BIOLOGY LIBRA** G A LABORATORY COURSE IN SERUM STUDY THE MACMILLAN COMPANY NEW YORK BOSTON CHICAGO DALLAS ATLANTA SAN FRANCISCO MACMILLAN & CO., LIMITED LONDON BOMBAY CALCUTTA MELBOURNE THE MACMILLAN CO. OF CANADA, LTD. TORONTO A LABORATORY COURSE IN SERUM STUDY BACTERIOLOGY 208 BEING A SERIES OF EXPERIMENTS AND DIAGNOSTIC TESTS IN IMMUNOLOGY CARRIED OUT IN AN OPTIONAL COURSE GIVEN TO MEDICAL AND GRADUATE STUDENTS IN THE DEPART- MENT OF BACTERIOLOGY, COLLEGE OF PHYSICIANS AND SURGEONS COLUMBIA UNIVERSITY NEW YORK, BY THE WRITERS HANS ZINSSER, M.D. \\ J. G. HOPKINS, M.D. REUBEN OTTENBERG, M.D. SECOND EDITION REVISED THE MACMILLAN COMPANY 1921 AH rights reserved - - . *..> - . a COPYRIGHT, 1916, 1921, BT THE MACMILLAN COMPANY. Sot up and f lectrotyped. Published February, 1916. New Edition Revised. Published January, 1921. PREFACE TO SECOND EDITION THE present issue of the Laboratory Course in Serum Study does not represent a new edition in that it has been found impos- sible to completely revise the book at this time. There are, how- ever, a number of important changes in protocols and technique which have been made on the basis of experience hi giving the course to medical and public health students. Most of these changes are in the form of alterations hi the plan of experiments, making them more easy to perform and more illustrative of under- lying principles. In preparing these changes for the new printing, we have been greatly aided by Dr. Samuel Cochran, Professor of Bacteriology at the Shantung Christian University hi China, who helped us hi giving this course at Columbia last year. iva INTRODUCTION THE course here outlined is given by the authors at Colum- bia University. The prerequisite theoretical knowledge is pre- sented in a series of lectures based on the textbook " Infection and Resistance/' by the senior author. Immunity, like other branches of science, cannot be taught without experiment and demonstration. For this reason we have, for several years, supplemented our lecture course on Infection and Resistance by an optional course on Serum Tech- nique. Our purpose in this has been not so much to teach beginners to carry out practical diagnostic tests as to allow the student to carry out fundamental experiments, and, in drawing conclusions from his results, to learn to reason from protocols and in this way discover the basic principles for himself. It has been our contention for a number of years that thorough instruction in the phenomena of immunity consti- tuted a logically necessary preparation for the clinic on infec- tious diseases. For this reason our courses have been offered as optionals to second and third year medical students. Con- trary to ordinary belief, students at this stage of preparation have found no difficulty in comprehending the work, and have, we think, derived benefits in experimental methods and reason- ing far beyond the actual gain in new facts. Though optional now, these courses we hope may eventually become integral, required parts of the regular medical curriculum the lectures and demonstrations correlated with the laboratory course fol- lowing the course in Bacteriology. This, however, we realize may have to await the lengthening of the medical course as a whole. Meanwhile such a course can certainly be optionally Vi INTRODUCTION available for students who have the desire to take it and our experience at two medical schools has taught us that there are always a good many who do. The little volume has been compiled in the first place for our own convenience from the protocols given to our students. If incidentally it is of service to others in planning similar courses, or to laboratory workers in repeating experiments, we shall be pleased. The book as it stands is, of course, not a manual of immunity. The course should follow or be syn- chronous with lectures, reading, and demonstrations on the principles of infection and immunity. In our own work the course is offered to students who have had work in general Bacteriology and have taken the lecture course on Infection and Resistance, and reading is assigned in the textbook on this subject. Since the manual has grown out of the course after some four years of experimentation and adaptation to classroom pos- sibilities, it in no way represents an " ideal " formula but de- scribes only work actually done with students. For this reason we have often simplified the experiments in a way which would be undesirable in actual research work. But we have not done this at the expense of exactitude. Thus we have in many cases adapted the technique to the possibilities of a single after- noon with a class not yet fully trained. In order to facilitate the giving of such a course by other teachers we have added a time schedule of the whole course, and have given in each exercise a list of the materials needed. TIME SCHEDULE OF LESSONS SUBJECT OF LESSON I. IMMUNIZATION OF ANIMALS. II. BACTERICIDAL AND H.EMO- LYTIC POWER OF NORMAL SERUM. III. H^MOLYSIS. IV. QUANTITATIVE RELATIONS OF AMBOCEPTOR AND COMPLEMENT. V. QUANTITATIVE RELATIONS (Continued). VI. REVERSIBILITY AND SPEED OF UNION OF IMMUNE BODY AND