observe the changes 
 in color. This "oxidation-reaction" or "fading purple test" 
 for strychnine is most delicate and characteristic. It is said 
 to be capable of detecting i /20000th of a grain of the alka- 
 loid. The order of the color changes is very important in 
 making the test. The purple, which should first appear, 
 changes to a crimson, which turns to a cherry red that is 
 fairly persistent. 
 
 Xb. Carry out the same experiment as above, using 
 manganese dioxide instead of potassium bichromate. Com- 
 pare the results with Exp. Xa. 
 
 This reaction is obtained with almost any oxidizing agent, 
 such as potassium permanganate, potassium f erricyanide, or 
 lead dioxide. Manganese dioxide is one of the best as the 
 changes in color take place very slowly, in some cases lasting 
 some minutes. 
 
io8 
 
 LABORATORY GUIDE IN 
 
 If much brucine is present in the preparation, this color 
 test is much interfered with and it may be impossible to get 
 the correct changes. 
 
 Xc. After testing your own alkaloid with the two re- 
 agents named, make the same tests on some pure strychnine 
 w^hich will be furnished you. Compare the colors with 
 those obtained with your preparation. 
 
 XL Dissolve the rest of the strychnine hydrochloride 
 (Exp. Ill) in about 20 cc. distilled water and put the solu- 
 tion into 5 test tubes, diluting with a little water if neces- 
 sary and test them with the various alkaloidal reagents. 
 
 
 Reagent 
 
 Precipitate? 
 
 Limit of delicacy of 
 reagent 
 
 a 
 
 b 
 
 c 
 
 d- 
 
 e 
 
 Tannic acid. 
 
 Picric acid. 
 
 Iodine in potassium iodide. 
 
 Mercury-potassivim iodide. 
 
 Phosphotungstic acid. 
 
 
 1-20,000 
 1-100,000 
 1-150,000 
 1-200,000 
 
 Therapeutic uses. 
 
 On account of their intensely bitter taste, preparations of 
 liux vomica are employed in cases of loss of appetite and 
 malnutrition. By their action on the taste organs they 
 probably increase reflexly the secretion of the gastric juice 
 and thus aid digestion. 
 
 Strychnine has been largely used in the past as a stimulant 
 to the central nervous system in various conditions in which 
 depression of the brain or cord was present. It was em- 
 ployed in shock or collapse, and in failure of the respiratory 
 or of the vaso-motor center. In recent years considerable 
 
EXPERIMENTAL PHARMA CO LOGY 
 
 109 
 
no LABORATORY GUIDE IN 
 
. EXPERIMENTAL PHARMACOLOGY 
 
 III 
 
 doubt has been expressed as to whether the drug exerts 
 any beneficial action in such conditions when administered 
 to man in therapeutic doses. This doubt has led to the 
 restriction of its use but it is still employed to a certain ex- 
 tent. By stimulating the activity of the spinal cord to a 
 slight extent it increases the "tone" of the muscles and of 
 the blood vessels whose muscular coats are controlled by 
 the spinal centers. 
 
112 LABORATORY GUIDE IN 
 
 Camphor. 
 
 Camphor is a white, translucent, crystalline substance ob- 
 tained from the volatile oil of the camphor tree (Cin- 
 namomum Camphora). In addition to its local irritant ac- 
 tion, it exerts a very marked effect upon the central nervous 
 system producing symptoms which are quite characteristic 
 and which may well be studied in connection with those 
 produced in mammals by strychnine. 
 
 I. Weigh a cat or rabbit and then administer to it 
 through a stomach tube camphor dissolved in olive oil (2 G. 
 camphor for each kilo of body weight). Replace the ani- 
 mal in a cage and observe the early symptoms and the con- 
 vulsions which should come on in about half an hour. Com- 
 pare them carefully with those produced by strychnine and 
 try to control them with chloroform. (After observing 
 them sufficiently the animal should be killed with the anaes- 
 thetic.) 
 
 A similar convulsion is also caused by thujon which is 
 an isomere of camphor and the active poisonous principle 
 derived from the volatile oil of sage, tansy, and absinth. It 
 may be used in place of the camphor in the above experi- 
 ment if desired. It is administered to cats by means of the 
 stomach tube in doses of about 2 cc. (Some preparations of 
 thujon are active in i cc. doses while others require 5 cc.) 
 Note the marked change in respiration which occurs be- 
 fore the convulsions come on. 
 
 It will be seen that the convulsions caused by thujon are 
 also of a distinctly different type from those following a 
 poisonous dose of strychnine. Those caused by the latter 
 drug are tonic in type and are due to an action on the spinal 
 
EXPERIMENTAL PHARMACOLOGY 113 
 
ij^ LABORATORY GUIDE IN 
 
EXPERIMENTAL PHARMACOLOGY 
 
 115 
 
 cord. On the other hand, the thujon (and camphor) con- 
 vulsions are "clonic" and are caused by stimulation of some 
 unknown part of the cerebrum or lower part of the brain. 
 
 Picrotoxin, the active principle of a climbing shrub, 
 Anamirta paniculata, indigenous to India, also has a phar- 
 macological action much like the drugs mentioned above, 
 and the clonic type of convulsion may be very easily dem- 
 onstrated by its use. 
 
 II. Inject into a frog i mg. picrotoxin and replace the 
 animal under the bell jar. When the convulsions come on 
 study them carefully indicating by a drawing the position 
 the animal takes. Remove in order the different portions 
 of the central nervous system by successive cuts as has been 
 done in previous experiments and thus determine the point 
 of action of the drug. 
 
 III. Inject subcutaneously into a guinea pig 5 mg. of 
 picrotoxin. Study the convulsions as before. 
 
 Thujon and picrotoxin are not employed in therapeutics. 
 Their pharmacological action would indicate that they might 
 be of value as stimulants to the central nervous system and 
 especially to the respiratory center. As such they might be 
 of value in shock and collapse and perhaps in narcotic pois- 
 oning. Camphor has long been used in shock and collapse. 
 
ii6 LABORATORY GUIDE IN 
 
 Opium. 
 
 Opium is the inspissated juice obtained from the fruit- 
 capsules of the white poppy (Papaver somniferum). The 
 capsules are incised while yet unripe and the milky juice 
 allowed to exude and dry by spontaneous evaporation until 
 it becomes a brownish gummy mass. 
 
 The poppy is grown in Turkey, Asia Minor, Persia, India 
 and China, but that obtained from Smyrna and Turkey is by 
 far the most valuable. Persian and Indian opium is im- 
 ported mainly as sources of the opium alkaloids and the 
 Chinese opium is consumed entirely in that country. 
 
 Opium contains about twenty alkaloids of which Mor- 
 phine and Codeine are the most important, and, indeed, it 
 may be said that the value of opium depends upon the per 
 cent of morphine which it contains. The U. S. Pharmacopoeia 
 specifies that the crude opium when moist shall contain not 
 less than 9.5 per cent of crystallizable morphine, while the 
 dried product must assay from 10 to 10.5 per cent. Opium 
 from the different localities varies exceedingly in its mor- 
 phine content. 
 
 Morphine was discovered in 181 7 by Seitiirner, of Eim- 
 beck, Germany, and was named by him morphium. It exists 
 in the opium principally in combination with meconic acid, 
 although some may be present as a lactate or a sulphate, both 
 of these acids being found in the crude drug. Meconic acid 
 is of most importance as it is strictly characteristic of 
 opium and its preparations, and its positive detection is 
 proof of the presence of some opium preparation. 
 
 In their solubilities morphine and codeine differ widely 
 from the other alkaloids, for morphine is relatively insoluble 
 
. UXPURIMBNTAI PHARMACOLOGY 117 
 
ii8 LABORATORY GUIDE IN 
 
EXPERIMENTAL PHARMACOLOGY 
 
 119 
 
 in chloroform and even less soluble in ether ( 1-6250) ; 
 codeine, on the other hand, is more soluble in water than 
 any other alkaloid except caffeine. 
 
 Morphine Reactions. 
 
 I. Put a few crystals of morphine on a porcelain cover 
 and add a drop or two of pure concentrated sulphuric acid 
 and mix the two with a clean dry glass rod. No color 
 should be formed if the morphine is absolutely pure, but oc- 
 casionally a faint pink tint is seen. Heat the mixture very 
 S^ently. At about 150° a dirty green or rose-red color is de- 
 veloped, and on still further heating, the solution becomes al- 
 most black. Allow it to cool and add some water, when a 
 greenish blue color appears which changes to green when 
 ammonia is added in excess. 
 
 II. Put a few crystals of morphine on a cover and mix 
 them with a drop or two of concentrated sulphuric acid as in 
 Exp. I. To this add a drop of distilled water which will 
 heat the mixture. Now treat with a drop of concentrated 
 nitric acid, which should give a rose-red color, changing to 
 brown. 
 
 (This reaction for morphine is very delicate, being well 
 shown by o.oi milligram of the alkaloid.) 
 
 III. To a drop of concentrated nitric acid on a porcelain 
 cover add a minute amount of morphine. An orange-red 
 color is obtained, which changes to yellow on heating. 
 
 IV. (a) Mix a small amount of morphine with about 
 twice its weight of cane sugar. By means of a glass rod add 
 a drop of concentrated sulphuric acid. This should produce 
 a purple color, changing gradually to blood-red and brown- 
 ish-red and becoming an olive-brown on dilution with water. 
 
I20 
 
 LABORATORY GUIDE IN 
 
 (b) The above test can be modified as follows : — To a 
 solution of morphine add cane sugar until the solution is 
 saturated. Now pour this solution carefully down the side 
 of a second test tube in the bottom of which is concentrated 
 sulphuric acid. By inclining the second tube the solution 
 can be poured in so that no mixture of the two fluids takes 
 place and a purple or rose-red ring will form at their junc- 
 tion. This ring becomes more intense on standing. 
 
 V. Dissolve some morphine sulphate in a little water and 
 add it to a solution of iodic acid. What color does it turn? 
 Add to the resulting mixture some thin starch paste. What 
 was the first color due to? 
 
 Note. — In order to color the starch paste blue morphine must be present in 
 the strength of i to looo. If no blue is obtained mix the solution well and 
 pour down the side of the test tube some very dilute ammonia. A double ring 
 will be seen at the junction of the two fluids; it will be blue below and brown 
 above. 
 
 VI. To a few cubic centimeters of water add some mor- 
 phine sulphate and to this solution add a few drops of ferric 
 chloride solution. A deep blue color is obtained. 
 
 VII. Meconic acid reaction. To a cubic centimeter of 
 tincture of opium diluted with a little water add two drops 
 of ferric chloride solution. What color does the solution 
 become? If the color is obscured by a precipitate, filter 
 tiie solution. 
 
 VIII. Inject into the anterior lymph sac of a frog 15 mg 
 of morphine sulphate. Replace the animal under the bell 
 jar and examine it from time to time, comparing the ef- 
 fects with those caused by chloral or strychnine, and with 
 those seen in the frog injected with codeine in Exp. IX. 
 
 Note. — This experiment (VIII) should be begun early in the laboratory ses- 
 sion as it sometimes takes two or three hours for the full effects to appear. 
 
. nXPURIMBNTAL PHARMACOLOGY . 121 
 
122 LABORATORY GUIDE IN 
 
, EXPERIMENTAL PHARMACOLOGY 
 
 123 
 
 IX. Inject in the anterior lymph sac of a frog i cc. of 
 a 1.5% solution of codeine phosphate and replace the animal 
 under the bell jar. Compare the symptoms in this animal 
 with those seen in the morphine frog noting especially the 
 relative time of onset of the symptoms in the two. 
 
 Among the alkaloids found in opium which are of less 
 importance than those studied is thebaine, which resembles 
 strychnine very closely in its pharmacological action. In 
 fact, many of the alkaloids of opium may be arranged in a 
 series with the depressant, morphine, at one end of the series 
 and thebaine at the opposite end. A comparison of the ef- 
 fects of thebaine with those produced by morphine and 
 codeine may be made by injecting i cc. of a 1.5% solution 
 of thebaine into a frog and comparing it with the animals 
 studied in Exp. VIII and IX. 
 
 X. Inject subcutaneously into a dog 50 mg. of morphine 
 sulphate and observe the effects keeping an accurate protocol 
 of the symptoms. 
 
 XIa. Inject 30 to 40 mg. of morphine sulphate sub- 
 cutaneously into a cat. Put the animal in a wire cage and 
 compare the effects with those seen in the dog. (Note un- 
 der Exp. VIII appHes here also.) 
 
 Xlb. Instead of injecting a cat a dose of from 5 to 10 
 mg. of morphine sulphate may be given a white mouse. 
 Symptoms much like those produced in the cat are seen, the 
 main difference being that they appear in from 5 to 10 min- 
 utes. 
 
 The peculiar action of morphine on the cat is not confined 
 to that animal, but is found in all the members of the cat 
 tribe, and also in the horse and ass. 
 
124 LABORATORY GUIDE IN 
 
 Examine a. crude opium, 
 
 b. powdered opium, 
 
 c. extract of opium, 
 
 d. tincture of opium (Laudanum), 
 
 e. camphorated tincture of opium (Paregoric), 
 
 f. powdered opium and ipecac (Dover's pow- 
 
 der), 
 ♦ g. codeine, 
 
 h. and herome. 
 
 Note the odor of all the opium preparations and taste d, 
 e, and f. 
 
 Therapeutics. 
 
 The symptoms induced by opium in man resemble more 
 closely those in the dog than those in the other animals. It 
 depresses the brain, and for this reason is used in cases of 
 sleeplessness when this condition results from pain, because 
 in depressing the cerebrum it acts on the pain centers, for 
 which it seems to have a special affinity. In the dog the 
 respiration was slowed in the later stages of the action from 
 depression of the respiratory center, and opium prepara- 
 tions are often employed in cough mixtures to allay exces- 
 sive irritability of this part of the medulla. The numerous 
 other purposes for which opium and morphine are used 
 cannot be illustrated experimentally. 
 
nXPBRIMBNTAL PHARMACOLOGY 125 
 
126 LABORATORY GUIDE IN 
 
. EXPERIMENTAL PH'ARMACOLOGY 127 
 
 Curara. 
 
 Curara, the South American arrow poison, varies in com- 
 position with the different locaHties from which it is ob- 
 tained. The best known product comes from Guiana and is 
 prepared from the bark of Strychnos toxifera, a tree native 
 to that country. Curara as obtained in commerce consists of 
 dark brown, shining, brittle masses which contain vegetable 
 extracts in addition to two alkaloids, tubocurarine and 
 curine. The characteristic action of the drug is dependent 
 principally upon the first alkaloid mentioned, the latter hav- 
 ing quite a different action and being much weaker. 
 
 Not only do solutions of curara^ deteriorate on keeping, 
 but, what is of greater importance, the crude drug, as ob- 
 tained in the market, varies greatly in strength, many of the 
 preparations being entirely inert. There is no chemical 
 method by which the activity of curara can be estimated ; 
 ihe only test is pharmacological. Each solution to be used 
 has to be "standardized" by tests on animals and the dose 
 necessary to produce characteristic effects ascertained. 
 
 I. Inject into a frog a poisonous dose of a standardized 
 solution of curara. Study the eff"ect on spontaneous move- 
 ments, reflexes, ability to turn over when placed on its back, 
 and length of time leg muscles will remain contracted when 
 stimulated by applying the tetanizing current to the lumbar 
 spine. If paralysis should occur see if it is due to central or 
 peripheral action (see Soporifics, Exp. Ill, page 92). This 
 animal is to be replaced under the bell jar and kept moist, as 
 it will probably recover in a few days. 
 
 ^ The curara solution should be made up in physiological salt solution to 
 which some thymol has been added. 
 
128 LABORATORY GUIDE IN 
 
 II. Pith a frog, tie it on the frog board and expose the 
 sciatic nerve and artery along the back of the thigh by a 
 short longitudinal incision through the skin, and careful 
 separation of the muscles with the dissecting needle. Sepa- 
 rate the sciatic nerve from the surrounding tissues along a 
 short distance of its course and then pass a thread under 
 the nerve and tie it tightly around the rest of the thigh as 
 high up as possible. 
 
 Free the animal, inject curara as in I, and from time to 
 time compare the reflexes in the hind legs. Stimulate the 
 lumbar cord with the electric current and note any differ- 
 ences between the two sides. See whether the reflexes re- 
 main equal in the two legs and whether cross reflexes can be 
 obtained. 
 
 If the experiment is successful the operated leg should not 
 be affected by the poison and should respond to electrical 
 stimulation. In such a case cut off the leg just above the 
 knee, leaving attached to it the sciatic nerve which has been 
 carefully separated from the thigh muscles and cut toward 
 the upper part of its course. Holding the leg preparation 
 by the severed end of the femur lay the hanging sciatic 
 nerve for a few moments in the curara solution. Remove it 
 and stimulate it with the electric current. Does curara affect 
 the nerve trunk? Dip the muscle in the solution and after 
 a few moments stimulate the sciatic nerve again. Is there 
 any change? If so, test the activity of the muscles by direct 
 stimulation. Where does curara act? 
 
 Examine the heart of the animal in Exp. II. 
 
 Examine Curara. 
 
EXPERIMENTAL PHARMACOLOGY. 129 
 
130 LABORATORY GUIDE IN 
 
. EXPERIMENTAL PHARMACOLOGY 131 
 
 Therapeutics. 
 
 The main importance of curara is in physiological experi- 
 ments. It has been suggested, and in some few cases used, 
 to allay the convulsions due to tetanus or to strychnine 
 poisoning. It is not recommended for such conditions as 
 its action is too uncertain and too dangerous. 
 
132 LABORATORY GUIDE IN 
 
 Nicotine. 
 
 The alkaloid, nicotine, is obtained from the dried leaf of 
 the tobacco plant (Nicotianum Tabacum) in which it is 
 found to the extent of from 2 to 8 per cent. The pure alka- 
 loid is a colorless, oily fluid, which, on exposure to air, be- 
 comes yellowish or brown. It has a strong, unpleasant odor 
 resembling somewhat that of tobacco. 
 
 In an aqueous solution the alkaloid gives a strong alkaline 
 reaction and it unites with various acids to form salts. 
 
 I. Inject 0.5 cc. of nicotine chloride solution^ into the 
 anterior lymph sac of a frog. Observe the effects, noting 
 especially the posture of the animal in the early stages of 
 poisoning as this is very characteristic of nicotine action. 
 In addition, watch for any twitching of the muscles, and if 
 paralysis should come on see whether it is due to central or 
 peripheral action. 
 
 Make a drawing of the animal in the early stage of the 
 nicotine action when the legs are drawn up over its back. 
 
 The action of nicotine upon the sympathetic nervous sys- 
 tem is studied later (pages 248, 260). 
 
 Therapeutics. 
 Nicotine is not employed in therapeutics. 
 
 ^ The nicotine chloride solution is formed by neutralizing a small quantity of 
 nicotine with 1-20 normal hydrochloric acid. Each cubic centimeter will then 
 contain about 4 mgs. of nicotine. 
 
EXPERIMENTAL PHARMACOLOGY. 
 
 133 
 
134 LABORATORY GUIDE IN 
 
- BXPBRIMBNTAl PHARMACOLOGY 135 
 
 Veratrine. 
 
 Vcratrine is an alkaloid prepared from the seeds of Asa- 
 groea officinalis. It is a grayish powder which causes very 
 marked irritation when even a minute quantity is inhaled. 
 
 The alkaloid is not of great medical importance but is of 
 pharmacological interest, owing to a peculiar action it pos- 
 sesses which can be best studied in cold-blood animals, al- 
 though the same effect may be elicited in mammals. 
 
 I. Inject I mg. of veratrine sulphate (i cc. of 0.1% sol.) 
 into a frog. Watch and describe the effects. Observe the 
 awkwardness of its movements which appears in a few min- 
 utes. When the clumsiness is well developed locate the 
 point at which the drug acts by removing the cerebrum, optic 
 lobes, and finally destroying the spinal cord if the symptoms 
 do not disappear earlier. Stimulate the sciatic plexus with 
 single shocks from the induction coil, comparing the results 
 with those seen in a normal frog. Repeat the stimulation 
 of the sciatic nerve several times at short intervals study- 
 ing the effect of fatigue upon the veratrinized muscle. 
 Finally, after allowing the muscle to rest, stimulate the 
 muscle directly, using single shocks. At what point does 
 the drug act? 
 