CELLS. VII. PFEIFFER PHENOMENON. WORK PRELIMINARY TO SUBSE- QUENT LESSONS Preparation of B. typhosus. B. coli. Staphylococcus au- reus. Vibrio choleras. Sheep serum. Ascitic serum. Sheep red cells. Cat red cells. Horse serum. Human serum. Injection of rabbits. Repetition of injections of Lesson I. Repeat injections of Lesson I. Commence collection of guinea pig hearts in absolute alcohol for Wassermann antigen (Lesson XIX). Repeat injections of Lesson I. Repeat certain injections of Les- son I. Trial bleeding of animals which re- ceived their last injections at 4th session. Bleeding of animals of Lesson I. 1 Arranged for two afternoons a week for a term of three and a half months. vii Vlll TIME SCHEDULE OF LESSONS SUBJECT OF LESSON VIII. BACTERICIDAL TESTS IN VITRO. IX. AGGLUTININS. X. AGGLUTINATION MICRO- SCOPIC METHOD. EF- FECT OF SALT. XI. ABSORPTION OF AGGLUTI- NINS. XII. IN Vivo EFFECT 6r H^EM- AGGLUTINATIVE AND H^MOLYTIC SERA. XIII. ISOH^MOLYSIS AND IsO- AGGLUTINATION. XIV. PRECIPITINS. XV. FORENSIC TEST. PRECIPITIN XVI. BORDET-GENGOU PHENOM- ENON. XVII. ALEXIN FIXATION BY SPE- CIFIC PRECIPITATES. XVIII. FORENSIC COMPLEMENT FIXATION. XIX. WASSERMANN ANTIGEN : PREPARATION AND Ti- TRATION OF ANTIGEN. XX. WASSERMANN REACTION : TECHNIQUE OF TESTS. XXI. COMPLEMENT FIXATION WITH BACTERIAL EX- TRACTS. WORK PRELIMINARY TO SUBSE- QUENT LESSONS Titrate hemolytic sera made by class. Inoculate flasks with virulent diph- theria strain for production of diphtheria toxin (Lesson XXII). Filter diphtheria toxin (Lesson XXII). Begin preparation of Wassermann antigens for Lesson XIX. Inject guinea pigs with diphtheria toxin for determination of mini- mal lethal dose (Lesson XXII). Finish preparation of simple alco- holic Wassermann antigen and Noguchi antigen. Inject guinea pigs with diphtheria toxin-antitoxin mixtures for de- termination of L dose (Lesson XXII). TIME SCHEDULE OF LESSONS IX SUBJECT OF LESSON XXII. DIPHTHERIA TOXIN : Ti- TRATION OF AN UN- KNOWN ANTITOXIN. XXIII. TETANUS TOXIN; TET- ANOLYSIN AND ANTI- TETANOLYSIN J TET~ ANOSPASMIN. XXIV. COBRA VENOM, KICIN, SAPONIN. XXV. ANTITRYPSIN. XXVI. NORMAL OPSONINS. XXVII. OPSONIC INDEX: WRIGHT'S TECHNIQUE. XXVIII. HEAT STABILITY OF OPSONINS. XXIX. TlTRATION OF XORMAL OPSONINS BY DILU- TION METHOD. XXX. TlTRATION OF IMMUNE OPSONINS BY DILU- TION METHOD. XXXI. PREPARATION OF BAC- TERIAL VACCINES. XXXII. ANAPHYLAXIS. WORK PRELIMINARY TO SUBSE- QUENT LESSONS Inject guinea pigs with serum for Lesson XXXII. TO BE PREPARED OR OBTAINED BY INSTRUCTOR BEFORE COURSE. BEFORE LESSON I. BEFORE LESSON IL BEFORE LESSONS III, IV, V, AND VI. BEFORE LESSON VII. BEFORE LESSON VIIL BEFORE LESSON IX. BEFORE LESSON X. BEFORE LESSON XI. BEFORE LESSON XII. Haemolytic (antisheep) serum. Immunize a guinea pig to cholera spirillum. Inoculate students against typhoid. One day before lesson plant agar slants of B. typhosus, B. coli, Sta- phylococcus aureus, Vibrio cho- lerae. Obtain sheep serum, ascitic fluid, horse serum, sheep's blood, two cats, ten rabbits, one guinea pig. One day before, inoculate broth cul- ture of B. typhosus. Fresh dog serum. Washed guinea pig cells. Washed human cells. Sheep's blood (washed). Fresh guinea pig serum. Diluted haemolytic serum. Plant culture of virulent cholera vibrios. Guinea pig immunized to cholera. 24 hours before, plant broth culture of B. typhosus. Obtain sterile blood from normal rabbit. 24 hour broth culture typhoid. Virulent diphtheria strain. 24 hour broth culture B. typhosus. Slant cultures of B. typhosus and B. coli. Two cats, one rabbit. Xll TO BE PREPARED OR OBTAINED BY INSTRUCTOR BEFORE LESSON XIII. BEFORE LESSON XV BEFORE LESSON XVI. BEFORE LESSON XVIII. BEFORE LESSON XIX. BEFORE LESSON XXIII. BEFORE LESSON XXIV. BEFORE LESSON XXV. BEFORE LESSON XXVI. BEFORE LESSON XXVII. BEFORE LESSON XXVIII. BEFORE LESSON XXIX. Beef hearts. Absolute alcohol for Wassermann antigens. Prepare blood stains to be tested. Typhoid slants. Obtain standard diphtheria anti- toxin. Horse meat. Give to class at previous exercise. Syphilitic and normal human sera. Tetanus toxin and antitoxin. Test haemolytic power of toxin. Guinea pig brains. Guinea pigs. Cobra venom. Saponin, ricin, leci- thin. Guinea pig's blood. Sheep blood. Prepare alkaline casein solution and trypsin solution. Obtain blood from carcinoma and other patients. Inject guinea pigs with aleuronat. Slants of Staphylococcus aureus. Guinea pig serum. Inoculate slants with Staphylococ- cus aureus. Glass tubing for making Wright's capsules and pipettes. Rubber nipples. Triangular files. Citrate solution. Staphylococcus immune guinea pig serum. Staphylococcus emulsions. Guinea pigs for preliminary work for Lesson XXXII. Horse serum. Sheep serum. Guinea pig serum. Same materials as for Lessons XXVII and XXVIII. TO BE PREPARED OR OBTAINED BY INSTRUCTOR Xlll BEFORE LESSON XXX. BEFORE LESSON XXXI. BEFORE LESSON XXXII. BEFORE LESSON XXXIII. Serum of rabbit immunized against staphylococcus. Inject guinea pig with aleuronat. * Serum of normal rabbit. Other materials same as for three preceding lessons. Agar slants of Staphylococcus au- reus. Sterile tubes with glass beads. Hsemocytometers. Methylene blue solution. Wright's stain or Jenner's stain. Hopkins centrifuge tube. Horse serum. Sheep serum. Immune anti-horse serum injected into guinea pigs day before lesson. Horse serum. A LABORATORY COURSE IN SERUM STUDY 2 LABORATORY COURSE IN SERUM STUDY LESSON I STUDENTS in this course will prepare their own materials for injec- tion, will immunize and bleed animals themselves, in fact will do all the manipulations necessary in the preparation of materials for the experi- ments that are done in the class. In some cases students will work in groups of two or more in order to save time and animals. It is suggested that each student be required to enter his results on a joint tabulation sheet prepared for each day's exercise and that these results be discussed by the instructor at the beginning of the following exercise. PREPARATION OF MATERIALS FOR INJECTION 1. SHEEP ERYTHROCYTES The students who are to immunize their rabbit to blood cells should obtain the blood from the sheep, and at subsequent lessons other students will be given experience in this procedure. Bleeding of Sheep. The animal is held by two assistants with the head thrown back and one side of the neck is clipped and carefully shaved. A rubber tourniquet is then placed around the neck as near as possible to the shoulders and tied tightly so as to compress the external jugular vein. The vein distends and will be seen to stand out as a ridge the size of a finger. A small part of the shaved area of the skin is painted with tincture of iodine, and a large sterile needle with four inches of rubber tubing attached is plunged through the skin into the vein. The blood is allowed to flow into small Erlenmeyer flasks containing about 20 large beads, and when the desired amount of blood is obtained the flask is shaken thoroughly until coagulation is completed, this procedure defibrinating the blood. If preferred, the blood may be received into citrate solution, containing 1 per cent sodium citrate and 0.5 per cent sodium chloride (not more than 1 part of blood to 1 part of solution) or into ammonium oxalate solution (1 per cent). To obtain washed erythrocytes and serum for injection the defibrinated blood is poured into sterile centrifuge tubes. The cotton plugs of these tubes should be fastened by turning the free edges back over the neck of the tube and securing them there by means of rubber bands; this will prevent the 4 LABORATORY COURSE IN SERUM STUDY plug being thrown to the bottom of the tube during centrifuga- tion. The cotton plug may also be replaced by a cap of sterile tin foil. After careful balancing, the tubes are centrifugalized at high speed until the sediment is thrown down. The super- natant fluid is removed with a sterile pipette and in this case should be preserved for the injection of rabbits for the production of precipitins. The tubes are then filled with sterile salt solution, the blood cells resuspended by drawing them in and out of a pipette and again thrown down in the centrifuge. This process is repeated three times. The washed sediment is then trans- ferred to another tube with a graduated pipette and an equal amount of salt solution added to make the 50 % suspension which is used for injection. 2. PREPARATION OF BACTERIAL EMULSIONS Ten c.c. of sterile salt solution are added by means of a pipette to a 24-hour culture on slant agar of the particular organism to be used. Surface growth is scraped from the agar by means of a platinum loop and the bacteria suspended in salt solution by gentle shaking. The suspension is then poured off into a sterile test tube, carefully flaming the mouths both of the culture tube and the sterile test tube before pouring. The second tube may then be drawn out and sealed in a blowpipe flame, and the tube entirely immersed in a water bath for sterilization ; or the upper portion of the tube may be carefully heated in a Bunsen flame to kill any bacteria which may be adherent to the side of the upper portion, and when the tube has cooled the lower portion is then immersed in a water bath, taking care that the level of the water comes well above the portion which has not been sterilized by flaming. The bacteria should be heated at 60 C. for one half hour and are then ready for injection. 3. PREPARATION OF SERUM FOR INJECTION The serum or ascitic fluid used for the production of precipitins should be clear and sterile and is less toxic if heated for 30 minutes at 56 C. before injection. It requires no other preparation. 