 Therapeutics. 
 
 The phenomena seen in this experiment give no hint as to 
 the use of the drug in therapeutics ; it is employed for ex- 
 ternal application, and internally to a limited extent for its 
 effects on the circulation. 
 
136 
 
 LABORATORY GUIDE IN 
 
 Caffeine. 
 
 Caffeine differs from the other alkaloids in the fact that it 
 is contained in a number of plants belonging to different 
 species. It is obtained from the berry of the coffee (Coffea 
 Arabica) ; from the leaves of tea (Thea Chinensis), besides 
 being present in the kola nut of Central Africa; in the 
 Paraguay tea and Guarana paste of South America and in 
 the Apalache tea of Virginia and Carolina. 
 
 In some of these plants it is associated with two closely 
 related alkaloids, TJieobromine and Theophyllin, which re- 
 semble caffeine very much in some of their actions. All 
 three alkaloids are derivatives of xanthine, which in turn 
 is closely related to uric acid. 
 
 Caffeine is a very feeble base and forms salts only with 
 difficulty and these salts, as a rule, are very unstable, being 
 easily decomposed, even by water. 
 
 I. Test the solubility of caffeine m 
 
 Reagent. 
 
 Solubility. 
 
 Water, cold. 
 " hot. 
 Alcohol. 
 Chloroform. 
 
 II. Put 100 mg. caffeine in a test tube and add 4 cc. 
 cold water to it. Shake. Now add 100 mg. sodium benzoate 
 and shake. What therapeutic use could be made of this 
 reaction ? 
 
 III. Compare in like manner the solubility of theo- 
 bromine with that of the double salt, theobromine sodium 
 salicylate, and note the advantage the latter might possess. 
 
. EXPERIMENTAL PHARMACOLOGY. 137 
 
138 
 
 LABORATORY GUIDE IN 
 
. EXPERIMENTAL PHARMACOLOGY 
 
 139 
 
 IV. Apply the various alkaloidal reagents to a 1% solu- 
 tion of caffeine. 
 
 
 Reagent. 
 
 Precipitation? 
 
 a 
 
 Tannic acid. 
 
 
 b 
 
 Picric ncid. 
 
 
 c 
 
 Iodine in potassium iodide. 
 
 
 d 
 
 Mercury-potassium iodide. 
 
 
 e 
 
 Phosphotungstic acid. 
 
 
 Note. — In its reaction to c and d caffeine differs from all other alkaloids 
 except theobromine and colchicine. 
 
 V. Murexoin test. To a few crystals of caffeine on the 
 porcelain cover add a drop of concentrated nitric acid. Hold 
 the cover with a pair of forceps some distance above the 
 flame of a Bunsen burner and thus gently evaporate the 
 mixture to dryness. After cooling the porcelain cover al- 
 low the residue to come in contact with strong ammonia 
 fumes and note the color obtained. 
 
 This reaction is given by the members of the xanthine 
 series and is practically identical, with that given by uric 
 acid. 
 
 VI. Inject 5 nig. of caffeine (i cc. 0.5% soi.) into a 
 frog. Study and describe the symptoms comparing them 
 with those induced by strychnine and veratrine. See whether 
 the peculiar change in the muscles is due to an action on 
 the nervous system by destroying its various parts as was 
 done with veratrine. Excise a small piece of the affected 
 muscle and tease it in salt solution on a microscopic slide. 
 Examine it under the microscope, using the low power lens, 
 and compare its appearance with normal muscle prepared 
 in the same way. (For an explanation of the changes con- 
 sult Pharmacology and Therapeutics, Chapter on Caffeine.) 
 
I40 LABORATORY GUIDE IN 
 
 The action of caffeine and its allies on the heart and 011 
 the kidney will be studied later (pages 207, 240). 
 Examine the Coffee bean (Coffea Arabica) ; 
 
 Tea leaves (Thea Chinensis) ; 
 
 Caffeine citrate; 
 
 Theobromine. 
 
 Theobromine sodium salicylate. 
 
 Therapeutic Uses. 
 
 Caffeine is largely employed as a central nervous stimu- 
 lant. The stimulant action on the highest parts of the brain 
 is more noticeable in man than in the frog as the cerebrum 
 ill the latter is not as highly differentiated and the principal 
 symptoms are due to cord action. In man, however, caffeine 
 (usually taken in the form of coffee or tea) stimulates the 
 cerebrum and the medulla and, to a less extent, the spinal 
 cord. It is of great use in cerebral depression, such as is 
 found in shock and collapse. In narcotic poisoning, also, its 
 action on the medullary centers is especially valuable. Both 
 caffeine and theobromine are largely used for their stimu- 
 lant action on the kidneys by which they increase the urine. 
 They are, therefore, of service in removing abnormal col- 
 lections of fluid from the body, such as oedema or ascites, 
 whether of cardiac or renal origin. 
 
EXPERIMENTAL PHARMACOLOGY. 141 
 
142 LABORATORY GUIDE IN 
 
, nXPBRIMBNTAL PHARMACOLOGY 143 
 
 Cocaine. 
 
 Cocaine is the principal alkaloid derived from the leaves 
 of Erythroxylon coca, a shrub which is a native of Peru and 
 Bolivia and of other parts of South America. It is very 
 unstable, being quite prone to undergo chemical change, so 
 tiiat preparations of the drug frequently contain the products 
 of its decomposition. For this reason there are no very 
 delicate or distinctive chemical tests by which the drug may 
 be identified. Being an alkaloid, it is thrown out of solution 
 by the various precipitants of the group, and in its behavior 
 to solvents it resembles in general the other alkaloids. 
 
 Cocaine has an important local action upon the termina- 
 tions of sensory nerves, which makes it of very great use in 
 medicine. 
 
 I. Take a piece of filter paper 2 or 3 cm. square and 
 moisten it with a few drops of a 4% cocaine hydrochloride 
 solution and place it upon the tip of the tongue. Retain it 
 in this position for two or three minutes and then examine 
 the condition of that part of the tongue exposed to the drug, 
 testing it especially as to the sense of touch, temperature, and 
 taste. Contrast its effect on the taste of sugar and of nux 
 vomica. 
 
 II. Place a cat in a cat box allowing the head to pro- 
 trude. After examining the corneal reflex and the reaction 
 of the iris to bright and dim light, put into one eye 2 drops 
 of a 4% solution of cocaine hydrochloride, keeping the 
 other eye as a control. After 5 minutes examine the eye 
 testing the reflexes to touch and to both dim and bright light 
 and comparing the size of the pupil with the control. If 
 
144 
 
 LABORATORY GUIDE IN 
 
 the drug effects are not sufficiently well marked make a 
 second or even third application of cocaine allowing 5 or 
 ID minute intervals to elapse to allow of absorption. What 
 therapeutic uses may be made of these actions of cocaine? 
 
 In addition to these local actions cocaine has also a marked 
 effect on the central nervous system which may be illustrated 
 by the following experiment: 
 
 III. Inject subcutaneously into a white mouse^ from 
 3 to 5 mg. of cocaine. Place the animal in a suitable con- 
 tainer and observe. 
 
 Therapeutic Uses. 
 
 Cocaine is moat largely employed in medicine for its action 
 in paralyzing the sensory nerve endings causing a local 
 anaesthesia, permitting of operations being performed with- 
 out pain. It is also employed to dilate the iris in order that 
 examinations of the interior of the eye may be made. It 
 is rarely used for its action on the nervous system. 
 
 ^ Somewhat similar symptoms may be obtained in the dog by the injection 
 of cocaine in doses of from 20 to 25 mg. per kg. If convulsions come on 
 they may be controlled with chloroform. 
 
. EXPBRIMBNTAL PHARMACOLOGY 
 
 145 
 
j^g LABORATORY GUIDE IN 
 
. nXPBRIMBNTAL PHARMACOLOGY 147 
 
 The Belladonna Series. 
 
 Several plants belonging to the Solanacese furnish alka- 
 loids which are very closely related, not only chemically, but 
 also in their pharmacological effects. Chief among these 
 alkaloids is Atropine which is contained in the leaves and 
 roots of the deadly nightshade (Atropa Belladonna). Next 
 in importance to atropine are Hyoscyamine and Hyoscine. 
 These alkaloids are all obtained from other sources than 
 that named, viz.; henbane (Hyoscyamus niger), the thorn- 
 apple (Datura Stramonium) and other plants of less im- 
 portance. They are very hard to isolate in absolutely pure 
 form because hyoscyamine is very prone to change to atro- 
 pine during the manipulations. 
 
 As stated above, they resemble one another closely in theii 
 ]")roperties and actions, so that atropine alone will be studied. 
 Examine Belladonna leaves and roots. 
 
 I. Prepare the alcoholic extract of belladonna leaves ac- 
 cording to the U. S. P., using 50 G. of the powdered drug. 
 
 Define an "extract" of a drug. 
 
 II. Take some of the extract, an amount about the size 
 of a pea and rub it up in 5 cc. of salt solution and put a drop 
 of the watery extract in a cat's eye and watch for any 
 changes comparing it with the normal eye. The cat is best 
 put in a cat box (footnote, page 12) with the head exposed 
 during the experiment; drop the solution near the outer 
 canthus of the eye so that it will have to cross the eyeball 
 before escaping by the nasal duct. 
 
 This action of the atropine series upon the pupil is known 
 as mydriasis and the drugs producing it are mydriatics. It 
 
148 LABORATORY GUIDE IN 
 
 is the most delicate test known for the group, although a 
 few other drugs, of which cocaine is the most important, act 
 as mydriatics, though to a less marked degree. 
 
 III. Isolate the atropine from the extract of belladonna 
 by the "shaking-out" process as described below. Observe 
 the precautions mentioned under the isolation of strychnine 
 (page lOo). 
 
 Mix the extract thoroughly with about lOO cc. of distilled 
 water and render it alkaline with ammonia. Put the mix- 
 ture in a separating funnel and add 20 cc. of chloroform. 
 Shake gently for 3 to 5 minutes and set the funnel aside to 
 allow the chloroform to separate, drawing it off into a flask. 
 Add 15 cc. more chloroform to the watery extract and shake 
 as before, and draw off the chloroform into the flask with 
 the first lot. The watery extract can now be thrown away. 
 
 Put the chloroform containing the alkaloid in the sepa- 
 rating funnel and add about 50 cc. of water rendered dis- 
 tinctly acid with sulphuric acid. Agitate the mixture several 
 minutes as before, drawing off the chloroform from the 
 water when separation has taken place. Repeat the process 
 with the chloroform and 50 cc. of fresh acidulated water. 
 (The waste chloroform is to be placed in a jar on the side 
 table for redistillation.) If the watery solution is colored, 
 even slightly, shake it in a flask with powdered charcoal and 
 filter it. The filtrate containing the atropine is now ren- 
 dered alkaline with ammonia and shaken out again with 20 
 cc. of chloroform, which is drawn off into a glass evaporat- 
 ing dish. Repeat the process with 10 cc. of chloroform and 
 add it to the 20 cc. in the dish and set the whole amount 
 aside, protected from the dust, to allow the chloroform to 
 evaporate and the atropine to crystallize. 
 
EXPERIMENTAL PHARMACOLOGY 149 
 
I50 LABORATORY GUIDB IN 
 
. nXPBRIMBNTAL PHARMACOLOGY 151 
 
 IV. Place a few crystals of atropine (from Exp. Ill) 
 on a porcelain cover and add to them a drop of concentrated 
 nitric acid and evaporate it to dryness over the water bath. 
 Allow the cover to cool and then touch the residue with a 
 glass rod which has been dipped in an alcoholic solution of 
 caustic potash. A rich violet color is produced, changing to 
 a dark red, which finally fades away, but can be reproduced 
 by the addition of fresh alcoholic potash. This reaction 
 (known as Vitali's test) is almost peculiar to the atropine 
 series and is said to be given by o.oooi mg. of the alkaloid. 
 
 V. Inject into a frog 15 mg. of atropine sulphate and de- 
 scribe all the symptoms which are observed following the in- 
 jection of the drug. Keep the animal for several days until 
 complete recovery takes place. Compare the effects with 
 those induced by other drugs used earlier in the course. 
 
 VI. Count the heart and respiratory rates in a dog 
 which is resting quietly and then inject hypodermically i 
 mg. atropine sulphate per Kg. body weight. After the 
 drug has had time to be absorbed repeat the observations 
 previously made and if necessary take further counts later 
 keeping a record of the findings. Explain the results. 
 
 VII. Take o.ooi G. of atropine sulphate (which will be 
 furnished you) and describe all the symptoms experienced. 
 Count the rate of your heart before and after taking the 
 drug and see if there is any change. 
 
 VIII. Dissolve the remainder of the atropine isolated 
 in about 20 cc. of distilled water which has been rendered 
 acid with sulphuric or hydrochloric acid. Divide the solu- 
 tion into five parts, put it in test tubes and add the alkaloidal 
 precipitants as follows : 
 
152 
 
 LABORATORY GUIDE IN 
 
 
 Eeagent. 
 
 Precipitate? 
 
 a 
 
 Tannic acid. 
 
 
 b 
 
 Picric acid. 
 
 
 c 
 
 Iodine in potassium iodide. 
 
 
 d 
 
 Mercury-potassium iodide. 
 
 
 e 
 
 Phosphotungstic acid. 
 
 
 The action of atropine on the innervation of the heart is 
 studied on the turtle (page 196) ; and on the frog (page 
 187). 
 
 Its action on secretions will be studied on the salivary 
 gland (page 263). 
 
 Its mydriatic effect when applied locally was shown in 
 Exp. II, and the same effect from internal administration 
 is studied under the heading "Cervical Sympathetic Nerves," 
 (page 251). 
 
 Chemically atropine consists of a combination of tropine 
 with tropic acid. Other alkaloids have been made artificially 
 by the substitution of other acids in the place of tropic acid. 
 Ihe most important of these synthetic alkaloids is Homa- 
 tropine in which tropine is combined with mandelic acid. 
 ,In many of its actions it resembles atropine quite closely, 
 the main point of difference being that its effects are much 
 more transient. Its main use is in ophthalmology where it 
 is employed to dilate the iris and paralyze the accommoda- 
 tion by acting on the ciliary body. 
 Examine Atropa Belladonna, 
 Hyoscyamus niger. 
 Datura Stramonium, 
 
 the alcoholic extract of belladonna leaves, 
 tincture of belladonna leaves and 
 the extract and tincture of hyoscyamus. 
 Homatropine hydrobromide. 
 
£XPBRIMBNTAL PHARMACOLOGY 153 
 
154 LABORATORY GUIDE IN 
 
MXPBRIMBNTAL PHARMACOLOGY 155 
 
 Therapeutic Uses. 
 
 Owing to the fact that atropine has such a wide range ot 
 activities, its uses in medicine are very numerous. Among 
 these a few may be mentioned as being indicated by the va- 
 rious experiments in which the drug is employed. It is used 
 as a stimulant in the depressed state of the central nervous 
 system occurring in shock, collapse and in narcotic poison- 
 ing. (Compare strychnine and caffeine.) For its peri- 
 pheral action it is employed for many purposes. In cases of 
 extreme slowness of the heart (Bradycardia) due to over- 
 activity of the vagus, atropine may be employed. It is used 
 to dilate the pupil of the eye to allow of ophthalmoscopic 
 examination, or as a therapeutic agent in the treatment of 
 many eye diseases. 
 
 In excessive secretion of the saliva such as occurs in 
 poisoning with mercury, and in the night sweats of tuber- 
 culosis, atropine is very valuable. 
 
 Finally it is used in cases of poisoning by pilocarpine or 
 muscarine, the latter being an alkaloid resembling pilocar- 
 pine in many of its actions. ' 
 
156 LABORATORY GUIDE IN 
 
 Cinchona. 
 
 In the plants belonging to the cinchona family are a num- 
 ber of species the bark of which yields some twenty-five 
 alkaloids. Quinine is by far the most important of these. 
 The Cinchonacas are natives of South America and, as far 
 as is known, were introduced into medicine about 1630. 
 
 Quinine may be regarded as a typical alkaloid and its be- 
 havior towards various solvents and precipitants has already 
 been examined (page 20). 
 
 In addition to those properties, which it shares in common 
 with the other alkaloids, it shows a few reactions of its own 
 which are more or less characteristic. 
 
 Taste quinine and quinine hydrochloride. 
 
 I. Weigh out 0.1 G. quinine, the same amount of quinine 
 sulphate, and of quinine hydrochloride and put them sepa- 
 rately in three test tuDes. Add 10 cc. of water to each test 
 tube and shake. Which is the most soluble? 
 
 II. To 0.1 G. quinine sulphate in a test tube, add about 
 10 cc. distilled water and about i cc. of dilute sulphuric acid. 
 Note the change in solubility compared with I and describe 
 the solution formed. Solutions showing this peculiar fluor- 
 escence are also formed by a few of the other cinchona 
 alkaloids. 
 
 III. Make a solution of quinine sulphate in a test tube, 
 adding a drop or two of sulphuric acid if necessary. Add 
 very carefully potassium hydroxide to make the solution as 
 nearly neutral as possible (not alkaline!). Add a few drops 
 of chlorine water and after shaking add ammonia to excess. 
 
nXPBRIMUNTAL PHARMACOLOGY 157 
 
158 
 
 LABORATORY GUIDE IN 
 
.BXPBRIMBNTAL PHARMACOLOGY 159 
 
 Observe the greenish coloration of the solution or the green 
 precipitate which will be formed if enough quinine is present. 
 This reaction is of importance, as it can be employed in 
 testing for quinine in the urine in cases in which it is im- 
 portant to know that the drug is being absorbed. 
 Examine Cinchona bark. 
 
 Tincture of cinchona. 
 
 Compound tincture of cinchona. 
 
 Quinine tannate (Tasteless quinine). 
 
 Euquinine (Quinine ethyl carbonate). 
 
 Therapeutic Uses. 
 
 The tinctures of cinchona are largely employed in medi- 
 cine on account of their bitter taste ; they are given espe- 
 cially in cases of lack of appetite. The other therapeutic 
 uses of cinchona and its alkaloids cannot be demonstrated 
 very satisfactorily in the laboratory. 
 
 The tannate of quinine and euquinine may be used in 
 case tasteless preparations are desirable as in children. 
 
i6o LABORATORY GUIDB IN 
 
 Anaesthetics. 
 
 The most important members of the group of drugs used 
 to produce general anaesthesia are Chloroform and Ether. 
 Their depressant action on the central nervous system will 
 be observed in some of the experiments in which they will 
 be used to supplement the other drugs used as anaesthetics 
 in animals (chloretone, etc.). The cardiac effects are 
 studied in the frog, turtle and dog. In these animals it 
 will be noticed that the heart dilates and becomes very weak 
 before there is very much change in the rate. The im- 
 portance of this in practical anaesthesia is that information 
 as to the condition of the heart cannot be derived entirely 
 from the pulse rate, as the heart may be seriously affected 
 and yet show very little change in the number of its con- 
 tractions per minute. The quality of the pulse must also 
 be noted. 
 
 The fact that chloroform acts very much more strongly 
 on the heart than ether, will also be demonstrated in the 
 experiments mentioned above. 
 
 Some of the early eff'ects of the anaesthetics are shown in 
 the following experiment : — 
 
 I. Place a rabbit on the table and hold it by placing your 
 hand under its chest in such a way that you can count its 
 heart and respiration. Pour some chloroform on a little 
 absorbent cotton and hold it to the animal's nose for a few 
 moments and observe any change in the rhythm of the heart 
 or respiration. 
 
 Repeat the experiment using ether and afterwards am- 
 monia. It will be seen that the same effeccs are produced by 
 all three drugs. They are caused by a reflex stimulation of 
 
. EXPERIMENTAL PHARMACOLOGY. i6i 
 
j52 laboratory guide in 
 
EXPBRIMBNTAL PHARMACOLOGY 163 
 
 the vagus and respiratory centers from the action of the irri- 
 tant fumes on the mucous membranes : 
 
 Other drugs that are used as general anaesthetics are 
 Nitrous oxide gas and Bthyl chloride. 
 