6 LABORATORY COURSE IN SERUM STUDY CARE OF ANIMALS The animals used for the routine work of the course are mainly guinea pigs and rabbits ; in some cases dogs or cats are used, and for demonstration a sheep is used on one or two occasions. Animals used should be selected from among the healthy stock ; animals having suppurations, diseased eyes, or skin diseases should be discarded. They should be well nourished and before immunization is begun should be weighed and the weight recorded. During the course of treatment students should, if possible, supervise the feeding and caging of their animals. Care should be taken that the cages are kept reasonably dry, that the animals have plenty of water to drink and the food is abundant, and that not too much green feed is given. If any of the rabbits under treatment show a catarrhal discharge from the nostrils, a condition spoken of by animal dealers as "wet mouth", these animals should be segregated and their nostrils and forepaws washed daily with weak bichloride solution. Great care should be taken that the food soiled by these animals is not put into cages of the healthy ones. Rabbits and guinea pigs are best preserved in a warm place, and wood bottoms on the floors of bins and cages are much better than either cement or tin unless these are covered. They should not be allowed to live for days on floors wet and soiled with their own discharges. During the immunization the animals should be weighed periodically and their weight recorded, and if great emaciation and loss of weight ensues in an individual case, the animal should be given a rest and carefully fed. Unless this is done many of the animals will die in the course of immunization. There is certain to be a considerable mortality among the animals even if the greatest care is used, and it is wise to start im- munizing two individuals for each sort of substance used as antigen. METHODS OF INJECTION 1. INTRAVENOUS INJECTION When a rabbit is to be injected the animal is held by an assist- ant. The outer border of the hairy side of the ear is shaved and 8 LABORATORY COURSE IN SERUM STUDY rubbed vigorously with alcohol until the marginal vein distends. The needle of the syringe is then introduced into the vein, point- ing toward the base of the ear, and the material injected, care being taken to avoid the introduction of bubbles. When the needle is withdrawn a small piece of absorbent cotton is pressed over the puncture to stop bleeding. It is well to leave this cotton adherent to the puncture when the animal is returned to the cage. In injecting guinea pigs intravenously it is necessary to incise the skin of the neck and expose the external jugular vein, and unless a very small needle is used the vein must be tied off after injection has been made. Etherization is needed for this procedure. The injection of mice and rats intravenously is difficult, but can be accomplished by rubbing the tail with xylol and holding the mouse so that the tail bends sharply over the edge of the jar. The four parallel veins will then stand out and with care a very fine needle can be introduced into one of them. 2. INTRAPERITONEAL INJECTIONS A rabbit or guinea pig is held back down by an assistant and a small area of the abdominal wall median line clipped and the skin disinfected with alcohol or iodine. A fold of skin and muscle is pinched up with the left hand, and the needle, which should not be too sharp, is cautiously introduced directly into the peritoneal cavity and the injection made. The safest point is the median line below the umbilicus, as in this area there is little danger of puncturing the stomach or large intestine. 3. SUBCUTANEOUS INJECTIONS Subcutaneous injections are made in a similar way, after clipping the hair of the area selected and pinching up a fold of skin, taking care that the needle does not pass into the muscles. Rabbits and guinea pigs are usually injected into the surface of the abdomen, rats and mice at the root of the tail. If there is danger of the substance injected causing ulceration, as in the case of red cells, it is best to inject rabbits under the skin of the back, as this area is less likely to become infected. 10 LABORATORY COURSE IN SERUM STUDY For injection a syringe is used which will withstand boiling. For general work all glass syringes of the Luer type are the most satisfactory. A tightly fitting needle of gauge 20 to 22 should be used for "Inrra venous injections; for intraperitoneal injections larger needles may be used if desired. If glass syringes are used the needle may be attached to the barrel of the syringe, the plunger withdrawn, and the two parts boiled separately for 5 minutes in water containing about 1 per cent of sodium carbonate. A pair of forceps should be placed in the sterilizer with the points under the water. The barrel is first removed with the forceps, the plunger then removed and inserted. The fluid to be injected is drawn into the syringe by tilting the test tube or container and placing the beveled tip of the needle closely against the lower side of the wall. If air is drawn into the syringe at the same time, it should be expelled by holding the syringe vertically, needle up, covering the tip of the needle with a small piece of cotton wet with alcohol or some other disinfectant and expelling the air. After use the syringe should be resterilized if any infected material has been used, washed with water, and the needle washed with water, then with alcohol and then with ether to dry it thoroughly. If care is not taken to clean the needles thoroughly after use, they are certain to become clogged. If blood or serum have been injected, the needle and syringe must be cleaned with cold water at once. Otherwise if alcohol or boiling water is used, the protein will be coagulated and the needle clogged. Animals should be labeled by attaching a number tag by means of a metal staple to the ear, should be placed in a cage with a tag recording the number of the animal, the name of the student in charge and the nature of the material injected, and the date of each injection. SCHEME FOR INJECTIONS (APPROXIMATE DOSES AND INTERVALS) 1. Rabbit immunized to B. typhosus by intravenous injection. 