 Nitrous Oxide. 
 
 The action of nitrous oxide may be observed by placing 
 a frog, white mouse, or small guinea pig in a wide mouth 
 bottle which is fitted with a cork through which pass two 
 pieces of glass tubing. One of these tubes is connected 
 with a tank of nitrous oxide gas. When the animal has 
 been placed in the bottle and the cork adjusted, nitrous oxide 
 gas may be admitted by opening the valve very gradually. 
 The outlet glass tube should be left open. 
 
 Observe the animal closely as the percentage of gas in- 
 creases in the jar, noting especially changes in the rate of 
 respiration. Keep a record of the time it takes to produce 
 complete ansesthesia and when that stage is reached turn off 
 the gas and remove the animal from the jar. Note the 
 time it takes for the animal to recover. 
 
 This experiment may be varied by connecting a tank of 
 oxygen to the inlet tube in addition to the nitrous oxide 
 and admitting small percentages of oxygen to the bottle at 
 the same time as the nitrous oxide is passing in. Note the 
 difference in symptoms from those seen when nitrous oxide 
 alone was given. 
 
 Bthyl Chloride. 
 
 The action of ethyl chloride in producing general anses- 
 thesia may be demonstrated as follows : — 
 
 Place a white mouse in a small beaker which has a little 
 absorbent cotton in the bottom of it, and then spray a few 
 
i64 LABORATORY GUIDE IN 
 
 drops of ethyl chloride (Kelene) upon the cotton. Note 
 the symptoms shown by the animal and the length of time 
 it takes to produce full anaesthesia. Immediately this stage 
 is reached remove the animal and observe the time it takes 
 to recover. 
 
 Magnesium Sulphate Anesthesia. 
 
 Depression of the central nervous system and general 
 anaesthesia may be produced in rabbits by the subcutaneous 
 or intramuscular injection of magnesium sulphate in doses 
 of 1.7 G. per kilo of body weight. This dose can be con- 
 siderably lessened if sodium oxalate is administered at the 
 same time as the magnesium is given. The anaesthesia and 
 depression may be entirely recovered from if a calcium salt 
 be given later as calcium antagonizes the magnesium action. 
 
 Experiment : — 
 
 Inject intramuscularly into a rabbit 1.2 G. magnesium 
 sulphate per Kg. body weight and 0.2 G. sodium oxalate 
 per Kg. In from i^ to 2 hours the animal should be in a 
 state of complete anaesthesia. Now inject slowly either 
 into an ear vein or into a jugular 6 or 8 cc. of a 3% solu- 
 tion of calcium chloride. Note the immediate effect upon 
 the respiration and upon the general condition of the animal. 
 
 Local Anesthesia. 
 
 Ethyl chloride may also be used to produce local anaes- 
 thesia. This action is explained by the great volatility of the 
 substance so that when it is sprayed upon the skin it evap- 
 orates very rapidly causing extreme chilling and finally 
 freezing of the part. 
 
 Holding a tube of ethyl chloride in one hand direct a 
 fine spray of the drug against the back of the other hand. 
 
EXPERIMENTAL PHARMACOLOGY. 165 
 
l56 LABORATORY GUIDE IN 
 
nXPBRIMBNTAL PHARMACOLOGY 167 
 
 The tube will have to be held at a distance of 18 inches or 
 2 feet from the object sprayed. In a very few moments 
 the area of skin will become white and examination of its 
 sensitiveness by means of a pin will show complete anaes- 
 thesia as compared with the surrounding normal area. 
 
 The action of other local anaesthetics was illustrated with 
 cocain. The action of this drug and of its alhes, novocaine, 
 etc., is entirely different from ethyl chloride as it is due to 
 a paralysis of the nerve endings conveying the sensations 
 of pain and touch. 
 
i68 LABORATORY GUIDE IN 
 
 The Digitalis Series. 
 
 This series embraces a large number of drugs which re- 
 semble each other in their pharmacological action. While 
 they act on various organs in the body, the cardiac action is 
 characteristic of the group and distinguishes it from all 
 others. The members of the series are derived from plants 
 widely distributed in nature and as widely separated in their 
 botanical relationships. 
 
 The most important is digitalis, which is obtained from 
 Digitalis purpurea (purple fox glove) ; strophanthus from 
 Strophanthus hispidus ; and squills from Scilla maritima. 
 
 I. Prepare the tincture of digitalis according to the U. S. 
 P., using 25 G. of the powdered drug and the proportionate 
 amount of 75% alcohol. (Made by adding one volume of 
 water to three of alcohol.) 
 
 Define a tincture. 
 
 This tincture is to be used in the following experiments : 
 
 II. To 5 cc. of the tincture add 15 cc. of distilled water, 
 is a precipitate formed? Cork and set aside the mixture 
 
 'and examine it after a few days. 
 
 III. Examine the tincture for the presence of tannic 
 acid by adding ferric chloride. 
 
 IV. To 5 cc. of the tincture add two drops of mercury- 
 potassium iodide. Is a precipitate formed? What does the 
 result indicate? 
 
 V. Evaporate over a water bath 10 cc. of the tincture to 
 about one-half its volume and then make up the concen- 
 tiated tincture to the original bulk by adding physiological 
 salt solution. 
 
EXPERIMENTAL PHARMACOLOGY. 169 
 
j-O LABORATORY GUIDE IN 
 
- EXPERIMENTAL PHARMACOLOGY 171 
 
 Now, pith a frog and tie it on a board. Expose the heart 
 and inject into a lymph sac i cc. of the modified tincture. 
 Record all changes in the heart. 
 
 Describe any change in the rate of the organ and also any 
 differences in the size of the various chambers or in their 
 manner of contracting or dilating. 
 
 VI. Biological assay of tincture of digitalis. 
 
 Digitalis preparations vary considerably in their strength 
 and it is impossible to standardize them by any chemical 
 means such as can be employed in the cases of those drugs 
 whose activity depends upon alkaloids which may be isolated 
 in pure form and weighed. 
 
 It is very hard to isolate the glucosides in pure form as 
 they are likely to break up, making the results obtained very 
 unreliable. For this reason a pharmacological or biological 
 estimation of the strength of a preparation is employed as 
 follows : — • 
 
 Evaporate 10 cc.^ of the tincture over a water bath to 
 about half volume- and then make it up to the original vol- 
 ume by the addition of physiological salt solution. Select 
 3 frogs of about the same size, weigh them carefully (within 
 0.5 G.) and copy the markings on the back of each animal 
 so as to be able to identify them later. Into the anterior 
 lymph sacs of the frogs inject by means of a glass pipette 
 doses of the modified tincture as follows : — ■ 
 
 The doses to be given have to be calculated according to 
 the weight of the frogs used. Frog No. i is to receive 
 
 ^ All measurements must be made with the same attention to accuracy 
 as in quantitative chemical analyses. 
 =" Why? 
 
172 LABORATORY GUIDE IN 
 
 0.004 cc/ multiplied by the number of grams body weight 
 of the animal; No. 2 receives 0.0055 cc. per gram body 
 weight, and No. 3 0.007 cc. per gram body weight. (These 
 small quantities may be most accurately measured by dilut- 
 ing the modified tincture still more with salt solution and 
 then injecting the proportionately larger dose. The amount 
 of fluid given each animal should measure about 0.5 cc.) 
 
 Keep a jrecord of the time when each injection is made. 
 Replace the frogs under the bell jar and at the end of one 
 hour from the time of each injection examine the condition 
 of each frog's heart. Pith the animal if necessary, tie it on 
 the frog board and expose the heart according to the usual 
 method. To have a correct "end reaction'' the heart should 
 have just ceased beating with the ventricle in systole and the 
 two auricles markedly distended with blood. Light me- 
 chanical stimulation of the ventricle may call out a local 
 contraction but there should be no general beat of the 
 ventricle. (Compare Exp. V.) If all three hearts are still 
 beating when they are examined the doses were too small 
 and must be increased in fresh frogs of corresponding 
 .weight. If, on the other hand, the frogs have been dead 
 for some time, the doses were too large and must be made 
 smaller in other frogs. 
 
 Having ascertained now by this method the toxic strength 
 of a tincture it may be adjusted by dilution or concentration 
 so that its strength may correspond to any standard which 
 may have been adopted. 
 
 The method of assay described is of considerable im- 
 portance as it or some of its modifications is employed by 
 
 ^ Three section of the class may assay tne same tincture by varying the 
 doses to be administered by 0.0005 cc. so as to fill in the gaps in the figures 
 given above. In this way a complete assay may be obtained. 
 
EXPERIMENTAL PHARMACOLOGY. 173 
 
jy^ LABORATORY GUIDE IN 
 
.EXPERIMENTAL PHARMACOLOGY ij^ 
 
 pharmaceutical firms for the standardization of the cardiac 
 remedies of the group (see page 272). 
 
 The remainder of the tincture is to be saved to be used in 
 subsequent experiments on blood pressure, etc. 
 
 The effects of digitalis on the mammalian heart, both 
 when normal and also when in a state of auricular fibrilla- 
 tion and on the arterial blood pressurv^ are studied later. 
 (Exps., pages 207 and 200.) Its effect on the excretion of 
 urine (diuretic action) may be studied in the rabbit (note, 
 page 239), and the vascular changes in the intestinal and 
 renal vessels according to directions given on pages 299 and 
 304- 
 
176 LABORATORY GUIDB IN 
 
 Infusion of Digitalis. 
 
 Another preparation of digitalis which is largely used in 
 some parts of the world in place of the tincture and which 
 it resembles in most of its actions is the Infusion. 
 
 I. Prepare the infusion of digitalis acording to the U. 
 S. P. 
 
 Define an infusion. 
 
 II. Test the infusion for glucosides with tannic acid. 
 
 III. Fill a test tube about half full with the infusion, 
 place the thumb over the mouth of the tube and shake it up 
 thoroughly. Note the soap-like character of the foam. The 
 infusion contains a glucoside, digitsaponin, which possesses 
 the property of forming a frothy solution and of holding in- 
 soluble bodies in suspension. 
 
 IV. Pith a frog, tie it on the board and expose the heart 
 as usual. Inject into a lymph sac 0.5 cc. of the infusion. 
 Compare the efifects with those noticed in the experiments 
 in which the tincture was employed. 
 
 V. The infusion of digitalis may be assayed by the meth- 
 od described under "Tincture" (Exp. VII, page 171), em- 
 ploying, of course, correspondingly larger doses of the in- 
 fusion. Also, in making the assay, it will not be necessary 
 to evaporate the preparation as was done with the tincture. 
 Why? 
 
 VI. Save the remainder of the infusion and examine it 
 from time to time and note any changes taking place in it. 
 
 Examine digitalis leaves (Digitalis purpuras) ; 
 
 strophanthus seeds ( Strophanthus hispidus) ; 
 and squills, the sliced bulb (Scilla maritima). 
 
EXPERIMENTAL PHARMACOLOGY 177 
 
178 LABORATORY GUIDE IN 
 
. EXPERIMENTAL PHARRIACOLOGY 179 
 
 Also examine the following preparations : — 
 Tincture of strophanthus ; 
 Tincture of squills; 
 Syrup of squills. 
 
 Therapeutic Uses. 
 
 Digitalis and its allies are employed principally in certain 
 affections of the heart. When a valve of the heart is diseased 
 so that it does not perform its functions properly, the heart 
 has an increased amount of work to do. In most cases its 
 walls increase in thickness (hypertrophy) to allow of the 
 extra work being done, in other words the heart becomes 
 "compensated." But if an unusual demand is made on the 
 heart before it has time to hypertrophy, the walls stretch on 
 account of their weakness, and the heart is said to become 
 "dilated." When this occurs the contractions are weak and 
 imperfect and the result is that the circulation becomes 
 slowed and the blood tends to collect on the venous side of 
 the vascular system, resulting in a passive congestion of the 
 organs, and in marked cases a generalized oedema of the 
 body may follow. 
 
 In such conditions digitalis is of the greatest value. As 
 was seen in its effect on the frog's heart, it increases the 
 strength of the contractions and in some cases lessens the 
 extent of dilatation. The heart begins to pump more blood 
 into the arteries, thus improving the nutrition of the body 
 cells. One of the first organs to be benefited is the heart 
 itself through its coronary arteries. From its improved 
 blood supply it becomes stronger and may be able to 
 hypertrophy and thus carry on its increased work without 
 the further use of digitalis. The improved circulation re- 
 
i8o LABORATORY GUIDE IN 
 
 suits in a disappearance of the congestion of the organs and 
 of the oedema. 
 
 The removal of the oedema is also aided by the diuretic 
 action of the drugs belonging to this series ; this appears to 
 be due mainly to the cardiac action (cardiac diuretic). 
 
 The use of the drug in conditions of auricular fibrillation 
 is demonstrated in a later experiment. 
 
EXPERIMBNTAL PHARMACOLOGY i8i 
 
i82 LABORATORY GUIDB IN 
 
. EXPERIMENTAL PHARMACOLOGY 183 
 
 Aconite. 
 
 Several alkaloids are derived from plants belonging to the 
 Aconitum genus, and among them Aconitine is the most im- 
 portant. It is obtained from the root of the monk's hood 
 (Aconitmii Napellus). 
 
 The most important preparation containing aconitine is 
 the tincture of aconite, the alkaloids being very rarely used 
 as different preparations vary considerably in activity, owing 
 to the fact that aconitine decomposes with great readiness. 
 
 I. To 2 cc. of the tincture of aconite add an equal amount 
 of water. Try to dissolve the precipitate formed by adding 
 some 95% alcohol. The precipitate in this case consists of 
 resinous bodies (extractives) which are thrown out of so- 
 lution by the water but which are redissolved by the alcohol. 
 
 II. Add 10 drops of tincture of aconite to about i cc. of 
 distilled water in a clean beaker. Take the solution in the 
 mouth and retain it there withovit swallowing for about a 
 ri]inute, after which it may be ejected and the mouth rinsed 
 with water. Observe whether any unusual sensation comes 
 on in from five to ten minutes in that part of the mouth 
 with which the aconite solution has come in contact. 
 
 These symptoms, which are due to stimulation of the sen- 
 sory nerve endings, are characteristic of two drugs, aconite 
 and veratrine. 
 
 III. Pith a frog, tie it on the board and expose the heart 
 by the usual method (page 14). Inject into a lymph sac 
 0.5 cc. of tincture of aconite and keep a record of all changes 
 taking place in the organ, noting not alone changes in rate, 
 but also in the character of the contractions. 
 
184 LABORATORY GUIDE IN 
 
 After some minutes (10) the heart may become very ir- 
 regular, this irregularity showing especially in the ventricle. 
 If this change in the rhythm does not develop in a reason- 
 able time it may be necessary to give a second injection of 
 the aconite. The observation of this phenomenon should 
 not be omitted. Keep the heart moist with salt solution. 
 
 IV. Biological assay of aconite. 
 
 The class may carry out a biological assay of the tincture 
 of aconite according to the method described in the Phar- 
 macopceia. Three guinea pigs may be injected after they 
 have been carefully weighed. The dose given the first shall 
 be the standard dose_ demanded in the Pharmacopoeia, viz. : 
 0.0004 cc. per gram body weight of the animal ; for the sec- 
 ond 0.0002 cc. per gram ; and for the third 0.0006 cc. per 
 gram. These doses will give a rough estimate of the strength 
 of the tincture, and this estimate may be still further nar- 
 rowed down by subsequent injections into fresh animals 
 employing doses determined by the results obtained in the 
 first series of animals. 
 
 Examine the root of Aconitum Napellus. 
 
 Therapeutics. 
 
 Aconite is occasionally used in cases of neuralgia on ac- 
 count of its action on sensory nerve endings. 
 
 It has been used more or less extensively in the past to 
 slow the heart and to lower the temperature in cases of 
 fever. It is doubtful whether it accomplished either of 
 the effects when given in therapeutic doses and its use has 
 therefore been largely given up in favor of the newer and 
 stronger drugs of the antipyrine group. 
 
. UXPBRIMBNTAL PHARMACOLOGY 1 85 
 
1 86 LABORATORY GUIDE IN 
 
nXPBRIMBNTAL PHARMACOLOGY 1S7 
 
 The Effect of Drugs on the Frog's Heart. 
 
 Pith a frog, tie it on a board and expose the heart as 
 usual. Dissect out the vagus nerve and stimulate it with a 
 weak tetanizing current from the induction coil. To find the 
 vagus nerve in the frog: expose the heart, split the pectoral 
 girdle entirely and draw the anterior limbs well apart. Care- 
 fully remove the skin and connective tissue in the neck and 
 two large nerves (9" and 12'') will be seen on either side 
 passing forward. Trace these backward and they will be 
 found to take a turn outward to enter the base of the skull. 
 The vagus in this position lies between them, running par- 
 allel to them in this part of their course. The electrical 
 stimulation of the nerve should be with a weak current and 
 not unduly prolonged as the nerve is fatigued very easily. 
 
 Describe the changes in the heart caused by vagus stimu- 
 lation. 
 
 Note. — In a certain number of cases the vagus in the frog is not active 
 and it will therefore be impossible to affect the heart by its electrical stimula- 
 tion. See that the heart is kept moist with salt solution and that it is not 
 injured by instruments or rough handling as it is very easily damaged. 
 
 In this frog or in a second one if necessary, examine the 
 action of chloroform by putting the animal, with the heart 
 exposed, under the bell jar in which there is a piece of ab- 
 sorbent cotton soaked in chloroform. 
 
 Record all changes seen in the heart, whether changes in 
 rate or in the condition of the various chambers. 
 
 If the effects become very marked, remove the animal 
 from the bell jar and allow the heart to recover. Keep it 
 moist by dropping on it a small amount of physiological salt 
 solution. 
 
 When the organ has recovered repeat the experiment, us- 
 ing ether instead of chloroform. 
 
1 88 LABORATORY GUIDE IN 
 
 Compare the actions of the two drugs. (See Anaesthet- 
 ics, page i6o). Also compare the action of chloral on the 
 heart. (Page 92.) 
 
 After the heart has returned once more to normal, put a 
 drop of a pilocarpine (or muscarine) solution on it and note 
 any change in rate. After a few minutes add a few more 
 drops of the same solution and note any further change. 
 Compare the appearance of the heart with its condition un- 
 der vagus stimulation. If the heart should stop contracting, 
 put two drops of an atropine solution on the organ and if 
 necessary, after a few minutes make a second or third ap^ 
 plication of atropine. 
 
 If the heart begins to beat once more apply pilocarpine 
 solution again and see if it will stop it. Also try stimulation 
 of the vagus nerve. If pilocarpine will not stop the heart, 
 try chloroform by exposing the organ to the fumes as be- 
 fore. 
 
 From your knowledge of the anatomy and physiology of 
 the vagus nerve try to account for the effects induced by 
 the drugs as seen above, considering them in the following 
 order, pilocarpine, atropine, pilocarpine and chloroform. 
 
 The effects of digitalis, aconite, chloral and paraldehyde 
 on the heart are studied elsewhere. (Pages 171, 183, 92.) 
 
nXPBRIMBNTAL PHARMACOLOGY 189 
 
igo LABORATORY GUIDB IN 
 
. BXPBRIMBNTAL PHARMACOLOGY 191 
 
 The Effect of Drugs on the Turtle's Heart. 
 