1st day 1st dose 0.05 agar slant 6th day 2d dose 0.1 agar slant heated as llth day 3d dose 0.2 agar slant indicated 16th day 4th dose 0.2 agar slant below 24th day 5th dose 0.2 agar slant 12 LABORATORY COURSE IN SERUM STUDY 2. Rabbit immunized to B. typhosus by subcutaneous injections. Injections at five or six day intervals, followed by intravenous injections. 1st dose 0.1 agar slant 2d dose 0.2 agar slant 3d dose 0.3 agar slant 4th dose 0.5 agar slant 5th dose 0.5 agar slant 3. Rabbit immunized to B. typhosus and to B. coli communior by intravenous injection. Intervals as above. 1st dose 0.05 agar slant of B. typhosus 2d dose 0.1 agar slant of B. typhosus 3d dose 0.2 agar slant of B. typhosus 4th dose 0.2 agar slant of B. typhosus, plus 1 0.05 agar slant of B. coli communior J 5th dose 0.2 agar slant of B. typhosus, plus 0.05 agar slant of B. coli communior 6th dose 0.2 agar slant of B. typhosus, plus j 0.1 agar slant of B. coli communior J 7th dose 0.2 agar slant of B. typhosus, plus 1 0.2 agar slant of B. coli communior J 4. Guinea pig immunized to Sp. cholerse by intraperitoneal injec- tion. An old laboratory strain of the cholera vibrio should be used for immunization. Intervals as above. 1st dose 0.05 agar slant 2d dose 0.1 agar slant 3d dose 0.2 agar slant 4th dose 0.2 agar slant 5th dose 0.2 agar slant 5. Rabbit immunized to micrococcus aureus by intravenous injec- tion. Intervals as above. In the case of this microorganism the intervals should be lengthened if there is any indication of illness. Great care is necessary in avoiding overdosage. 1st dose 0.05 agar slant 2d dose 0.1 agar slant 3d dose 0.2 agar slant 4th dose 0.2 agar slant 5th dose 0.2 agar slant 14 LABORATORY COURSE IN SERUM STUDY 6. Rabbit immunized to sheep serum by intravenous injection. Five-day intervals. 1st dose 2.0 c.c. 2d dose 2.0 c.c. 3d dose 2.0 c.c. 7. Rabbit immunized to human serum (ascitic fluid) by intravenous injection. Five-day intervals. 1st dose 4.0 c.c. 2d dose 4.0 c.c. 3d dose 4.0 c.c. 8. Rabbit immunized to horse serum. (Immunize noteless than three in order to obtain sufficient serum for later exercises.) 1st dose 2.0 c.c. 2d dose 2.0 c.c. 3d dose 2.0 c.c. 9. Rabbit immunized to sheep erythrocytes by intravenous injec- tion. Four or five day intervals. 1st dose 4.0 c.c. of 50 % suspension 2d dose 4.0 c.c. of 50 % suspension 3d dose 4.0 c.c. of 50 % suspension A fourth injection may be given in some cases, but is not often necessary. 10. Rabbit immunized to sheep erythrocytes by intraperitoneal injections. Intervals as above. 1st dose 4.0 c.c. of 50 % suspension 2d dose 8.0 c.c. of 50 % suspension 3d dose 8.0 c.c. of 50 % suspension 4th dose 8.0 c.c. of 50 % suspension 11. Rabbit immunized to cat erythrocytes by intravenous injec- tions of 2 c.c. at 4-day intervals. See Lesson XII. A preliminary titration of the serum done with a specimen obtained from an ear vein should be done about the eighth or ninth day after the last injection. If this is satisfactory, the bleeding of the animal is usually done the ninth or tenth day. No absolute rule can be established as to exact period at which the serum has the highest antibody concentration, this depending to 16 LABORATORY COURSE IN SERUM STUDY some extent on the health of the animal, the size of the injec- tions, and the intervals. It is a safe rule to assume that the high point in the curve is apt to lie between the eighth and the twelfth day after the last injection and to be guided by this in preliminary titrations. THE BLEEDING OF ANIMALS Before the immunization of the animal is begun a sample of blood should be taken from the ear vein of the animal and the serum stored in the refrigerator for use as a control at a later exer- cise when the serum of this animal is tested for immune bodies. The blood can be obtained from the marginal vein in the ear in the following manner. The animal is firmly held with the head down by an assistant. The lateral portion of the hairy surface of the ear is carefully shaved and the skin rubbed vigorously to cause the veins to dis- tend. If necessary the ear may be rubbed with xylol. The skin over the vein is washed over with alcohol and a cut made in the vein with a razor blade or other sharp instrument. The blood is allowed to drip into a clean centrifuge tube until about 4 c.c. (60 drops) are obtained. It is then allowed to coagulate and the edges of the clot are freed from the side of the tube by means of a sterile platinum wire. The tube is centrifugalized and the clear serum thus expressed is transferred to a sterile test tube by means of a capillary pipette with a rubber nipple attached. It is then labelled carefully and stored in the icebox for future use. Later bleeding from the ear veins of a