 Destroy the brain of a turtle by a blow with a hammer, 
 and turning the animal on its back, tie it on an operating 
 board. Draw the head out so as to put the neck on the 
 stretch and fasten it in this position by means of a nail 
 driven through it. Remove the lower shell (plastron), cut- 
 ting the lateral attachments with bone forceps or a saw; 
 after cutting the hard parts the shell can be raised slightly 
 and the underlying soft parts severed with a scalpel, taking 
 care to cut toward the shell. Carefully cut away the skin 
 and fascia near the base of the neck, and if the coracoids 
 (G) and clavicles (F) are in the way they may be cut back 
 with bone forceps and a scalpel. Large white muscles 
 (B. B.), the long retractors of the head (Retrahens capitis 
 collique) will now be exposed, running«back from the head 
 and placed deeply in the neck on either side of the median 
 line. Two other large white muscles (coraco-hyoideus. A) 
 will also be seen passing on either side from the front legs 
 forward toward the median line of the neck. Two nerves 
 (C, D) may be found emerging from under the long re- 
 tractors just posterior to the point where the coraco-hyoidei 
 unite in the median line. In the upper part of their course 
 they lie internal to the long retractors, then wind around 
 the muscle to reach its upper surface (in this position of the 
 animal) and pass down the neck, lying on the muscle. One 
 of these nerves is the vagus (D), which must be identified 
 by gently separating it from the surrounding structures and 
 stimulating it with the electric current, watching the efifect 
 on the heart. When it is found, place a ligature around it. 
 In dissecting out the nerve be careful not to take hold of it 
 
192 
 
 LABORATORY GUIDE IN 
 
 Vvith forceps as this injures it. If it is necessary the fascia 
 in the vicinity of the nerve may be grasped with the forceps 
 and then torn away from the nerve by means of the dissect- 
 ing needle. 
 
 Expose the heart by cutting away the pericardium. Thread 
 a small curved needle with about six or eight inches of 
 
 Fig. No. 6. Turtle with plastron removed and vagi dissected out. A. Coraco- 
 
 hyoideus muscle, B. Retrahens capitis collique muscle (right muscle is 
 
 cut across), C. Sympathetic nerve, D. Vagus, E. Carotid artery, 
 
 F. Clavicle, cut end, G. Coracoid, cut end, H. Heart. 
 
EXPERIMENTAL PHARMACOLOGY. 193 
 
^^^ LABORATORY GUIDB IN 
 
EXPERIMENTAL PHARMACOLOGY 
 
 195 
 
 medium thread and take a stitch in either side of the 
 ventricle, trying to include between them the area of great- 
 est contraction. Tie the thread in the heart with a loose 
 knot, leaving the two free ends which are to be attached to 
 the myocardiograph (Fig. 7). 
 
 Fig. No. 7. Myocardiograph for turtle's heart. 
 
 Place the board with the turtle on the stand of the myo- 
 cardiograph and arrange the instrument so that the ends of 
 the two levers (A and B) shall be beside the ventricle. Tie 
 the threads attached to the ventricle to the ends of the two 
 levers. In making this attachment see that the heart is tied 
 up close to the levers and that considerable space is not 
 allowed between because the threads are not drawn tightly. 
 The writing lever now begins to move downwards with 
 each contraction and upwards with each relaxation of the 
 
196 LABORATORY GUIDE IN' 
 
 ventricle. Vary its height so that it will record the move- 
 ments of the heart on the smoked surface of a drum properly 
 placed. Arrange the stationary lever (D) to draw a base 
 line just below the tracing of the cardiac movements. 
 - After taking a short tracing showing the normal con- 
 tractions of the heart, take one showing the effects of stimu- 
 lation of the vagus. 
 
 Note. — Mark on the tracing when any stimulation is begun or stopped or 
 when any drug is applied. 
 
 After the heart has recovered and while the record is 
 continuing apply slowly 8 or lo drops of a 0.5% nicotine 
 solution near the base of the heart where the large vessels 
 enter. This should be sufficient to cause distinct slowing 
 of the heart. 
 
 After the heart has recovered from the nicotine try the 
 effect of vagus stimulation again. 
 
 Continuing the tracings, apply a few drops of a pilocar- 
 pine solution (0.5%) to the heart. When the contractions 
 become very infrequent (and not until then) or stop entirely, 
 apply atropine (0.1%) to the heart, adding a drop or two at 
 a time to the organ. 
 
 When the contractions return to normal, stimulate the 
 vagus with the electric current as before. 
 
 Apply pilocarpine again to the heart. Compare the trac- 
 ings with those obtained earlier in the experiment. 
 
 Shake i cc. of chloroform in 20 or 30 cc. of physio- 
 logical salt solution and pour some of this solution from 
 time to time over the heart. When the cardiac contractions 
 become very weak, remove the chloroform solution with a 
 piece of absorbent cotton and assist the recovery of the heart 
 by bathing it in fresh salt solution. 
 
EXPERIMENTAL PHARMACOLOGY. 
 
 197 
 
198 
 
 LABORATORY GUIDE IN 
 
- EXPERIMENTAL PHARMACOLOGY 199 
 
 Note that pilocarpine does not stop the heart after atro- 
 pine, nor has vagus stimulation any effect. Chloroform, on 
 the other hand, acts after atropine. How is this to be ex- 
 plained? How did nicotine produce its effect? 
 
 Remove carefully the paper with the tracings from the 
 drum and fix it by passing it through a solution of shellac 
 in alcohol. 
 
 Study the tracings according to directions given on 
 page 215. 
 
200 LABORATORY GUIDE IN 
 
 The Effect of Drugs on Blood Pressure. 
 
 Anzesthetize a dog, cat or rabbit according to the methods 
 described earlier (page ii), and when the animal is com- 
 pletely under the influence of the anaesthetic, tie it on its 
 back on the operating board and remove the hair from the 
 neck region. Make an incision about four inches long in 
 the median line of the neck and expose the trachea ; insert 
 a tube (page iS) to allow of artificial respiration should it 
 become necessary. 
 
 Expose the jugular vein on one side and insert a cannula 
 (page i6). On the opposite side expose the carotid artery 
 and in like manner tie a cannula in it (page 17). 
 
 Fill the venous cannula with physiological salt solution 
 and the carotid cannula with a solution made by mixing 
 equal volumes of water and a saturated solution of sodium 
 sulphate. (See note 2, page ry.) 
 
 The ordinary form of mercury manometer, which is so 
 well known as to need no description, is used. Fill the 
 proximal limb of the U-shaped tube, i. e., the limb which is 
 to be connected with the blood vessel, with the sodium 
 sulphate solution, using for this purpose a pipette with a 
 long tapering point. The rubber tube, which is connected to 
 the side tube of the proximal limb of the manometer, is to 
 be completely filled with the sulphate solution, taking great 
 care to see that all air is expelled from the tube and manom- 
 eter. Close the clamp on the tubing which connects the 
 rubber tube and manometer. With a short piece of rubber 
 tubing connect a large pipette (25 cc. ) filled with the sul- 
 phate solution to the upper opening of the proximal limb of 
 the manometer and blow in the pipette so as to raise the 
 
- EXPERIMENTAL PHARMACOLOGY. 201 
 
202 LABORATORY GUIDE IN 
 
BXPBRIMBNTAL PHARMACOLOGY 203 
 
 pressure until there is a difference of 10 or 12 cm. in the 
 height of the mercury in the two hmbs.^ Retain this pres- 
 sure by closing the piece of rubber tubing with a spring or 
 screw clamp and disconnect the pipette. 
 
 Connect the distal end of the rubber tube which is at- 
 tached to the side tube of the manometer to the carotid can- 
 nula and open the clamps so as to allow the carotid pulsa- 
 tions to be transmitted to the mercury. Arrange the writing 
 lever so that it will record the pulsations on a blackened 
 drum with a time marker writing below the tracing. Keep 
 the writing lever in light contact with the drum by means 
 of a small weight suspended by a thread from a support 
 above the drum, taking care that the thread does not press 
 too hard against the lever. 
 
 Administer the following, drugs in the manner described, 
 taking a short piece of normal tracing before each is given, 
 and waiting until the effects of one drug have passed oft' 
 Ijefore applying the next. Observe the precautions as tc 
 the injection of any air. 
 
 1. Amyl nitrite: 3 drops on absorbent cotton applied to 
 the animal's nose or to the tracheal tube if that has been in- 
 serted. 
 
 2. Chloroform: given on absorbent cotton as above. 
 
 3. Ether: given on absorbent cotton. 
 
 4. Suprarenal extract. Adrenalin chloride (i-iooo) ; T 
 drop in salt solution injected into the vein. Wash the can- 
 nula and syringe thoroughly before injecting the next drug. 
 
 5. Nicotine chloride i mg. diluted with salt solution. 
 
 ^ The pressure may also be raised by means of a pressure bottle partially 
 filled with sulphate solution and suspended from the ceiling. A tube from 
 this bottle may be connected with the manometer in the manner described 
 above. 
 
204 LABORATORY GUIDB IN 
 
 6. Pituitary gland extract (Pituitrin 0.5 cc.) in salt so- 
 lution. 
 
 7. Digitalis, i cc. of the tincture from which the alcohol 
 has been evaporated (given intravenously). 
 
 8. Barium chloride 20 mg. in salt solution. 
 
 Allow the drum to continue rotating a short distance after 
 the heart has stopped, so that the writing lever traces a 
 straight line from which measurements may be made to 
 ascertain the absolute blood pressure. Remove the tracings 
 from the drums and fix them by passing them through a 
 solution of shellac in alcohol. 
 
 The blood pressure tracings are to be analyzed according 
 to the directions given on page 215. 
 
EXPERIMENTAL PHARMACOLOGY. 205 
 
2o6 LABORATORY GUIDE IN 
 
.EXPURIMUNTAL PHARMACOLOGY 
 
 207 
 
 Effect of Drugs on the Mammalian Heart and Blood 
 Pressure. 
 
 Anaesthetize a dog (page 12) and when anaesthesia is 
 complete, tie the animal on its back on the operating table. 
 Make an incision through the skin in the median line of the 
 i.ieck, and after exposing the trachea insert a cannula in the 
 usual way (page 18). Expose a vein, either one of the jug- 
 ulars or a saphenous in the leg, and insert a venous cannula. 
 
 Also dissect out an artery, either one of the carotids or 
 a femoral and insert a cannula into it and arrange it for 
 blood pressure tracing according to the method described 
 in the previous experiment. 
 
 Dissect out the vagus nerve on one side, tie a ligature 
 around it and cut the nerve between the ligature and the 
 head. 
 
 Continue the skin incision in the median line from the 
 neck down the thorax to an inch or two below the end of the 
 sternum, and deepen the cut to the bone. Stop all bleeding 
 points with artery forceps. 
 
 Connect the tubing of the bellows with the tracheal tube 
 and start artificial respiration. 
 
 Saw through the entire length of the sternum, keeping ex- 
 actly in the median line and complete the division with bone 
 forceps; draw the two sides of the thorax well apart and 
 secure them to the sides of the table with sharp hooks. 
 
 Regulate the amount of air entering the lungs by means 
 of a screw clamp placed on a piece of rubber tubing on the 
 arm of the tracheal cannula. 
 
 Note. — During the process of sawing through the sternum and opening the 
 thorax the animal must be very deeply under the anaesthetic, otherwise the 
 heart may go into delirium. 
 
2o8 
 
 LABORATORY GUIDE IN 
 
 Cut both phrenic nerves as they pass down to the di- 
 aphragm lying in plain sight on either side of the pericardial 
 sac. 
 
 Open the pericardium along its entire length with a pair 
 of scissors and fasten it to the cut edges of the sternum by 
 means of four or five stitches. 
 
 Fig. No. 8. IVlyocardiograph for dog's heart. 
 
 Take a stitch in either side of the right ventricle with 
 small curved needles and thread, and tie the thread into the 
 heart in the same way as you did with the turtle. Similarly 
 take stitches in the right auricle. If bleeding should occur 
 from the needle passing completely through the wall of 
 either chamber take up the bleeding point with a pair of dis- 
 secting forceps and tie a ligature around it. 
 
 Attach the two sets of levers of the dog's myocardiograph^ 
 
 * The dog myocardiograph (Fig. 8) is constructed on exactly the same prin- 
 ciples as is the myocardiograph for the turtle's heart (Fig. 7), excepting 
 that, as is shown in the figure, it is double, so that it will record the move- 
 ments of both auricle and ventricle. The apparatus has other modifications as 
 is seen in the plate, but the principle is not essentially altered. A descrip- 
 tion of that part of the apparatus which is connected to the heart is found in 
 the Jour, of Physiol., 1897, XXI, P- 213. 
 
■ nXPURIMBNTAL PHARMACOLOGY 209 
 
2IO 
 
 LABORATORY GUIDB IN 
 
BXPERIMBNTAL PHARMACOLOGY 
 
 211 
 
 tightly (Fig. 8) to the heart by means of the threads and 
 arrange the writing levers to record the cardiac movements 
 on a blackened drum. Arrange a time marker to write just 
 below the cardiac levers, and the blood pressure writing point 
 at a convenient location on the drum and nearly in a line 
 with the levers. 
 
 Fig. No. 9. Anaesthetic inhaler for dogs. By rotating handle of lever either 
 air or anaesthetic may be forced into the animal's lungs. 
 
 Start the kymograph and take a normal tracing, after 
 which take tracings to show the effects of the following: 
 
 1. Vagus stimulation, weak and strong currents. 
 
 2. Chloroform (given with special inhaler. Fig. 9) ; 
 continue the tracing so as to show the recovery. 
 
 3. Ether (given with special inhaler, Fig. 9) ; continue 
 the tracing so as to show the recovery. 
 
 4. Suprarenal extract. (Adrenalin chloride, i-iooo), 
 I drop added to 4 cc. salt solution and injected into the vein 
 with a syringe. 
 
 5. Alcohol, 10 cc. of 50% given intravenously. 
 
 6. Pituitary gland extract (Pituitrin) 0.5 cc. in salt 
 solution. 
 
212 LABORATORY GUIDE IN 
 
 7. Amyl Nitrite. 3-5 minims dropped into the side of 
 the tracheal cannula. 
 
 8. Nicotine chloride. 2 mg. in salt solution. 
 
 9. Digitalis. 2 cc. of the tincture which has been evap- 
 orated to one-half volume and made up to original bulk 
 with salt solution. 
 
 10. Digitalis. Toxic doses. Give 3 cc. doses repeatedly 
 until the animal dies allowing about 4-minute intervals be- 
 tween injections. 
 
 Mark the tracings carefully so that they can all be identi- 
 fied and fix them in shellac as before. Analyze them ac- 
 cording to directions given on page 219. 
 
 Cut off the head of the animal and as soon as convenient 
 dissect out the submaxillary ducts according to the directions 
 given on page 255. This dissection will allow more speedy 
 operating on the living animal in the experiment on the sali- 
 vary secretion. 
 
.nXPERIMENTAL PHARMACOLOGY 213 
 
214 LABORATORY GUIDE IN 
 
. nXPBRIMUNTAL PHARMACOLOGY 215 
 
 Analysis of Tracings. 
 
 Graphic records have been obtained in three experiments 
 (turtle's heart, dog's heart and blood pressure) and it is of 
 first importance that these records should be carefully 
 analyzed in order to properly study the changes induced by 
 the various drugs. A few hints are given below to call at- 
 tention to some of the more important points to be looked 
 for, and some suggestions are made as to the best way of 
 studying the tracings. 
 
 Tracings Obtained from the Turtle's Heart. 
 
 It is rarely necessary in these records to take accurate 
 measurements, as the changes induced by the drugs are gen- 
 erally quite marked. 
 
 Describe the effect of stimulation of the vagus, noting 
 whether the heart stops in systole or diastole. Likewise, 
 describe the condition of the heart under pilocarpine (before 
 and after atropine administration) and also the effect of the 
 atropine. 
 
 Under chloroform note especially the strength of the heart 
 as indicated by the extent of contraction and of dilatation, 
 which is measured by the distance of the tracing from the 
 base line. 
 
 Summarize the actions of the different drugs. 
 
 Tracings from the Blood Pressure Experiment. 
 
 Study these first with regard to changes in pressure. Take 
 careful measurements of the distance of the tracing from 
 the base line at various points along the course of the curve ; 
 
2i6 LABORATORY GUIDE IN 
 
 viz., before the drug is injected to get the normal; after the 
 action of the drug becomes apparent ; and later at short in- 
 tervals of time which will depend upon the drug used. 
 Where the curve is fairly uniform readings every thirty sec- 
 onds are frequent enough. 
 
 Measuring in this way from the base line will only give 
 relative changes in pressure, which is all that is necessary in 
 these experiments. To get the absolute pressure, the zero 
 line which was marked on the tracing would have to be used, 
 measuring its distance above or below the base line and add- 
 ing or subtracting this distance from the figures obtained 
 above. This result must then be doubled as there are two 
 arms of mercury in the manometer. 
 
 The changes in the rate of the heart are studied in the 
 following manner: Draw two lines parallel to each other 
 and perpendicular to the base line so that they shall intersect 
 the tracing. The distance of the lines from each other is 
 determined by the rate of the drum as indicated by the time 
 marker; they are usually drawn to include between them a 
 space equal to five or ten seconds. Count the heart beats in 
 the tracing included between the two lines and estimate the 
 rate of the heart per minute. These estimations are made 
 along the curve at the same points as the pressure changes 
 are measured. 
 
 Also describe any other changes in the curves, such as ir- 
 regularities of the heart or of the respiration, etc. 
 
 Summarize the effects of the different drugs upon the 
 pressure of the blood and upon the rate of the heart. 
 
EXPBRIMBNTAL PHARMACOLOGY 217 
 
2i8 LABORATORY GUIDE IN 
 
.EXPERIMENTAL PHARMACOLOGY 219 
 
 Tracing from the Dog's Heart. 
 
 These are to be studied and analyzed by a combination of 
 the methods outlined above. The rate of the heart is ascer- 
 tained as described under the directions for studying the 
 blood pressure tracings, while the strength of the organ as 
 indicated by the extent of systole and diastole is measured 
 from the base line. Describe all other changes in the trac- 
 ings, and in your notes make a full summary of the actions 
 of the different drugs. 
 
220 LABORATORY GUIDU IN 
 
 Digitalis in Auricular Fibrillation. 
 
 One of the most important disturbances of the heart in 
 which digitaHs is employed and in which it probably exerts 
 its most favorable action is in Auricular Fibrillation. In this 
 condition there is no orderly contraction in the auricle, but 
 due perhaps to excessive irritability of the muscle there is 
 a constant fibrillary twitching in all parts of the auricular 
 wall. These fibrillary contractions not only prevent the 
 auricle from emptying itself properly, but also incessantly 
 bombard the auricular-ventricular bundle. These stimuli 
 coming to the bundle so rapidly, only a certain number of 
 them can get through and some of these, coming at irregular 
 intervals, will strike the ventricle when it is in its refractory 
 stage. The net result of these irregular stimuli upon the 
 ventricle is to make it very irregular with consequent marked 
 arhythmia of the pulse. At first these attacks of auricular 
 fibrillation may be quite transient and occur at long in- 
 tervals, but later on they may be more frequent and grad- 
 ually the condition becomes permanent. 
 
 In this form of cardiac disturbance, as stated, digitalis is 
 of great value and it will frequently restore the ventricular 
 rhythm to a state of almost complete regularity. This may 
 be accomplished either by an effect upon the ventricle itself, 
 altering it in such a way that it does not respond so readily 
 to the rapid and irregular stimuli coming down from the 
 auricle or it may interfere with the passage of the stimuli 
 through the Bundle of His so that fewer reach the ventricle. 
 In any case the net result of the administration of digitalis 
 is a disappearance of the cardiac arhythmia and an im- 
 provement in the circulation. There is no change in the 
 
■ BXPBRIMBNTAL PHARMACOLOGY 221 
 
222 LABORATORY GUIDE IN 
 
MXPBRIMBNTAL PHARMACOLOGY 223 
 
 fibrillary contractions of the auricle only their influence on 
 the ventricle is removed. 
 
 This condition of Auricular Fibrillation can be produced 
 experimentally and the action of digitalis upon it demon- 
 strated. 
 
 The early part of the experiment is carried out and the 
 dog prepared in the same manner as was outlined in the 
 work upon the dog's heart (page 207). After the carotid 
 has been prepared for a pulse tracing or blood pressure and 
 the myocardiograph has been attached to the heart, the two 
 wires from a secondary coil are attached to the two poles 
 of that portion of the myocardiograph which is attached to 
 the auricle, and a weak tetanizing current is passed through 
 the auricle. The chamber responds immediately by passing 
 into a condition of fibrillation. The electric current must 
 not be too strong or it may spread to the ventricle and set 
 up delirium in that chamber also. When a satisfactory 
 strength of current is found, the auricular recording lever 
 should show merely an irregular twitching while that from 
 the ventricle should record a strong but very irregular con- 
 traction. The pulse tracing would also naturally be ir- 
 regular. 
 