rabbit can be facilitated by strapping the animal down on a flat, hot water bag. A techni- cal point of great simplicity but which helps to make the sort of cut in the vein which favors free bleeding, consists in taking a pointed steel writing pen, breaking off one of the prongs and plunging the stilette thus made straight into the vein in a direc- tion toward the heart. The entry thus made through the wall of the vein does not easily collapse and very free bleeding results. To bleed a rabbit from the carotid the rabbit is strapped down on its back on a dissecting board and ether administered. A 18 LABORATORY COURSE IN SERUM STUDY central incision along the neck above the trachea is made, and the carotid will be found lying very close to the trachea on either side. A ligature is applied distally, the artery raised on the handle of a forceps and a bulldog clamp applied toward the ridge of the neck. A sterile glass canula can be inserted, but we have found that with a little skill it is quite easy to lift up the artery and hold it by its wall with a forceps in such a way that the blood stream is not blocked. The vessel is then cut, the clamp removed, and by aiming the artery with the forceps in which it is held the stream of blood can be directed into a sterile test tube. In this way rabbits are usually bled until distinct respiratory distress ensues and the stream of blood ceases, the blood coming in drops. The clamp is then applied, the vessel tied and the rabbit sewed up aseptically. Such a rabbit can be kept alive and further used after recovery for continuation of the immunization. 1 The bleeding of guinea pigs from the carotid is in every way similar, though a little more difficult than the bleeding of rabbits. When guinea pigs are bled, as they will be for the purpose of ob- taining complement or alexin, it is necessary to do this carefully in order to obtain the best results. The blood is best taken into large sterile test tubes with as little violence as possible in order that there may be no breaking up of blood cells with consequent hemolysis. It is best to take the blood the evening before it is to be used, to allow it to clot in the test tube and then to separate the clot very gently from the sides of the tube. The tube should then be set aside in the refrigerator at a low temperature (1-4 C.). The rapidity with which complement degenerates at higher tem- peratures is generally underestimated. The following day the serum is pipetted away from the clot. Some observers believe that complement increases in potency on standing for some hours on the clot. This was the con- tention of Henderson-Smith, though it has recently been con- tradicted by Addis and others. It is a good practice to obtain it in this way, however, because blood so taken is apt to be clearer and freer from haemolysis than blood centrifugalized immediately after clotting. Although a slight tinge of haemolysis does not 1 Intravenous injections of 10 to 20 c.c. of warm salt solution will often help to keep such animals alive. \ 20 LABORATORY COURSE IN SERUM STUDY f-'- 1 - v/ ^bes ^ u~ render blood useless for haemolytic purposes, nevertheless the products of haemolysis do affect the haemolytic reaction and it is t to have an entirely clear alexin for use. The technique of bleeding of sheep or other large animals has been described on page 2. LESSON II BACTERICIDAL POWER OF NORMAL SERUM NORMAL blood plasma or serum possesses the power of killing bacteria. Not all bacteria are equally susceptible to this effect. Some, like the Gram-positive cocci, the anthrax bacillus, and others, are probably killed in the circulation only by the co- operation of serum and leucocytes in phagocytosis. When the serum is heated to 56 C., its bactericidal power is suspended. Reagents : 1. Fresh normal rabbit serum, unheated. 2. Bacillus typhosus 24-hour broth culture diluted 1-5000 in sterile salt solution. 3. Sterile salt solution. The materials in this experiment must be measured with sterile pipettes and placed in sterile test tubes, with care to avoid con- tamination during the process of the experiment. (a) Place 0.5 c.c. of normal rabbit serum in each of two sterile test tubes and heat one tube for half an hour at 56 C. Then set up the following preparations in sterile plugged test tubes : 1. Normal rabbit serum 0.5 c.c. + Typhoid broth (1-5000) 0.5 c.c. 1 (fresh) 2. Normal rabbit serum 0.5 c.c. + Typhoid broth (1-5000) 0.5 c.c. (heated) 3. Salt solution 0.5 c.c. + Typhoid broth (1-5000) 0.5 c.c. 1 To make dilutions for this and other purposes proceed as follows : 1. 1 c.c. of original substance to 9 c.c. of salt solution = 1-10 2. 1 c.c. of dilution (1.) to 9 c.c. of salt solution = 1-100 3. 1 c.c. of dilution (2.) to 4 c.c. of salt solution = 1-500 4. 1 c.c. of dilution (3.) to 1 c.c. of salt solution = 1-1000 5. 1 c.c. of dilution (4.) to 4 c.c. of salt solution = 1-5000 This illustrates the general method, which can be shortened or lengthened by longer or smaller intervals as the particular occasion requires. 