 The tincture of digitalis (from which the alcohol has 
 been evaporated and its original volume made up by salt 
 .solution) may now be injected intravenously in 3 cc. doses, 
 the injections not being repeated at shorter intervals than 
 about four minutes. After a few of these injections have 
 been given in the manner described, the heart will show the 
 effect of the digitalis in a strong regular beat of the ventricle, 
 regular pulse, and probable increase in blood pressure. The. 
 auricle will still show its fibrillary contractions. 
 
224 LABORATORY GUIDB IN 
 
 The importance of the action of digitalis in this condition 
 is shown by the fact that it is said that of all forms of 
 cardiac arhythmias auricular fibrillation constitutes about 
 40%. 
 
-EXPERIMENTAL PHARMACOLOGY 225 
 
226 LABORATORY GUIDE IN 
 
BXPBRIMENTAL PHARMACOLOGY 227 
 
 Perfusion of Blood Vessels. 
 
 .The experiment can be carried out either upon the kid- 
 ney or other organ such as a spleen removed from a dog, 
 cat or rabbit or upon the vessels of a frog. The latter 
 animal proves very successful and that method will there- 
 fore be described first. Select a fairly large frog (30 G.- 
 40 G.), pith it and expose the heart and insert a cannula in 
 one aorta, pointing it away from the heart including the 
 second aorta in the ligature with which the cannula is tied. 
 Make a small cut in the sinus venosum so that the fluid 
 which is perfused through the vessels can escape. Suspend 
 the frog by its jaw and place under it a receptacle in which 
 the outflow can be caught. About a foot above the frog 
 place a small pressure bottle (500 cc), the lower opening 
 of which is fitted with a piece of rubber tubing upon which 
 is a snap clamp. Fill the aortic cannula with Locke's solu- 
 tion or physiological salt and fill the pressure bottle about 
 half full of the same solution. When all the air is excluded 
 from the rubber tubing connect it with the aortic cannula. 
 Open the snap clamp which has been placed on the rubber 
 tube and allow the perfusion to proceed, measuring the out- 
 flow every five minutes. After about three readings there 
 should be a fairly uniform outflow and 200 mg. of sodium 
 nitrite are added to the fluid in the bottle and the solution 
 mixed thoroughly. The readings of the outflow are con- 
 tinued and after one or two the amount obtained should 
 be very considerably increased. If it is not increased within 
 a reasonable time further nitrite may be added. Now re- 
 place the nitrite solution in the flask with fresh salt solu- 
 tion and wash out the frog's vessels, seeing whether the 
 
228 LABORATORY GUIDE IN 
 
 outflow will return to normal. After two or three readings 
 add to the solution in the flask i cc. of a i-iooo solution of 
 adrenalin. Continue the readings until a marked decrease 
 in outflow is obtained. In place of the adrenalin tincture 
 of digitalis in the strength of 3 cc. of tincture to 100 cc. of 
 salt solution may be used. 
 
 If a kidney or other organ is to be used in place of the 
 frog's vessels the experiment is carried out in essentially the 
 same manner. 
 
 The kidneys which have been removed from a dog, cat 
 or rabbit should not be kept any longer than is absolutely 
 necessary after being removed from the animal, certainly 
 not more than twenty-four hours, as after that time post- 
 mortem changes have advanced so far as to interfere with 
 the action of drugs on the vessel walls. 
 
 In preparing the kidneys for the experiment it is usually 
 best not to remove the capsule, as in so doing the paren- 
 chyma is frequently torn. Remove the fat and connective 
 tissue from around the vessels and insert a glass cannula in 
 the artery. Fill the artery and cannula with salt solution. 
 Fasten a small glass reservoir (say 250 cc.) about 1.5 meters 
 above the table and connect a long piece of rubber tubing 
 with its lower orifice, and fill reservoir and tube with Locke's 
 solution and clamp the tube. 
 
 Connect the lower end of the rubber tube with the arterial 
 cannula taking care that all air is expelled. Remove the 
 clamp from the tubing and place a receptacle under the 
 kidney to catch the fluid after it has passed through the 
 vessels and continue the experiment as described above for 
 occasions where a frog is used. 
 
nXPBRIMBNTAL PHARMACOLOGY 229 
 
230 LABORATORY GUIDE IN 
 
.EXPERIMENTAL PHARMACOLOGY 231 
 
 What do the changes in the rate of outflow under the 
 two drugs indicate in regard to their action on the blood 
 vessels? How do the results help to explain the observa- 
 tions made in the experiment on blood pressure (page 200). 
 
232 LABORATORY GUIDE IN 
 
 Action of Drugs on Isolated Tissues. 
 
 The action of many drugs in the body may be studied to 
 advantage by observing the effects of these drugs upon iso- 
 lated tissues or organs, which during the time that they are 
 in use are under as nearly normal conditions as possible as 
 far as temperature and oxygen supply are concerned. Those 
 structures which are most commonly employed in this work 
 are the uterus, or strips of intestine or blood vessels. 
 
 While more or less complicated apparatus is needed if 
 quantitative results are desired, yet if the requirements are 
 only qualitative quite a simple arrangement is all tha:t is 
 demanded. The Harvard muscle warmer and heart lever, 
 kymograph, oxygen or air supply, and warm Locke's solu- 
 tion will answer all purposes. 
 
 In case the uterus is to be used, a guinea pig (or rabbit 
 or cat) weighing 200 G. or 300 G. is killed by a blow on 
 the head and the abdomen opened and uterus exposed and 
 a fine thread tied around the ovarian end of one horn. After 
 the organ is freed from the posterior wall, another thread 
 is tied around it near its lower end, and it is then removed 
 from the body. Using the thread on the vaginal end of the 
 organ, the uterus is now tied to the end of the bent metal 
 rod of the muscle warmer. The thread on the ovarian 
 end is passed through the round opening in the plate disc 
 and fastened to a muscle lever so that the lever will record 
 the contractions of the uterus upon a lightly smoked drum. 
 Uteri differ considerably in activity as some are so active 
 that it is necessary to weight the lever in order to diminish 
 the extent of the contractions while other organs are quies- 
 
nXPHRIMBNTAL PHARMACOLOGY 233 
 
234 LABORATORY GUIDE IN 
 
BXPBRIMENTAL PHARMACOLOGY '235 
 
 cent unless aroused to action by some drug. The lower 
 end of the glass tube of the muscle lever is fitted with a 
 piece of rubber tubing which can be closed by a snap clamp, 
 and the tube is then adjusted in place around the piece of 
 uterus, and filled with Locke's solution at a temperature of 
 38° C. Air or oxygen is led to the bottom of this tube by 
 means of a capillary glass tube or a fine rubber catheter. 
 For long experiments or for any exact work the muscle 
 warmer should be enclosed in an outer bath of water main- 
 tained at body temperature, but for short experiments this 
 is hardly necessary as the Locke's solution is replaced at 
 short intervals, after the use of each drug, by fresh solu- 
 tion of the proper temperature. When the lever is record- 
 ing properly and the drum is moving at a slow rate, the 
 actions of the following drugs may be studied by adding 
 them in turn to the solution bathing the uterus. When the 
 climax of the action is reached, the used solution should 
 be drawn off and replaced by fresh and the uterus allowed 
 at least five minutes to recover before another drug is added. 
 
 Add to the solution bathing the uterus i drop of adrenalin 
 (i-iooo). When the maximum effect is obtained, draw off 
 the solution and replace it with fresh warm solution as 
 directed above. After giving the organ a rest, add i or 2 
 mg. of pilocarpine and then immediately after putting fresh 
 Locke's solution on the uterus give i to 2 mg. Atropine 
 sulphate. This may be followed in turn by pilocarpine 
 again ; Fl. Ext. Ergot 0.3 cc. ; Pituitary Extract (Pituitrin 
 2 drops) ; each in a fresh solution. 
 
 The doses given above may have to be modified to suit 
 the requirements of the individual organ. When the ex- 
 
236 LABORATORY GUIDE IN 
 
 periment is completed, fix the tracings with shellac and later 
 study them carefully, taking up the action of the individual 
 drugs, comparing the results with those obtained with these 
 drugs m other experiments. 
 
BXPERIMBNTAL PHARMACOLOGY 237 
 
238 LABORATORY GUIDE IN 
 
EXPERIMENTAL PHARMACOLOGY 239 
 
 Diuresis. 
 
 Anaesthetize a rabbit with paraldehyde (page 11) and 
 when anaesthesia is complete tie the animal on the operating 
 board. Cut the hair from the neck and also from the lower 
 part of the abdomen. Expose a jugular vein and insert a 
 cannula as usual. 
 
 To insert a cannula in the bladder to collect the urine, 
 make an incision about an inch and a half long in the median 
 line of the abdomen, beginning the incision at the symphysis 
 and extending it upward the distance named. Deepen the 
 cut until the peritoneal cavity is opened. The bladder is 
 usually found partially distended and lying amid folds of 
 fat. DraAV it out through the opening made and empty it by 
 gentle pressure, catching the urine in a dish as it flows from 
 the urethra. Save this urine to be tested for the presence of 
 sugar. 
 
 Tie a knot loosely in a piece of thread and lay it on the 
 abdomen so that the knotted thread will encircle the bladder 
 and be ready to tie the cannula in place when the latter shall 
 have been inserted. 
 
 After locating the position of the ureters, so as to avoid 
 including them in the ligature, take hold of the upper sur- 
 face of the bladder on the two sides with two pairs of for- 
 ceps and draw them gently up and outwards so as to exert a 
 slight tension on the organ. While one operator holds the 
 bladder in this way the other operator makes a cut with a 
 sharp pair of scissors midway between the forceps in the 
 fundus of the bladder. The cut will vary with the size of 
 the cannula flange (a, Fig. 10) and will probably average 
 about two centimeters in length. Into this incision now 
 
240 
 
 LABORATORY GUIDE IN 
 
 place the cannula so that its flange shall be entirely sur- 
 rounded by bladder tissue and tie it in place with the loose 
 ligature already mentioned. Examine it after it is tied to 
 see that the ureters are not included. 
 
 Fill the bladder and cannula with warm physiological salt 
 solution and connect the end of the cannula with the piece 
 of bent tubing (b, Fig. 10) which can now rest on a support 
 so as to extend over the end of the op- 
 erating board where small weighed' 
 dishes are to be placed to catch the 
 urine. 
 
 As soon as the urine begins to drop 
 from the tube a record of the time is to 
 be made, and thereafter to the end of 
 the experiment, every five minutes, the 
 dish with the urine is to be replaced 
 by a clean receptacle and the first one 
 weighed, and the weight of urine re- 
 corded. 
 
 During the course of the experiment 
 the animal should be kept covered with 
 a towel or other cloth in order to re- 
 tain as much of its body heat as possi- 
 ble. 
 
 Take two or three readings until a 
 fair regularity is obtained, and after 
 this normal is ascertained inject into the 
 
 Fig. No. io. Bladder 
 Cannula. A. Flange to Vein 40 Ulg. of thcocill^ dlsSolvcd in hot 
 be tied in bladder. B. 
 Bent delivery tube. watCr. 
 
 " 
 
 ^Theocin is artificial theophyllin. A like quantity of caffeine might be given 
 in place of the theocin except that the action of the caffeine lasts much longer 
 and may prolong the experiment unduly. 
 
. BXPBRIMBNTAL PHARMACOLOGY 241 
 
2^2 lABORA'tORY GUIDH IN 
 
nXPBRIMBNTAL PHARMACOLOGY 243 
 
 After this injection the urine will probably be increased 
 and the next drug must not be injected until the flow has 
 returned to about the normal. 
 
 Test some of the urine which was in the bladder at the be- 
 ginning of the experiment for sugar by means of the Fehling 
 test. If none is found make one or tw^o tests later in the 
 course of the experiment with urine which is excreted under 
 the action of the theocin or sodium nitrate until sugar shall 
 be found. 
 
 The excretion of iodide in the urine may also be studied 
 by the careful injection intravenously of 30 mg. sodium 
 iodide. The urine passed later is tested for iodide as fol- 
 lows : add a few drops of concentrated sulphuric acid to 
 the urine to be tested, and to this some 10% sodium nitrite 
 solution and some starch paste which if iodide is present 
 sliould be colored blue. 
 
 When the amount of urine has fallen, inject 0.250 G. 
 (5 cc. of a 5% solution) of sodium nitrate and collect the 
 urine as before. 
 
 Note. — If time allows, the diuretic effect of digitalis may be studied in the 
 same way. 
 
 Caffeine, sodium nitrate and digitalis are taken as repre- 
 sentatives of the large class of drugs known as Diuretics 
 from their action in increasing the amount of urine. 
 
 They may act either on the heart or on the kidney, and 
 they are known as cardiac or renal diuretics according to 
 the point of action. Digitalis is an example of the former 
 class, as it causes diuresis indirectly through its effects on 
 the circulation. Caffeine probably acts directly on the renal 
 epithelium, while various salts, of which sodium nitrate is 
 an example, induce diuresis by their salt action (sahne 
 diuretic) in addition to which there may be an action on the 
 kidney epithelium. 
 
244 LABORATORY GUIDE IN 
 
 Therapeutic uses of the Diuretics. 
 
 See also under Caffeine and Digitalis, Therapeutic uses, 
 (pages 140 and 179). 
 
 These drugs or their allies are chiefly employed in diseased 
 conditions in which the amount of urine excreted is below 
 the normal, a condition which may result from disease of 
 the kidneys or of the heart. In conditions of oedema or 
 ascites, there is an abnormal collection of fluid in the tis- 
 sues; its removal may be aided by the use of the diuretics. 
 In poisoning with an irritant drug which is eliminated by 
 the kidneys, the latter may be protected from the action of 
 the toxic substance by increasing the volume of urine 
 through the administration of these drugs. 
 
nXPBRIMBNTAL PHARMACOLOGY 245 
 
246 
 
 LABORATORY GUIDE IN 
 
- BXPBRIMENTAL PHARMACOLOGY 247 
 
 Action of Drugs on the Cervical Sympathetic Nerves and 
 on Intestinal Peristalsis.^ 
 
 Ansesthetize a rabbit (an albino if possible) with paralde- 
 hyde (page 11) and when anaesthesia is complete tie the 
 animal on the operating board and remove the hair from 
 the neck region. Expose the jugular vein on one side and 
 also the trachea and insert cannulas in each. Expose the 
 carotid sheath on the other side, open it, laying bare the 
 vessels and nerves. The largest nerve seen is the vagus and 
 the smallest is the depressor nerve, while the third is the 
 cervical sympathetic trunk, which is to be separated very 
 carefully from all connective tissues and a fine thread passed 
 under it. Make sure you have the correct nerve by gently 
 raising it and stimulating it with a weak tetanizing current, 
 observing meantime the effect on the pupil on the corre- 
 sponding side; it should dilate during the stimulation. Tf 
 the correct nerve has been isolated tie the ligature around it 
 as low down in the neck as convenient and cut the nerve 
 trunk below the ligature. Hold the animal's ears up gently 
 against the light so that the vessels may be clearly seen, and 
 observe the difference in their size on the two sides ; note 
 also the difference in the temperature of the two ears. The 
 differences in the vessels in the two ears may be best seen 
 by comparing the smaller branches rather than the main 
 arteries. Compare the size of the pupils on the two sides. 
 How would you explain these changes ? 
 
 Make a longitudional incision about 10 cm. long in the 
 middle line of the abdomen extending the cut completely 
 
 1 The expjeriment on intestinal peristalsis may be run as a separate experi- 
 ment (see page 292) or as directed here in conjunction with the cervical 
 =;-vmpathetic experiment. 
 
248 LABORATORY GUIDE IN 
 
 through the wall. Pulling the two sides of the wall apart 
 insert in the incision a glass evaporating dish measuring 
 about 10 cm. in diameter. Place the dish between the skin 
 and muscle layers so that it will be held in place by the 
 wall and thus serve as a window. It may be necessary to 
 pull the caecum to one side in order to permit of a free 
 view of the small intestine. Observe closely the normal 
 movements of the intestine. 
 
 Now raise the trunk of the cut cervical nerve carefully by 
 the ligature and stimulate it with the electric current as be- 
 fore. During this stimulation look especially for any 
 changes in the calibre of the ear vessels on that side; and 
 for any changes in the position of the eyeball, or of the eye- 
 lids, or in the size of the pupil. After the changes have been 
 seen and described, inject into the jugular vein 3 mg. of nico- 
 tine chloride. Immediately after the injection notice the ef- 
 fect of the drug on the animal's respiration (make sure the 
 animal starts breathing again, otherwise start artificial res- 
 piration), and also look for any movement in the anim^al's 
 whiskers. 
 
 Again stimulate the cut cervical sympathetic comparing 
 ' the results with those obtained at the first stimulation. 
 (Should they be the same as before, it will be necessary to 
 inject more nicotine.) Now using very careful dissection 
 trace the trunk of the sympathetic toward the head until the 
 superior cervical ganglion is reached. It will be recognized 
 as a small whitish enlargement on the trunk of the nerve. 
 When it is found, stimulate the trunk of the sympathetic be- 
 yond the ganglion, that is, between the ganglion and the 
 head of the animal. Compare the results of stimulation at 
 this point with those obtained below the ganglion. 
 
EXPERIMENTAL PHARMACOLOGY 249 
 
250 LABORATORY GUIDE IN 
 
BXPBRIMENTAL PHARMACOLOGY 251 
 
 Note. — In nddition to the nerve fibres which run to the eye by way of the 
 cervical sympathetic and which are distributed to the radial mtiscle fibres of 
 the iris there are nerves which control the circular fibres. These come from 
 the motor oculi to the ciliary ganglia and run from that point to the iris. 
 The size of the pupil can be changed by drugs acting along the course of 
 these fibres also. 
 
 Inject into the jugular vein 5 mg. of pilocarpine (i cc. of 
 0.5% solution) and observe any change in the pupil and also 
 in intestinal peristalsis. (Note any effect on the heart rate.) 
 Now inject i mg. atropine sulphate (i cc. of 0.1% solution). 
 What effect has it upon the pupil, peristalsis and heart rate? 
 
 Finally inject a small dose of suprarenal extract 
 (adrenalin i-iooo solution, i drop in salt solution) note 
 changes in peristalsis, calibre of vessels and heart rate. 
 
 What effect has nicotine upon the sympathetic gangha? 
 
 Cocaine dilates the pupil by stimulating the terminations 
 cf the nerves from the superior cervical ganglia which end 
 on the radial fibres. 
 
 Physostigmine (Eserine) acts on the same point and in 
 the same manner as pilocarpine, i. e., on the nerve termina- 
 tions on the circular muscular fibres; atropine acts at the 
 same place, but instead of stimulating the nerve endings, its 
 action is to paralyze them, removing the pilocarpine effects. 
 
 Therapeutic Uses. 
 
 Physostigmine (eserine) and, less commonly, pilocarpine 
 are used in ophthalmological practice to contract the pupil of 
 the eye in certain diseased conditions, the most important of 
 which is glaucoma. For this purpose a solution of ^ to 1% 
 is dropped in the eye as often as necessary. Physostigmine 
 is also occasionally used to stimulate intestinal peristalsis in 
 cases of atony of the intestine. For this purpose it is given 
 by hypodermic injection. 
 
252 LABORATORY GUIDB IN 
 
 Atropine is used to dilate the pupil to permit of ophthal- 
 moscopic examinations, and as a therapeutic agent in many 
 ocular diseases. It is also used in cases of colic or griping 
 to overcome the abnormal contractions of the intestine. 
 Homatropine, an artificial alkaloid resembling atropine in 
 its action on the eye, is largely used as a substitute for the 
 latter, as its action is not nearly so prolonged. 
 
 Cocaine is also largely employed to dilate the pupil to 
 allow of examination of the interior of the eye. 
 