22 LABORATORY COURSE IN SERUM STUDY (6) Place the tubes in the incubator for two hours, and then pour into each tube a tube of agar which has been melted in boiling water and cooled to 42 C. Pour the entire contents of each tube into a sterile Petri dish. When the plates are hardened, place them in the incubator in an inverted position, to be observed at the following lesson. The plates made from tubes 2 and 3 will show an enormous number of minute typhoid colonies after incubation. The plate from tube 1 will be sterile or may show a few large colonies. ILEMOLYTIC AND ILEMAGGLUTINATIVE POWER OF NORMAL SERUM The normal blood serum of many animals has haemolytic and haemagglutinative action upon the red blood cells of animals of some other species. This hsemolytic action, like the bacteri- cidal action, is lost when the serum is heated to 56 C. The haemagglutinating property, on the other hand, is relatively resistant to heat and is not destroyed until the serum is heated to 70 C. or above. Such a hsemolytic normal serum acts only on the erythrocytes of certain other species, sometimes upon 2 or 3 varieties with differing intensity. Thus goat serum is strongly haemolytic for rabbit cells less so for those of guinea pigs. Dog serum contains a relatively large amount of normal haemo- lysin for most cells used in laboratory work. Guinea pig cells are highly susceptible, and human cells relatively resistant to haemolysis by dog serum. Reagents : 1. Fresh dog serum. 2. 2 % suspension of washed guinea pig cells. 3. 2 % suspension of washed human cells. 4. Salt solution. Place 0.25 c.c. of dog serum in a test tube and heat for one half hour in water bath at 56 C. Bring up volume to 2.5 c.c. with normal salt solution. Prepare a similar 1-10 dilution of unheated dog serum. Set up the following experiment in half-inch tubes : 24 LABOKATORY COURSE IN SERUM STUDY 1. Dog serum unheated (1-10) 0.5 c.c. + Guinea pig cells 2 % 0.5 c.c. 2. Dog serum unheated (1-10) 0.5 c.c. + Human cells 2 % 0.5 c.c. 3. Dog serum heated (1-10) 0.5 c.c. + Guinea pig cells 2 % 0.5 c.c. 4. Dog serum heated (1-10) 0.5 c.c. + Human cells 2 % 0.5 c.c. 5. Salt solution 0.5 c.c. + Guinea pig cells 2 % 0.5 c.c. 6. Salt solution 0.5 c.c. -f Human cells 2 % 0.5 c.c. Incubate the tubes one hour at 37 C. in water bath, observing changes in cells at intervals of 5, 10, 15, 30, and 60 minutes. Tabulate the results. Tube 1 should show prompt haemolysis; tube 3 should show agglutination of the cells. In tubes 2 and 4 there should be no observable change in the cells in most samples of dog serum. Each student needs: 10 J^-inch test tubes, test tube rack, 5 one-c.c. pipettes, 1 five-c.c. pipette. LESSON III HAEMOLYSIS. (EHRLICH AND MORGENROTH EXPERI- MENTS) 1. To a group of students is assigned the task of bleeding a sheep from the jugular vein into a flask containing glass beads. After defibrina- tion the cells are washed three times in salt solution and a 5 per cent suspension of the well-packed sediment is made (1.0 c.c. of sediment and 19.0 c.c. of salt solution). 2. To another group of students is assigned the bleeding of guinea pigs from the carotid artery into centrifuge tubes. The blood is allowed to clot, this clot is "rimmed" and the serum separated. The process may be hurried by centrifugation. The serum is then diluted, 1 part of serum to 9 parts of salt solution. 3. Immune rabbit serum (lytic for sheep cells) will be given out diluted. 1 This serum has been heated for one half hour at 56 C. before dilution, in order to destroy its alexin or complement. This is done so that no unknown amount of complement may be present in the final tests. All these materials must be prepared before the regular class period, as the experiments themselves consume several hours. Provided the guinea pig serum is kept very cold (0-^i C.), all the materials can be prepared the preceding day. 1 In order to save time in the course an immune serum previously prepared by the instructor is used here. The immune haemolytic serum whose preparation was begun by the students in Lesson I is to be titrated later and used for the lessons on complement fixation . 26 LABORATORY COURSE IN SERUM STUDY Experiment 1 To SHOW THAT RED BLOOD CELLS WILL ABSORB AMBOCEPTOR BUT NOT COMPLEMENT Set up two mixtures in centrifuge tubes as indicated below: A. Sheep cells and heated immune rabbit serum B. Sheep cells and fresh normal guinea pig serum. Incubate for 30 minutes at 37 C., then centrifugalize and remove the supernatant fluid, using a capillary pipette with rubber nipple. Wash the sediment once in salt solution. Resuspend the washed sediment in each tube in 3 c.c. salt solution. Add the reagents as indicated and incubate for one hour at 37 C., observing at short intervals. Tabulate the results. Tube A Sheep cells 5% 1.0 c.c. Inactivated immune rabbit serum l .... 1.0 c.c. Incubate 30 minutes at 37 C., centrifugalize, remove the superna- tant fluid to another tube and wash the sediment. We now have 2 tubes one (Tube 1) containing the supernatant fluid, the other (Tube 2) the sediment from the original Tube A. To these tubes add reagents as follows : * Tube 1 Tube 2 (Supernatant fluid from (Sediment from Tube A Tube A) 2.0 c.c. resuspended in salt Guinea pig serum (1-10) 1.0 c.c. solution) 3.0 c.c. Sheep cells 5% . . . 