 Mydriatics are drugs which dilate the pupil; myotics con- 
 tract it. 
 
EXPERIMENTAL PHARMACOLOGY 2 
 
 JO 
 
254 LABORATORY GUIDE IN 
 
BXPBRIMBNTAL PHARMACOLOGY 255 
 
 Salivary and Pancreatic Secretion.^ 
 
 Ansesthetize a large dog with morphine and chloretone 
 (page 12) and if necessary supplement them by the use of 
 chloroform. When anaesthesia is complete, expose a vein 
 in each leg and insert cannulas. As a matter of precaution, 
 the trachea may also be exposed and a cannula tied in it to 
 permit of artificial respiration should it become necessary. 
 
 The submaxillary duct and chorda tympani nerve are to 
 be dissected out according to the following directions. (This 
 dissection _ should have been previously carried out on the 
 head of a dead animal (from Exp., page 212) so as to allow 
 greater speed in operating on the living dog.) 
 
 "Make an incision 3 or 4 inches long through the skin and 
 platysma muscle along the inner border of the lower jaw, 
 beginning about the angle of the mouth and continuing back- 
 ward towards the angle of the jaw. Ligate doubly and 
 divide any vessels that come in your way. Divide the digas- 
 tric muscle about its anterior third and clear it from its at- 
 tachment. The broad-, thin mylo-hyoid will now" be seen 
 with its motor nerve lying on it. Divide the muscle across 
 its middle and dissect it up carefully. The lingual nerve is 
 seen emerging from under the ramus of the jaw, running, 
 transversely towards the middle line and then passing for- 
 ward parallel to the larger hypoglossal nerve. In its trans- 
 verse course it crosses the submaxillary and sublingual 
 ducts. The chorda tympani leaves the lingual nerve and 
 runs backward along the duct towards the gland. Put a 
 glass cannula into the duct in the same way as you would 
 
 ^ The salivary and pancreatic secretion experiments may be run separately if 
 preferred. 
 
256 
 
 LABORATORY GUIDE IN 
 
 put a cannula in a vein. Trace back the lingual and put a 
 ligature around it and tie it as far back as possible, and then 
 cut it centrally so as to permit of the chorda tympani being 
 put on electrodes." (Stewart.) Connect the cannula with 
 a long, horizontal tube on which a scale is marked. 
 
 Fio. No. II. Dog's head dissectpd to show Chorda Tympani. A. Chorda Tym- 
 pani, B. Wharton's duct, C. Hypoglossal nerve, D. Mylo-hyoid muscle 
 turned back, E. Lingual nerve, F. Digastric muscle cut, G. Ra- 
 mus of jaw cut away to show nerve emerging from 
 behind it. 
 
 While the dissection of the duct is being carried on, a 
 second operator is to dissect out the cervical sympathetic 
 trunk^ on the same side. In the dog the cervical sympathetic 
 is contained in the carotid sheath and closely connected with 
 the vagus, from which it may be separated by careful dis- 
 
 ^ This part of the experiment may be omitted if desired. 
 
EXPBRIMMNTAL PHARMACOLOGY 257 
 
2^8 LABORATORY GUIDB IN 
 
EXPERIMENTAL PHARMACOLOGY 259. 
 
 section, as the fibres run in distinct strands. For some dis- 
 tance below the superior cervical ganglion the two nerve 
 trunks are not connected. After the sympathetic is isolated 
 tie a ligature around it and cut the nerve below it. 
 
 If the experiment has been properly done, both nerves 
 (viz., the chorda and the sympathetic) can be stimulated by 
 placing them on electrodes, and any secretion from the gland 
 will pass into the horizontal tube, Avhere it can be measured 
 on the scale. 
 
 The pancreatic duct is exposed in the following manner. 
 An incision is made through the abdominal wall about 6 or 
 S cm. to the right of the median line and starting from the 
 costal border and extending caudalwards for a distance of 
 about 15 to 20 cm. The duodenum is located and pulled 
 out through the incision and the pancreas is found attached 
 to it for some distance. Starting at the point where the 
 tail of the pancreas leaves the intestine and following up to- 
 ward the pylorus for about 3 cm. the pancreas is separated 
 from its anterior attachment to the intestine by blunt dissec- 
 tion. At about this point the large duct of the pancreas will 
 be found entering the wall of the intestme. It is very short. 
 The surrounding tissue is separated from the duct and a 
 ligature placed around it. A V-shaped cut is made in it and 
 a glass cannula inserted and fastened by means of the liga- 
 ture. A small calibre tube is now connected to the cannula 
 by means of a piece of rubber tubing and the pancreatic 
 secretion observed either by means of a scale back of the 
 tube or by the drops falling from the end. The intestinal 
 loop is now returned to the abdomen or may be secured out- 
 side protected by warm moist towels and the abdominal in- 
 cision closed as far as possible. 
 
26o LABORATORY GUIDE IN 
 
 1. Stimulate the chorda tympani with a weak induction 
 current and watch the effect on the secretion. 
 
 2. When the effect of chorda stimulation has passed off 
 stimulate the sympathetic trunk (peripheral or head end) 
 and observe the difference in the quantity and quality of the 
 saliva when compared with the "chorda saliva." 
 
 3. Inject 5 mg. of nicotine chloride and observe closely 
 the effect upon the salivary secretion. Watch the effect 
 upon the respiration and heart rate. (Be sure the respira- 
 tion continues after the nicotine has been injected.) 
 
 4. Stimulate the chorda again and compare the results 
 v/ith those obtained in i. 
 
 5. Stimulate the sympathetic and compare with 2. 
 
 6. If the nerves are still active, repeat the injections of 
 nicotine, giving 20 mg. each time until no further effect is 
 
 Preparation of secretin. The secretin which is to be used in the experi- 
 nitni. is to be prepared from the intestine of one of the dogs which has been 
 used earlier in the course. Its mode of preparation is as follows: Immediately- 
 after the animal has been killed, open the abdomen and remove about 1.5 
 meters of the upper part of the intestine. Cut this piece open longitudinally 
 under running cold water and after it is cleansed, spread it flat on a piece 
 of glass and scrape off the mucosa by means of a second piece of glass. Put 
 this material in a large mortar with about an equal volume of fine clean 
 sand and from 100 to 300 cc. of 0.4% HCl, the amount depending upon the 
 bulk of sand and* mucosa. Grind this mass now thoroughly for ten minutes 
 sc as to get the tissue cells well broken up. Transfer the mixture to a large 
 evaporating dish and boil it for a minute or two, keeping it well stirred. Now 
 add a sufficient quantity of a fairly strong solution of NaOH so as to give 
 an alkaline reaction and follow this with sufficient 25% glacial acetic acid to 
 give a distinct acid reaction. Strain the mixture now through muslin, pressing 
 out all the fluid possible. This fluid, which should be rather strongly opalescent, 
 is now partially sterilized by placing the flask containing it over a water bath 
 for ten minutes and then plugging it with absorbent cotton. Keep it in a 
 cool place until it is needed. It may be injected in this strength or still bet- 
 ter diluted with salt solution. The normal pouring out of secretin into the 
 blood may perhaps be best simulated by diluting the stock solution of secretin 
 with many times its volume of physiological salt solution and then placing this 
 solution in a burette and connecting it with a femoral vein, allowing it to 
 enter the blood stream slowly, and continuously. Marked effects on the blood 
 pressure are thus avoided and a constant secretion from the pancreas obtained. 
 This latter method is the one to be used in this experiment. 
 
nXPERIMENTAL PHARMACOLOGY 261 
 
262 LABORATORY GUIDE IN 
 
MXPBRIMBNTAL PHARMACOLOGY 263 
 
 obtained on nerve stimulation. How do the effects of sub- 
 sequent injections compare with the primary injections? 
 
 7. Trace the trunk of the sympathetic to the superior 
 cervical ganglion and stimulate the nerve beyond, observing 
 if secretion can be obtained. 
 
 Inject the following drugs in the order named, observing 
 their effects upon both the salivary and pancreatic secretion : 
 
 8. Pilocarpine, 5 mg. 
 
 9. Atropine, i mg. 
 
 10. Pilocarpine, 5 mg., compare with 8. 
 
 11. Suprarenal extract, Adrenahn chloride, i-iooo sol., 
 5 drops in salt solution. 
 
 What effect has nicotine upon the sympathetic ganglia? 
 Compare also the experiment on the sympathetic ganglia in 
 the rabbit (page 247). 
 
 As was shown in this experiment, the salivary gland is 
 innervated from two sources, both of which are acted on by 
 nicotine. Pilocarpine and atropine aft'ect the salivary secre- 
 tion by acting on the cranial nerve supply of the gland. The 
 action of the suprarenal gland extract in causing secretion 
 is believed to be upon the terminations of the sympathetic 
 fibres in the gland cells. (It is not always possible to get 
 a secretion of saliva by the injection of suprarenal extract.) 
 
 12. After the effects of the suprarenal extract have 
 passed oft", start the infusion of the secretin, giving it quite 
 slowly. In a minute or two the pancreas should begin to 
 secrete actively. When the secretion is well established and 
 while the secretin is still entering the vein inject into the 
 opposite femoral vein suprarenal extract as was done in ii. 
 What effect has this upon the pancreatic secretion and what 
 
264 LABORATORY GUIDE IN 
 
 explanation can you suggest for it, knowing the effect of 
 the drug upon the blood vessels? 
 
 13. Tr} the effect of an injection of 10 mg. of nicotine. 
 The influence of this injection may not be very marked on 
 account of the earlier use of nicotine in the experiment. 
 
 14. Give an injection of pituitary extract (0.5-1 cc. 
 pituitrin). These last two drugs also cause marked con- 
 striction of the abdominal vessels. 
 
 The action of the hormone, secretin, upon the pancreas is 
 in marked contrast to that of pilocarpine which is so active 
 in producing a secretion from the salivaiy gland, and it 
 illustrates probably the relative importance of chemical and 
 nervous control over the secretion of this gland. Any drug 
 which constricts the vessels of the gland and thus cuts off 
 its blood supply will in this way inhibit its activity. 
 
BXPBRIMBNTAL PHARMACOLOGY 265 
 
266 LABORATORY GUIDE IN 
 
. EXPERIMENTAL PHARMACOLOGY 267 
 
 Antipyretics. 
 
 The temperature of the animal body is determined by the 
 relation between heat formation and heat dissipation. To 
 maintain a uniform temperature in warm blood animals, it 
 is necessary, therefore, to have some mechanism to preserve 
 a balance between these two activities. This heat regulating 
 mechanism has not been shown to be an anatomical or 
 physiological unit or definite center, but on the other hand 
 some of the well-known centers are closely connected with 
 heat control. 
 
 Temperature changes in the warm blooded animal are 
 usually brought about by a disturbance of the regulating 
 mechanism. For instance, the temperature may be lowered 
 by depressing the central nervous system as by ether, chloro- 
 form or chloral, under the influence of which less heat is 
 formed; or the temperature may be lowered by increasing 
 heat output either by dilating the peripheral vessels and ex- 
 posing more blood to the outside air or by increasing the 
 production of sweat. The typical antipyretics, such as 
 acetanihde and acetphenetidine, act after this manner in- 
 creasing output. They have practically no effect against 
 normal temperature, acting only in fever. Another drug 
 which lowers temperature is quinine and this drug acts di- 
 rectly upon metabolism lessening the formation of heat. 
 
 Drugs sucli as strychnine or atropine which increase 
 muscular movements, may raise the temperature, but the 
 most important agents to increase temperature are the 
 bacterial poisons and toxins. 
 
 The action of the antipyretics may be studied in the fol- 
 lowing experiments : — 
 
268 LABORATORY GUIDE IN 
 
 Select three rabbits of about the same size, weighing say 
 1500 G. to 2000 G., and take the rectal temperature of each. 
 The thermometer bulb should be vaselined in each case be- 
 fore use and should be inserted always about the same dis- 
 tance — say 2 inches. As the fever takes some time to de- 
 velop, for afternoon use, two of the three rabbits should be 
 injected subcutaneously at 9 A. M. with Witte's Peptone 
 I G. per kilogram of body weight. The temperature should 
 be taken each hour and plotted upon a chart. By afternoon 
 both animals should have a well developed fever. Inject 
 subcutaneously into one of these animals 200 mg. antipyrine 
 and use the other fe/ered animal as a control. Into the 
 third rabbit, vvhich is normal, inject 200 mg. antipyrine and 
 this animal will then serve as a further control in the ex- 
 periment for the effect of the drug. The temperatures of 
 the three animals should then be taken every half hour 
 during the afternoon. 
 
 In place of using peptone to produce the fever, it is often 
 possible to secure even more satisfactory results by the use 
 of the increase in temperature which occurs in the ana- 
 phylactic reaction. To utilize this phenomenon, two rabbits 
 should be sensitized by the injection of 2 cc. of beef serum 
 two weeks before they are needed for the experiment in 
 fever. On the day of the experiment at about 12 o'clock 
 (for afternoon work) each of the two rabbits should be in- 
 jected in the ear vein with i cc. of a i-io solution of beef 
 serum. By i :30 each of the rabbits will have fever. One 
 of these fevered animals and a third (normal) rabbit are 
 now injected with 200 mg. antipyrine and the temperature 
 of each animal is taken every half hour and plotted on 
 a chart as described in the earlier experiment. One of the 
 
BXPBRIMHNTAL pharmacology 269 
 
270 LABORATORY GUIDB IN 
 
EXPERIMENTAL PHARMACOLOGY 
 
 271 
 
 animals will show the normal fever curve; the second will 
 show the curve as modified by the antipyretic, and the third 
 curve will show the absence of effect in the case of the use 
 of an antipyretic in an animal with a normal temperature. 
 
 Taste acetanilide, acetphenetidine and antipyrine, and test 
 the solubility of each in water. How would they be pre- 
 scribed ? 
 
272 LABORATORY GUIDE IN 
 
 Biological Assay. 
 
 One of the essential requirements in any medicine which 
 is to be used in the treatment of disease is that not only shall 
 it possess the properties usually found in that drug, but also 
 that it shall be of a definite and uniform strength. With 
 a great many drugs, such as opium and nux vomica the 
 active principicc are alkaloids and are well known, and in 
 such cases unifoim preparations are obtainable by chemical 
 means as these alkaloids can be isolated in pure form and 
 weighed. For instance, the Pharmacopoeia directs that lOO 
 cc. of Tincture of Opium shall contain i Gram of anhydrous 
 morphine and lOO cc. of tincture of nux vomica shall con- 
 tain 0.25 grams of the alkaloids of the drug, etc. With other 
 drugs, however, the active principles are either not known, 
 or if known, it frequently happens that they cannot be 
 isolated without great difficulty and in the process of isola- 
 tion some of the active substance may be destroyed. To this 
 group of drugs to which chemical standardization cannot 
 well be applied belong the members of the digitalis group, 
 digitalis, stropnanthus, squills; ergot; cannabis indica; 
 aconite and extracts of the suprarenal and pituitary glands. 
 
 For such drugs in place of chemical standardization meth- 
 ods have been introduced by which the strength of prepara- 
 tions is ascertained by the effects which they produce upon 
 animal tissues. This method is known as Physiological or 
 Biological Assay or Standardization. 
 
 On account of the importance of the subject, a brief dis- 
 cussion may be given of the principal methods in use. Ful- 
 ler details and directions for the carrying out of the tests 
 may be found in the Pharmacopoeia. 
 
BXPBRIMBNTAL PHARMACOLOGY 273 
 
274 LABORATORY GUIDE IN 
 
, EXPERIMENTAL PHARMACOLOGY 275 
 
 For the standardization of digitalis and its allies frogs 
 are used more frequently than any other animals. One of 
 the most commori methods and the one recommended by 
 the Pharmacopoeia is to ascertain the minimum dose of 
 digitalis which will bring the heart of a frog to a systolic 
 standstill in one hour. This method has been carried out 
 in the experiment under the general subject of digitalis 
 (Exp. VI, page 171). Fuller details are to be found in 
 the Pharmacopoeia. 
 
 Another important method of assaying digitalis is to as- 
 certain the minimal fatal dose for a frog of a certain size, 
 and from the knowledge of the strength of the preparation 
 thus gained adjustments to a standard strength are made. 
 This is the so-called "Toxic" method. 
 
 In the Focke method the time of cessation of the circula- 
 tion is observed when a certain dose of digitalis is given. 
 Again the toxic dose for guinea pigs and also for cats are 
 obtained in still other methods of assay and adjustments 
 made accordingly. While the two frog methods which were 
 mentioned first are probably the most largely used of any, 
 yet each method enumerated has certain adherents who pre- 
 fer it to any other. 
 
 Brgot. 
 
 Ergot preparations are also standardized according to 
 biological metliods because on account of the complexity of 
 its chemical constitution a standardization by chemical 
 means is impossible. There are two principal methods in 
 vogue. The first is to test the strength of a preparation 
 upon an isolated uterus of a guinea pig or other animal 
 comparii:g its activity with that of some known standard. 
 
276 LABORATORY GUIDE IN 
 
 The method is described in the chapter on "Perfusion of 
 Isolated Organs" (Page 232). 
 
 The second method for ergot depends upon the darkening 
 or cyanosis which is produced in the cock's comb by ergot 
 preparations. 
 
 Experiment. Select preferably Leghorn roosters weighing 
 about 1000 G. to 1200 G. and inject into the breast muscle of 
 one a dose of the standard preparation which is known to 
 produce a mild degree of cyanosis of the comb. Into the 
 breast muscle of the second rooster inject a corresponding 
 dose of the unknown preparation and compare the intensity 
 of the discoloration in the combs of the two birds at the end 
 of one hour. This should give some idea as to ihe relative 
 activity of the two preparations. Two or three days later 
 inject the birds again reversing the order of the preparations 
 upon the two birds and giving doses modified by the experi- 
 ence gained by the first injection so that they shall give re- 
 sults which are more comparable. In subsequent injections 
 the doses are narrowed down still further until it is known 
 exactly how much of the solution of unknown strength it is 
 necessary to give to produce results analogous to those ob- 
 tained from a certain dose of the standard. Knowing this 
 relation it is easy to calculate how much the unknown solu- 
 tion must be diluted or concentrated to conform to the stand- 
 ard adopted. The results obtained by the isolated uterus 
 method and by the cock's comb method run roughly parallel 
 to each other. 
 
 Aconite. 
 
 This drug is frequently assayed biologically as its activity 
 depends upon the presence of alkaloids differing from each 
 other in strength. In order, therefore, to get an estimate 
 
BXPBRIMBNTAL PHARMACOLOGY 277 
 
278 LABORATORY GUIDB IN 
 
BXPBRIMBNTAL PHARMACOLOGY 279 
 
 of the activity of the total alkaloids biological methods are 
 necessary. The method employed is to ascertain the min- 
 imal fatal dose of the aconite preparation for a guinea pig. 
 The standard lethal dose adopted by the Pharmacopoeia for 
 the tincture of aconite is 0.0004 cc per gram of body weight 
 of guinea pig. The details of the assay as described in the 
 Pharmacopoeia were carried out in the experimental work 
 on Aconite (Page 183). 
 
 Cannabis Indica. 
 
 The assay of cannabis depends upon the fact that this drug 
 produces in dogs certain symptoms of muscular inco -ordina- 
 tion. The method therefore consists of ascertaining the 
 dose of a preparation which will produce these symptoms 
 and then adjusting its strength to the standard requirements. 
 The directions for carrying out the test are to be sought in 
 the Pharmacopoeia and a preparation of cannabis is to be 
 assayed according to those directions. 
 
 Suprarenal Gland. 
 
 The assay of products of the suprarenal gland is carried 
 out by means of a comparison of the blood pressure rise 
 produced in dogs by an extract of the glands with that pro- 
 duced by a solution of known strength of the active princi- 
 ple of the gland. According to the standard adopted each 
 gram of the dried suprarenals shall contain the equivalent 
 of 10 mg. of the active principle of the gland. 
 
 The directions for carrying out this test are given in the 
 Pharmacopoeia. 
 
28o LABORATORY GUIDE IN 
 
 Pituitary Gland. 
 
 The active principle of this gland has not been isolated in 
 pure form and therefore products made from the gland have 
 to be assayed biologically. The method which is used almost 
 exclusively is the isolated uterus method which as men- 
 tioned above is used for ergot assay and which is described 
 in the chapter on "Perfusion of isolated organs" (page 232). 
 
 The Pharmacopoeia directs that 1 cc. of the solution of 
 the Pituitary Gland shall have the same activity on the iso- 
 lated uterus of a virgin guinea pig as a i to 20 million solu- 
 tion of beta-iminazolyl-ethylamine hydrochloride. 
 
 A second method which is not so commonly employed is 
 similar to that in use for suprarenal glandular products. A 
 comparison is made of the rise in blood pressure produced 
 by a certain dose of the product to be a.-^sayed with that 
 produced by a standard preparation. 
 
nXPURIMBNTAL PHARMACOLOGY 281 
 
82 LABORATORY GUIDE IN 
 
 2»2 
 
EXPERIMENTAL PHARMACOLOGY 283 
 
 Pilocarpus. 
 
 Pilocarpine is an alkaloid derived from the leaves of sev- 
 eral species of Pilocarpus. It has been employed in certain 
 of the earlier experiments, which may be summarized in 
 order to give a more complete picture of the action of the 
 drug. 
 
 Describe the effects of pilocarpine on — 
 
 the frog's heart (Exp. page 187) ; 
 the turtle's heart (Exp. page 191) ; 
 the pupil of the eye (Exp. page 247) ; 
 the salivary secretion (Exp. page 255) ; 
 and on the intestinal peristalsis (Exp. page 247). 
 How does atropine affect the activity of the drug? 
 I. (These drugs will be furnished you.) Take c.003 G. 
 of pilocarpine hydrochloride and describe any symptoms 
 noted. Should any of these become unpleasant take atropine 
 sulphate 0.0005 G. and describe any changes. 
 
 Other drugs which resemble pilocarpine in many of their 
 actions are Muscarine, from one of the poisonous mush- 
 rooms (Agaricus muscarius), and Physostigmine or Hs- 
 serine, from the Calabar or Ordeal bean (Physostigma 
 venenosum) . 
 
 Examine the leaves of Pilocarpus jaborandi, and the 
 calabar bean. 
 
 Therapeutic Uses. 
 The employment of pilocarpine in diseases of the eye is 
 discussed elsewhere (page 251)." The drug is practically 
 never used for its cardiac action. 
 
 It is used to increase the activity of the sweat glands 
 (diaphoretic action) in various diseases in which there is 
 
284 LABORATORY GUIDB IN 
 
 an abnormal collection of fluid in the body. (Compare Di- 
 uretics, page 244). Through its action a very large amount 
 of fluid may be excreted so that in some cases the body may 
 lose from four to nine pounds in weight from a single dose 
 of the drug. 
 
 Pilocarpine is employed in some diseases of the kidneys in 
 which the urine excretion is lessened, with the idea of se- 
 curing the elimination of toxic materials by the sweat glands. 
 
EXPERIMENTAL PHARMACOLOGY 285 
 
286 LABORATORY GVIDB IN 
 
EXPBRIMBNTAL PHARMACOLOGY 287 
 
 The Nitrite Series. 
 
 This series comprises several drugs wliose action in the 
 body is confined almost exclusively to the blood vessels. The 
 principal members are Auiyl Nitrite, Nitroglycerin, Sodium 
 and Potassium Nitrite. 
 
 They have been employed in various experiments and the 
 results are to be collected and summarized. 
 
 Describe the effects of the nitrites on — 
 
 the mammalian blood pressure (Exp. page 207) ; 
 perfused vessels (Exp. page 227) ; 
 on the vessels of the intestine (Exp. page 299) ; 
 and on the kidney vessels (Exp. page 304). 
 
 Inhale 3 drops of amyl nitrite. 
 
 Therapeutic Uses. 
 
 The members of this series are employed in medicine to 
 lower the blood pressure and to relieve vascular spasm. 
 
288 LABORATORY GUIDE IN 
 
 Suprarenal Gland Extract. 
 
 The suprarenal glands contain a very active substance. 
 Epinephrine, which can be isolated, but only with consider- 
 able difficulty. 
 
 For experimental work the commercial adrenalin chloride (i-iooo) may be 
 used. From one to three drops of this preparation diluted with salt solution, 
 give good results. 
 
 Describe the effects of extract of suprarenal gland upon 
 
 the blood pressure (Exp, page 200) ; 
 
 the mammalian heart (Exp. page 207) ; 
 
 perfused vessels (Exp. page 227) ; 
 
 the salivary secretion (Exp. page 255) ; 
 
 the kidney vessels (Exp. page 304) ; 
 
 and upon intestinal vessels (Exp. page 299). 
 
 I. Examine the local effects of the drug by putting one 
 drop of adrenalin chloride (i-iooo) in the eye. Compare 
 with the normal eye. How would this experiment aid you 
 in explaining the results obtained in the experiment on blood 
 pressure? 
 
 Therapeutic Uses. 
 
 The powerful effects of the drug on the circulation are 
 limited in their application to therapeutics by their transitory 
 nature and by the fact that in order to act on the heart or 
 blood vessels (except the local action), the drug has to be 
 injected intravenously. In some cases of shock and collapse 
 it has been given in this way, but it requires constant admin- 
 istration as the effects pass off so rapidly. It is of most 
 value for local application in surgical operations to constrict 
 the vessels and thus render the field of operation bloodless. 
 
MXPBRIMBNTAL PHARMACOLOGY 
 
2go LABORATORY GUIDE IN 
 
nXPBRIMENTAL PHARMACOLOGY 291 
 
 Pituitary Extracts. 
 
 Extracts of the posterior lobe of the pituitary gland pos- 
 sess quite a powerful action which is exerted mainly upon 
 structures containing smooth muscle. Their most important 
 cfiect perhaps is upon the blood vessels where a stimulant 
 action leads to an increased blood pressure. They also 
 cause contractions in other organs especially in the uterus 
 and bladder. The action is probably not upon any nerve 
 structure but is direct upon the muscle cell. 
 
 The effect of extracts upon the circulation is studied in 
 the experiment on page 207. 
 
 The action on smooth muscle is studied in the experiment 
 on page 232. 
 
 Inasmuch as extracts of the gland differ considerably in 
 strength, it is very desirable that some method of assay and 
 of standardization should be utilized in their manufacture in 
 order to insure a uniform strength in the glandular products. 
 It is impossible to standardize them by chemical means at 
 present on account of our lack of knowledge of the chem- 
 istry of the active principle of the gland. However, biol- 
 ogical methods of assay have been adopted to a greater or 
 less extent, and these are described in the special chapter 
 devoted to the subject. Page 280. 
 
292 LABORATORY GUIDE IN 
 
 The following experiments are not suitable for the regu- 
 lar laboratory course, but may be carried out by advanced 
 students ; they will also serve very well for class demonstra- 
 tions by the instructor in charge. 
 
 Intestinal Peristalsis.^ 
 
 The peristaltic movements of the intestines and the effects 
 of various drugs upon them may be demonstrated very well 
 by submerging an animal in a saline solution, opening the 
 abdomen and keeping the intestines under the surface of the 
 water by means of a pane of glass. 
 
 For this experiment a metallic tank is needed which will 
 be large enough to allow a rabbit to be placed in it when the 
 animal is tied to an operating board. A tank measuring 
 70x30x30 cm. is large enough for most purposes and it 
 should be mounted on legs long enough to allow a Bunsen 
 burner to be placed under it. The operating board should 
 be weighted with lead so that it will sink in the water. A 
 pane of glass to cover the intestines and some small lead 
 piping, which can be bent over the sides of the tank to act 
 as a support for the glass, and a bath thermometer complete 
 the special apparatus necessary for the experiment. 
 
 Fill the tank about two-thirds full of water and add 
 sodium chloride to make a physiological salt solution (8 G. 
 per liter) . Light the Bunsen burner under the tank and heat 
 the solution until it is of the body temperature. 
 
 ^ This experiment may have been carried out in connection with the experi- 
 ment on the cervical sympathetic nerve in which case it would not be neces- 
 sary to repeat it. The directions herein given, however, serve very well in 
 case the experiment has not been carried out earlier in the course. 
 
MXPBRIMBNTAL PHARMACOLOGY 293 
 
294 LABORATORY GUIDB IN 
 
■EXPERIMENTAL PHARMACOLOGY 295 
 
 Operation. 
 
 In the meantime ansethetize a rabbit or cat in the usual 
 manner and when anaesthesia is complete insert a venous 
 cannula in the jugular vein and also insert a tracheal can- 
 nula. Place short pieces of rubber tubing on both free ends 
 of the tracheal cannula and close one by means of a clamp. 
 The second piece of tubing is to extend above the surface of 
 the water when the animal is submerged. 
 
 Place the animal in the Avarm salt solution which should 
 completely cover its body. Take great care no water enters 
 the tracheal tube, the open end of which is to be tied to a 
 support above the tank. Make an incision through the skin 
 and muscles in the median line of the abdomen from the end 
 of the sternum to the symphysis. The abdominal walls may 
 be drawn outward and secured to the operating board by 
 means of tacks. The intestines will float to the top of the 
 water and they must be held just beneath the surface by 
 placing the pane of glass over them supporting the latter by 
 means of the lead tubing. 
 
 The peristaltic movements may now be observed as w^ell 
 as the vascular changes which will follow the injection of 
 the drugs named below ; the changes in color of the intestines 
 indicate the alterations in the circulation of tlie biood. 
 
 Inject into the jugular vein the following drugs in the 
 order named : — • 
 
 Nicotine chloride, 5 mg., 
 Pilocarpine hydrochloride, 5 mg., 
 Atropine sulphate, i mg., 
 
 Physostigmine hydrochloride, 3 mg., injected 
 slowly. (Observe any changes in the skeletal 
 
296 LABORATORY GUIDE IN 
 
 muscles such as twitching. This injection may- 
 have to be repeated to secure well marked re- 
 sults.) 
 
 Adrenalin chloride (i-iooo sol.), 2 drops in 
 salt solution. 
 
 Amyl nitrite, 2 or 3 drops given by inhalation. 
 
MXPBRIMBNTAL PHARMACOLOGY 297 
 
LABORATORY GUIDE IN 
 
.EXPERIMENTAL PHARMACOLOGY 299 
 
 Vasomotor Changes in the Intestine. 
 
 The special apparatus needed for this experiment is, first, 
 some form of intestinal plethysmograph and second, an in- 
 strument to record the intestinal vascular changes upon a 
 blackened drum. 
 
 The plethysmograph may be made very easily of plaster 
 of Paris after the pattern devised by A. Edmunds.^ 
 
 With the plaster make a hemispherical basin ten centi- 
 meters in diameter and four deep with the walls about seven 
 millimeters thick and with an elliptical opening in the bottom 
 of the basin three centimeters by one and a half. Make a 
 small opening in one side of the apparatus and cement in it 
 a short piece of glass tubing which connects with the interior 
 of the basin and which extends outside three or four centi- 
 meters to allow of rubber pressure tubing being connected 
 with it. Smooth off the upper edge of the apparatus with 
 sand paper so that a piece of glass will fit fairly tightly on it. 
 A plethysmograph made in this way will answer all the pur- 
 poses of the experiment. Its more complicated forms may 
 be made as described in the article referred to. A glass plate 
 which will close the top of the plethysmograph is needed as 
 well as a piece of rubber tubing long enough to connect the 
 plethysmograph with the recording apparatus, which may be 
 either a tambour, a delicate piston recorder or a Brodie bel- 
 lows recorder. Either of these should be provided with a 
 side tube to allow of proper equalization of pressure on both 
 sides of the piston or membrance of the recorder. 
 
 'Jour, of Physiol., Vol. XXII, 189S, page 380. 
 
300 LABORATORY GUIDE IN 
 
 Vaseline is needed as well as a petrolate which melts at 
 a higher temperature than vaseline and which may be made 
 by melting the vaseline with paraffine using such proportions 
 that the resulting mixture is fairly hard when cold. 
 
 Operation. 
 
 Anaesthetize a cat or a rabbit as usual, tie it on an operat- 
 ing board and insert a venous cannula. 
 
 If it is desired to take a tracing of the carotid blood pres- 
 sure at the same time, the artery must be isolated, a cannula 
 inserted and connected with the mercury manometer accord- 
 ing to the directions given on page 200. By thus taking 
 simultaneous tracings of the general blood pressure and of 
 the vascular conditions in the intestinal area very instructive 
 records may be obtained. 
 
 It is best to warm the plethysmograph slightly before ap- 
 plying it and to have plenty of hot salt solution ready for use 
 as well as some absorbent cotton. Make an incision about 
 4 cm. long in the median line of the abdomen about midway 
 between the sternum and symphysis and with the fingers 
 carefully pull out of the abdominal cavity a short loop of the 
 small intestine. Place the elliptical opening of the plethys- 
 mograph over the incision and intestinal loop and support the 
 instrument in place with an iron ring. Draw the loop of the 
 intestine into the apparatus until a section from 20 to 30 cm. 
 long is lying in the basin. Cover the intestine temporarily 
 with absorbent cotton dipped in the hot salt solution. Ligate 
 doubly each end of the section of intestine and cut across it 
 between the ligatures, tearing down the mesentery as far as 
 possible and ligating any bleeding vessels. Now return to 
 abdominal cavity the upper and lower attached ends of the 
 
,nXPBRIMENTAL PHARMACOLOGY 301 
 
302 LABORATORY GUIDE IN 
 
■EXPERIMENTAL PHARMACOLOGY 303 
 
 the intestine, leaving the isolated loop in the plethysmograph 
 and connected to the animal by its section of mesentery con- 
 taining the vessels and nerves. 
 
 Pack absorbent cotton rubbed up w^ith the hardened 
 vaseline around the mesentery so as to completely close the 
 elliptical opening, using soft vaseline to make it air tight 
 near the vessels ; 'great care must be taken not to constrict 
 the vessels. Remove the cotton covering from the intestines ; 
 vaseline the upper rim of the plethysmograph and close it 
 with the pane of glass which has been dipped in warm salt 
 solution to prevent the condensation of moisture on its sur- 
 face. 
 
 Connect the instrument with the recorder, close the side 
 tube on the latter and take a tracing, which, if the experi- 
 ment has been properly carried out, will show both cardiac 
 beats and the respiratory waves. Arrange the lever to write 
 in line with the blood pressure pointer and the time marker 
 which has been placed below. 
 
 Take tracings to shoAV the effects of the following drugs 
 on the general arterial blood pressure and on the vessels of 
 the splanchnic area. 
 
 Adrenalin chloride (i-iooo), 1-3 drops in salt solution. 
 
 Digitalis, tincture, i cc. in salt solution. 
 
 Nicotine chloride, 2 mg. in salt solution. 
 
 Amyl nitrite, 2 drops tc be given by inhalation. 
 
 Pituitary extract (Pituitrin), 0.5 cc. in salt solution. 
 
 Chloroform to be given until the heart stops. 
 
 The tracing from this experiment should be studied in 
 connection with those obtained earlier in the course in the 
 experiment upon the dog's heart and with the results found 
 Vvhen the vessels of the frog were perfused Vv^ith salt solu- 
 tion. 
 
304 LABORATORY GUIDE IN 
 
 Vascular Changes in the Kidney. 
 
 To record the changes taking place in the size of the kid- 
 ney due to the differences in its blood content, an oncometer 
 and a recording apparatus are needed. The former can. be 
 made from plaster of Paris^ in the same manner as the intes- 
 tinal plethysmograph. Several sizes should be made to ac- 
 commodate the kidneys of dift'erent sized animals. A cylin- 
 drical-shaped oncometer with a flat base, having a diameter 
 of about 5 cm. and with walls about 3.5 cm. high is large 
 enough for the average sized cat or rabbit. One side should 
 have a cut in it about 0.5 cm. wide and extending nearly to 
 the bottom of the apparatus, where the cut should be widened 
 to allow greater room for packing around the pedicle of the 
 kidney. On the opposite side of the oncometer is a glass 
 tube communicating with the interior of the apparatus and 
 extending outward for the attachment of rubber pressure 
 tubing. A piece of glass is needed to fit tightly the top of 
 the instrument. Vaseline together with a mixture of vaseline 
 and wax (as described on page 300) is also necessary. 
 
 For recording the volume changes on the kymograph, a 
 tambour, piston recorder, or a bellows recorder as mentioned 
 under the experiment on the intestinal vessels (page 299) 
 is required. It should be fitted as before with a side tube. 
 
 ^ In place of the oncometer made of plaster of Paris in the manner de- 
 scribed a Roy kidney oncometer such as can be obtained on the market may be 
 used, thus saving considerable time as it can be more easily adjusted than the 
 plaster apparatus. Descriptions of the Roy oncometer may be found 
 in works on physiology. 
 
- BXPBRIMBNTAL PHARMACOLOGY 305 
 
3o6 LABORATORY GUIDE IN 
 
- nXPBRIMENTAL PHARMACOLOGY 307 
 
 Operation. 
 
 Anjesthetize a rabbit or a cat and insert one cannula in the 
 jugular vein and a second in the carotid artery. Connect the 
 latter with the mercmy manometer for blood pressure, 
 which has been arranged according to the directions given 
 on page 200. Either kidney may be employed in the experi- 
 ment, the choice largely depending upon the arrangement of 
 the apparatus and the light. If the left has been selected, 
 make an incision in the abdomen well over in the left flank, 
 extending from the costal margin downward, the length of 
 the incision necessary depending upon the size of the onco- 
 meter. Draw the side muscles outward, if necessary con- 
 tinuing the cut in the muscles along the costal margin, and 
 nail the reflected muscles to the operating board. The intes- 
 tines and stomach should be packed back with absorbent 
 cotton which has been dipped in hot salt solution and the 
 abdominal cavity closed in as far as possible with a hot 
 towel. This should leave the left kidney well exposed. 
 Loosen it very carefully from all its fibrous attachments and 
 remove the fat as far as possible, so that the kidney remains 
 connected only by the structures entering the hilus. 
 
 Place the organ in the w^armed oncometer so that the 
 pedicle passes through the widened end of the cut in the 
 side of the apparatus. Pack around the pedicle with ab- 
 sorbent cotton saturated with the hardened vaseline and 
 completely close the opening in the side in the same way, 
 employing the soft vaseline to make it entirely air tight. Of 
 course it is essential to see that no undue pressure is exerted 
 on the kidney vessels ; the circulation must remain perfectly 
 free in them. Close the top of the oncometer with the 
 pane of glass which is made air tight with vaseline. Con- 
 
3o8 LABORATORY GUIDE IN 
 
 nect the outlet tube of the instrument with rubber tubing,, 
 the other end of which is connected with the recorder. Ar- 
 range the writing point of the recorder so that it marlcs 
 directly above the pointer of the blood pressure manometer 
 and the time marker. Close the side tubes of the recorder 
 and the lever should show the distinct cardiac pulsations as 
 they are indicated by the changes in the kidney volume. If 
 the pulsations do not show, either the kidney vessels are con- 
 stricted or the apparatus is not air tight. 
 
 When all the apparatus is working satisfactorily inject the 
 following drugs into the jugular vein: 
 
 Adrenalin chloride, i-iooo, 1-3 drops in salt solution. 
 
 Tincture of digitalis, i cc. in salt solution. 
 
 Nicotine chloride, 3 mg. in salt solution. 
 
 Amyl nitrite, 3 drops, given by inhalation. 
 
 Pituitary extract (Pituitrin), 0.5-1 cc. in salt solution. 
 
 Chloroform to be given until the heart stops. 
 
 These tracings are to be studied after the manner outlined 
 in the section on intestinal vasomotor changes (page 303) 
 and in connection with the tracing obtained in that experi- 
 ment. 
 
BXPBRIMBNTAL PHARMACOLOGY 309 
 
iio LABORATORY GUIDE IN 
 
^ BXPBRIMENTAL PHARMACOLOGY 311 
 
 Perfusion of the Isolated Frog's Heart. 
 
 Pith the brain and the cord of a large frog weighing about 
 75 grams. Tie it on the frog board and expose the heart 
 in the usual manner. Pass a fine ligature around both aortae. 
 With a fine pair of scissors make a V-shaped incision in one 
 of the aortae, insert a cannula pointing towards the heart 
 and tie it in, tying both aortae at the same time. With a 
 fine glass capillary pipette wash the blood out of the cannula 
 with Ringer solution until it is clear. Divide the aortae. 
 Divide the connections of the heart with the posterior wall 
 of the thorax avoiding cutting any of the vessels. Pass a 
 ligature around all the remaining vessels and turn the heart 
 up so as to expose the ascending vena cava into view. Tie 
 a cannula into it as low down as possible, pointing towards 
 the heart, and tying off all the other vessels at the same time. 
 Fill the cannula with Ringer solution, and remove the heart 
 from the body. 
 
 The perfusion apparatus consists of a double reservoir 
 provided with Mariotte stoppers, rubber tubing, and a 3- 
 way stop-cock. Place 100 cc. of Ringer solution in one 
 reservoir, and the same amount of Ringer solution to which 
 2 to 3 drops of i-iooo adrenaline solution have been added, 
 in the other. Connect the venous cannula with the per- 
 fusion apparatus making sure that all air bubbles have been 
 excluded. The cannula inserted into the aorta is to be held 
 in position by a clamp. The tip of the ventricle is attached 
 by means of a fine spring clasp and silk thread to a light 
 recording lever. The pressure of the perfusion fluid should 
 not exceed 6 cm., and the flow of the perfusion fluid into the 
 
312 LABORATORY GUIDE IN 
 
 heart is adjusted by means of a screw clamp, and is to be 
 maintained constant throughout the experiment. A rate of 
 flow so that 1-2 drops are expelled with each systole will 
 probably be most satisfactory. The resistance against which 
 the heart is to contract is adjusted by properly tipping the 
 cutflow cannula, and this should likewise be kept constant. 
 
 After obtaining a normal record of the heart action turn 
 on the adrenalin perfusion fluid and note the effects of this 
 drug upon the heart. When this has been noted turn on 
 Ringer solution until the heart has returned to its normal 
 condition. Now add to the fresh Ringer solution 0.5 cc. 
 of a i-iooo solution of aconitine hydrochloride. Continue 
 the perfusion with this drug until the heart stops. 
 
 Other drugs that may be tried are : 
 
 Cocaine hydrochloride, 1-25000. 
 
 Calcium chloride, 1-500. 
 
 Digitalein, i -20000. 
 
 Ammonium chloride, i-iooo. 
 
nXPBRIMBNTAL PHARMACOLOGY 313 
 
ii4 LABORATORY GUIDE IN 
 
nXPBRlMENTAL PHARMACOLOGY 315 
 
 The Action of Drugs on the Isolated Mammalian Heart. 
 
 The apparatus required for this experiment consists of a 
 double reservoir suspended at a height of about 60 inches, 
 and connected by means of rubber tubing and a 3-way stop- 
 cock with a glass tube drawn out into a cannula and passing 
 through a warming chamber. The latter may conveniently 
 be made of a small Liebig condenser, the top and bottom of 
 which have been cut off. The bottom is closed with a rub- 
 ber stopper through which the inner cannula tube is passed. 
 Through the lower exit tube of the modified condenser a 
 brass rod is fitted (after the manner of Dale). The water 
 in the warming jacket is maintained at the desired tem- 
 perature by applying the Bunsen flame to the brass rod, and 
 this heats the perfusion fluid as it runs through the inner 
 tube. 
 
 Operation. 
 
 Anaesthetize a small rabbit or a cat with ether. Expose 
 the carotid artery and insert into it a cannula. Bleed the ani- 
 mal into a large evaporating dish and defibrinate the blood 
 thoroughly by whipping with glass rods. Quickly open the 
 animal's chest, open the pericardium, and while the heart is 
 still beating seize the aorta' with a fine pair of forceps and 
 divide it at the point where its branches begin to come oft'. 
 Sever all the other vessels, and at once transfer it to Ringer- 
 Locke solution. It will usually beat quite vigorously at this 
 time. If it beats feebly gentle massage with the fingers in 
 the Ringer-Locke solution will help to empty the heart and 
 its vessels of blood w^hich is essential to prevent clotting in 
 the coronaries. The heart is now left in the solution after 
 the aorta and the pulmonary artery have been identified, 
 
3i6 LABORATORY GUIDE IN 
 
 until such time as you are ready to proceed with the experi- 
 ment. 
 
 The defibrinated blood is strained through cotton and 
 diluted with warmed (38°C.) Ringer-Locke solution to 
 make a total volume of about 200 cc. (From 10 to 15% 
 blood will give best results.) Pass a stream of oxygen 
 through the diluted blood for a few minutes. To 100 cc. of 
 the oxygenated blood solution add 0.2 cc. of i-iooo solu- 
 tion of ouabain and transfer this to one reservoir. Into the 
 other reservoir place 100 cc. of normal oxygenated blood. 
 Fill the whole system with the perfusion fluids, excluding 
 rigorously all air bubbles. Tie the cannula of the perfusion 
 apparatus into the aorta, making sure that it does not pene- 
 trate the semilunar valves. Connect the apex of the heart 
 with a recording lever by means of a hook and silk thread, 
 and start the perfusion of the coronaries with the normal 
 blood solution at a constant temperature of 38° C. The 
 perfusion fluid is to be caught in a large beaker and is to be 
 returned to the reservoir as often as may be necessary. 
 At the beginning of the experiment the perfusion fluid will 
 run through rather rapidly, and the heart will probably beat 
 quite irregularly. After the heart has become perfectly 
 regular, and a record has been taken on the revolving drum, 
 2 to 3 drops of a i-ioooo solution of adrenalin may be added 
 to the normal perfusion fluid. Note its effects upon the 
 heart, especially with regard to the amplitude, rate of beat, 
 and the rate of the outflow of the perfusion fluid. 
 
 After the effects of adrenalin have been studied, turn on 
 the ouabain perfusion fluid, and study the various stages of 
 digitalis action upon the heart. Continue the perfusion with 
 this drug until the heart has stopped beating and for 5 to 10 
 minutes thereafter. 
 
EXPERIMENTAL PHARMACOLOGY 317 
 
LABORATORY GUIDB IN 
 
EXPERIMENTAL PHARMACOLOGY 319 
 
320 LABORATORY GUIDE IN 
 
DRUGS AND REAGENTS REQUIRED FOR EACH 
 EXPERIMENT. 
 
 Aneesthetics : Page ii. 
 Ether. 
 Chloroform. 
 Paraldehyde. 
 
 Chloretone in alcohol or olive oil. 
 Urethane. 
 
 Morphine Sulphate. 
 Anticoagulating Solutions: Page 17. 
 Sodium Sulphate, half saturated. 
 Sodium citrate. 5-10%. 
 Magnesium Sulphate, 25%. 
 General Reagents Needed : 
 Alcohol. 
 Chloroform. 
 Ether. 
 
 Dilute Sulphuric Acid. 
 Potassium Carbonate, 10%. 
 Potassium Hydroxide, 10%. 
 Alkaloidal Reagents. 
 
 Tannic Acid, 10%. 
 
 Picric Acid, Saturated. 
 
 Iodine in Potassium Iodide, U. S. P. 
 
 Mercury Potassium Iodide, U. S. P. 
 
 Phosphotungstic Acid, 2%. 
 Fehling's Solution, U. S. P. 
 Ferric Chloride Solution. 
 Concentrated Acids. 
 Alkaloids : Page 20. 
 Quinine. 
 
 Quinine Sulphate. 
 Glucosides : Page 28. 
 Amygdalin. 
 Bitter Almonds. 
 Volatile Oils : Page 32. 
 Oil of Turpentine. 
 Cotton Seed Oil. 
 Resins : Page 39. 
 
 Powdered resin. 
 
LABORATORY GUIDE IN 
 
 Saponins : Page 40. 
 
 Saponin, 2%. 
 
 Castor Oil. 
 Powders : Page 51. 
 
 Calomel. 
 
 Sugar of jMilk. 
 Capsules : Page 55. 
 
 Quinine Sulphate. 
 
 Oil of Turpentine. 
 Cachets : Page 59. 
 
 Quinine Sulphate. 
 Pills : Page 60. 
 
 Extract of Gentian. 
 
 Starch. 
 
 Lycopodium. 
 Emulsions : Page 67. 
 
 Bitter Almonds. 
 
 Asafoetida. 
 
 Castor Oil. 
 
 Powdered Acacia. 
 Ointments : Page 72. 
 
 Extract of Belladonna. 
 
 Benzoinated lard. 
 
 Hydrous wool fat. 
 Suppositories : Page 76. 
 
 Oil of Theobroma. 
 
 Tannic Acid. 
 
 Lycopodium. 
 Physiology of Frog : Page 80. 
 
 Acid fuchsin, 1.5%. 
 Soporifics : Page 88. 
 
 Chloral. 
 
 Sulphonal. 
 
 Paraldehyde. 
 
 Veronal. 
 Xux Vomica : Page 96. 
 
 Powdered nux vomica. 
 
 Strychnine Sulphate. 
 
 Strychnine Sulphate, i/io% Solution. 
 
 Potassium Bichromate. 
 
 Manganese Dioxide. 
 Camphor Group; Page 112. 
 
 Camphor. 
 
 Thujon. 
 
 Picrotoxin. 
 
EXPERIMENTAL PHARMACOLOGY 323 
 
 Opium : Page 116. 
 
 Morphine Sulphate. 
 
 Cane Sugar. 
 
 Iodic Acid, 5% solution. 
 
 Starch Paste. 
 
 Codeine phosphate, 1.5% solution. 
 Curara : Page 127. 
 
 Curara Solution. 
 Nicotine : Page 132. 
 
 Nicotine Chloride Solution. 
 
 Veratrine : Page 135. 
 
 Veratrine Sulphate, 0.1% solution. 
 
 Caffeine Group ; Page 136. 
 
 Caffeine. 
 
 Sodium Benzoate. 
 
 Theobromine-Sodium Salic3'late. 
 Cocaine :' Page 143. 
 
 Cocaine Hydrochloride. 
 
 Cocaine Hydrochloride, 4% solution. 
 
 Belladonna Group : Page 147. 
 
 Powdered Belladonna Leaves. 
 
 Atropine Sulphate. 
 
 Potassium Hydroxide in Alcohol. 
 
 Cinchona: Page 156. 
 Quinine. 
 
 Quinine Sulphate. 
 Quinine Hydrochloride. 
 Chlorine Water. 
 
 Anesthetics : Page 160. 
 Nitrous Oxide. 
 Ethyl Chloride. 
 Magnesium Sulphate. 
 Sodium Oxalate. 
 Calcium Chloride, 3% solution. 
 
 Digitalis Group : Page 16S. 
 
 Powdered Digitalis leaves. 
 Bruised Digitalis leaves. 
 
 Aconite : Page 183. 
 
 Tincture of Aconite. 
 
 Drugs on Frog's Heart : Page 187. 
 
 Pilocarpine Nitrate, 0.5% solution. 
 Atropine Sulphate, 0.1% solution. 
 
324 
 
 LABORATORY GUIDE IN 
 
 Drugs on Turtle's Heart: Page 191. 
 
 Nicotine Solution. 
 
 Pilocarpine Solution. 
 
 Atropine Solution. 
 Drugs on Blood Pressure : Page 200. 
 
 Amyl Nitrite. 
 
 Suprarenal Gland Extract. 
 
 Nicotine Chloride Solution. 
 
 Pituitary Gland Extract. 
 
 Tincture of Digitalis. 
 
 Barium Chloride, 2% solution. 
 Drugs on Mammalian Heart : Page 207. 
 
 Same as for "Blood Pressure." 
 Digitalis in Auricular Fibrillation : Page 220. 
 
 Tincture of Digitalis. 
 Perfusion of Blood Vessels : Page 227. 
 
 Sodium Nitrite. 
 
 Suprarenal Gland Extract. 
 Drugs on Isolated Tissues : Page 232. 
 
 Suprarenal Gland Extract. 
 
 Pilocarpine Solution. 
 
 Atropine Solution. 
 
 Fluid Extract of Ergot. 
 
 Pituitary Gland Extract. 
 Diuresis : Page 239. 
 
 Theocin. 
 
 Sodium Iodide. 
 
 Sodium Nitrite, 10% solution. 
 
 Fehling's Solution. 
 
 Sodium Nitrate, 5% solution. 
 Cervical Sympathetic System : Page 247. 
 
 Nicotine Chloride Solution. 
 
 Pilocarpine, 0.5% solution. 
 
 Atropine, 0.1% solution. 
 
 Suprarenal Gland Extract. 
 Salivary and Pancreatic Secretion : Page 255- 
 
 Same as for Cervical Sympathetic System. 
 
 Secretin. 
 Antipyretics : Page 267. 
 
 Peptone. 
 
 Beef Serum. 
 
 Antipyrine. 
 
 Acetanilide. 
 
 Acetphenetidine. 
 
EXPERIMENTAL PHARMACOLOGY. 325 
 
 Biological Assay : Page 272. 
 
 Fluid Extract Cannabis. 
 
 Fluid Extract Ergot. 
 
 Suprarenal Gland Extract. 
 
 Pituitary Gland Extract. 
 Intestinal Peristalsis : Page 292. 
 
 Same as for Cervical Sympathetic System. 
 
 Physostigmine Hydrochloride. 
 
 Amyl Nitrite. 
 Vasomotor Changes in the Intestine : Page 299. 
 
 Suprarenal Gland Extract. 
 
 Tincture of Digitalis. 
 
 Nicotine Chloride Solution. 
 
 Amyl Nitrite. 
 
 Pituitary Gland Extract. 
 Vasomotor Changes in the Kidney : Page 304. 
 
 Same as for the Intestine. 
 Isolated Frog's Heart: Page 311. 
 
 Cocaine Hydrochloride. 
 
 Calcium Hj'drochloride. 
 
 Digitalein. 
 
 Ammonium Chloride. 
 Isolated Mammalian Heart: Page 315. 
 
 Suprarenal Extract. 
 
 Ouabain. 
 
INDEX. 
 
 PAGE 
 
 Absinthe 112 
 
 Ac.id f uchsin 84 
 
 Aconite 183 
 
 Aconitine • • • 183 
 
 Adrenalin 288 
 
 Alkaloids ". 20 
 
 Alkaloidal precipitants 27 
 
 Amygdalin 31 
 
 Amyl nitrite 287 
 
 Anaesthetics 160 
 
 Ansesthetic for cat 12 
 
 for dog 12 
 
 for rabbit 11 
 
 Analysis of tracings 215 
 
 Antipyretics 267 
 
 Aqua 36 
 
 Assay of tincture of digitalis 171 
 
 " " aconite 184 
 
 Atropine I47 
 
 Auricular Fibrillation, Digitalis in 220 
 
 Balsams . 39 
 
 Belladonna series I47 
 
 Berberine 28 
 
 Biological assay 272 
 
 Blood pressure in animals, drugs on . . . .' 200 
 
 Brucine 96 
 
 Cachets 59 
 
 Caffeine 136 
 
 Camphor 112 
 
 Cat box 12 
 
 Cannabis assay 279 
 
 Cannulas, arterial and venous 16 
 
 tracheal 18 
 
 Capsules 55 
 
 Chemistry of drugs 20 
 
 Chloral 88 
 
 Chloralose 95 
 
 Chloretone 95 
 
 Chloroform 160 
 
328 INDEX. 
 
 PAGE 
 
 Cinchona 156 
 
 Cocaine - 143 
 
 Codeine ; 116 
 
 CofiFee 136 
 
 Coniine 28 
 
 Curara 127 
 
 Curine 127 
 
 Digitalis 168 
 
 Digitalis in Auricular Fibrillation : 220 
 
 Diuresis 239 
 
 Diuretic, cardiac 180 
 
 Dover's powder 124 
 
 Drugs and reagents required 321 
 
 Emulsions 6y 
 
 Epinephrine 288 
 
 Ergot assay 275 
 
 Eserine 25 1 
 
 Ether 160 
 
 Ethyl chloride • 163 
 
 Euquinine i59 
 
 Fehling's reaction 28 
 
 Fixed oil emulsion 68 
 
 Frog's heart, drugs on 187 
 
 " " " " isolated 311 
 
 Glucosides 28 
 
 Gums 40 
 
 Gum resins 40 
 
 Gum resin emulsion 68 
 
 Heroine 124 
 
 Homatropine 152 
 
 Hyoscine I47 
 
 Hyoscyamine I47 
 
 Hypnotics 88 
 
 Infusion of digitalis 176 
 
 Intestine, Vasomotor changes in 299 
 
 Isolated tissues, drugs on 232 
 
INDEX. 329 
 
 PAGE 
 
 Isolated frog's heart, drugs on • 311 
 
 Isolated mammalian heart, drugs on 3i5 
 
 Intestinal peristalsis 247, 292 
 
 Laudanum 124 
 
 Locke's solution I9 
 
 Magnesium sulphate anaesthesia 164 
 
 Mammalian heart, drugs on 207 
 
 " isolated .' 3i5 
 
 Meconic acid • 116 
 
 Meconic acid reaction 120 
 
 Morphine , • 116 
 
 ■ Muscarine 283 
 
 Mydriatics 252 
 
 Myotics \ 252 
 
 Nicotine 132 
 
 Nicotine chloride 132 
 
 Nitrite series 287 
 
 Nitrous oxide 163 
 
 Nux vomica 96 
 
 Oils, fatty 35 
 
 volatile 32 
 
 Ointments 72 
 
 Oleoresins 39 
 
 Opium 116 
 
 Operations on frogs, pithing 13 
 
 to inject in lymph sac 13 
 
 to expose the heart 14 
 
 Operations on mammals, to insert an arterial cannula 17 
 
 to insert a tracheal cannula iS 
 
 to insert a venous cannula 16 
 
 Pancreatic secretion 255 
 
 Paraldehyde 88 
 
 Paregoric 124 
 
 Percolation • 44 
 
 Perfusion of blood vessels 227 
 
 Peristalsis, drugs on intestinal 247, 292 
 
 Pharmacopoeia ^ 9 
 
 Physiological assay 272 
 
 Physiology of frog's nervous system 80 
 
330 INDEX. 
 
 PAGE 
 
 Phj'sostigmine 251 
 
 Picrotoxin 115 
 
 Pills 60 
 
 Pilocarpus 283 
 
 Pipette, glass injecting •. 14 
 
 Pithing frogs 13 
 
 Pituitary gland assay 280 
 
 extracts 291 
 
 Plethysmograph, intestinal 299 
 
 Powders 51 
 
 Quinine 156 
 
 Reagents and drugs required 321 
 
 Resins , 39 
 
 Ringer's solution 19 
 
 Sage 112 
 
 Salivary secretion, drugs on 255 
 
 Saponins ; 40 
 
 Secretin, preparation of 260 
 
 Solutions, physiological 19 
 
 Anticoagulating 17 
 
 Soporifics 88 
 
 Spiritus ■ 36 
 
 Squills 168 
 
 Stomach tube for cats 11 
 
 dogs 13 
 
 rabbits . 1 1 
 
 Strophanthus 168 
 
 Strychnine 96 
 
 Sulphonal 88 
 
 Suppositories 76 
 
 Suprarenal gland assay 279 
 
 extract 288 
 
 Sympathetic system, drugs on 247 
 
 Syrupus 2^ 
 
 Tansy 112 
 
 Tetronal 95 
 
 Theobromine 136 
 
 Theocin 240 
 
 Theophyllin 136 
 
INDEX. 
 
 531 
 
 PAGE 
 
 Thebaine 123 
 
 Thuj on 112 
 
 Trional 95 
 
 Turtle's heart, drugs on igi 
 
 Tubocurarine 127 
 
 Tyrode's solution _ 19 
 
 Urethane 95 
 
 Vasomotor effects in the intestine 299 
 
 in the kidney 304 
 
 Veratrine 135 
 
 Veronal ' 88 
 
 Vitali's test 151 
 

 ^^5 
 
 aP'305- £^s-