1.0 c.c. Guinea pig serum (1-10) 1.0 c.c. Incubate and observe. In which of the two does haemolysis take place? What conclu- sion as to the reaction that has taken place in the original mixture in Tube A can you draw from this? TubeB Sheep cells 5% 1.0 c.c. Guinea pig serum (1-10) 1.0 c.c. Incubate 30 minutes at 37 C., centrifugalize, remove supernatant fluid to another tube and wash sediment. 1 Diluted by the instructor so as to contain two hemolytic units in 1 c.o. 28 LABORATORY COURSE IN SERUM STUDY Then, as in the preceding case, we have two tubes to which the following additions are made : Tube 1 Tube 2 (Supernatant fluid from (Sediment from Tube B Tube B) about . . . 2.0 c.c. resuspended in salt Immune rabbit serum 1.0 c.c. solution) 3.0 c.c. Sheep cells 5% . . 1.0 c.c. Immune rabbit serum . 1.0 c.c. Incubate and observe. In which tube does haemolysis take place? Contrast this with results in Tube A and draw conclusions. Experiment 2 TO SHOW THAT AT C. CELLS WILL ABSORB AMBOCEPTOR ALONE FROM A MIXTURE OF AMBOCEPTOR AND COMPLEMENT The same reagents will be used in this as in the preceding experi- ment. The three reagents will first be cooled in ice water and then be mixed in a cold centrifuge tube, and allowed to stand at C. for an hour. With proper care this can easily be done in a battery jar filled with cracked ice and brine. The success of the experiment depends on having the reagents and the centrifuge tube and metal holder thoroughly cooled in ice water before the mixture is made and in centrifugalizing and removing the supernatant fluid before they have time to regain the temperature of the room. The protocol for this experiment is given below : Place centrifuge tube in holder in a cup of cracked ice and water and add: 5% sheep cells 1.0 c.c. Inactive immune rabbit serum (diluted) . . 1.0 c.c. Guinea pig serum (1-10) 1.0 c.c. After twenty minutes the tube is centrifugalized, the supernatant fluid removed and half of it placed in each of two small test tubes. The sediment is to be washed once in cold salt solution, resuspended in 4 c.c. of salt solution and half of this suspension placed in each of two small test tubes. We now have four tubes two of which contain washed sediment and two of which contain supernatant fluid of the original mixture. To these the following additions are now made : 30 LABORATORY COURSE IN SERUM STUDY Tube 1 Supernatant fluid 1.5 c.c. Fresh guinea pig serum 1-10 (complement) 0.5 c.c. Washed sheep cells 5 % 0.5 c.c. Tube 2 Supernatant fluid 1.5 c.c. Immune rabbit serum 0.5 c.c. Washed sheep cells 5 % 0.5 c.c. TubeS Sediment 2.0 c.c. Guinea pig serum 1-10 0.5 c.c. Tube 4 Sediment 2.0 c.c. Immune rabbit serum 0.5 c.c. Incubate one hour at 37 C., observing at frequent intervals. The results will show that the haemolytic amboceptor or sensitizer present in the original heated immune serum was bound by the cells at C. but at this low temperature the complement or alexin remained not only inactive but unbound and free. APPARATUS Each student will require : 3 graduated centrifuge tubes 8 half-inch test tubes 9 one c.c. pipettes 1 test tube rack 1 bottle of salt solution 1 jar of cracked ice (pint measure) 1 rubber nipple and capillary pipette The class will require : 1 outfit for bleeding sheep : needle, tourniquet, razor and scissors, bottle of tincture of iodine, sterile flask containing beads. 32 LABORATORY COURSE IN SERUM STUDY 1 or more outfits for bleeding guinea pigs : scissors, forceps, ether cone, centrifuge tubes. MATERIALS Each student will require : Washed sheep cells 5 % 6.0 c.c. Guinea pig serum 1-10 6.0 c.c. Inactive serum of rabbit immunized against sheep cells, diluted so that each c.c. con- tains 2-4 hsemolytic doses 6.0 c.c. LESSON IV QUANTITATIVE RELATIONSHIPS OF AMBOCEPTOR AND COMPLEMENT METHODS OF TITRATION THE haemolysis of red cells by specific sensitizer [or ambocep- tor] and alexin [or complement] is a delicately quantitative reaction in which very definite amounts of the three substances are necessary to produce a complete reaction. As a unit of red cells, one cubic centimeter of a 5 per cent suspension of red cells is conveniently taken. This means that the cells after washing are sedimented in the centrifuge and 1.0 c.c. of the sediment is added to 19.0 c.c. of salt solution. (Some investi- gators have worked with suspensions representing 5 per cent of the original blood. Assuming the cells to constitute 50 per cent of the blood volume, suspensions so made up are just half as concentrated as the ones here used. In all the work here de- scribed the term 5 per cent suspension is taken to represent 5 per cent by actual volume of erythrocyte sediment in salt solution.) Definite amounts of sensitizer and of alexin are necessary to lake completely one cubic centimeter of such a suspension, and it is necessary for accurate experimentation to determine for each set of reagents the minimal amounts necessary to accomplish 34 LABORATORY COURSE IN SERUM STUDY this purpose. It must be remembered that no two suspensions of red cells are exactly alike since it is impossible to measure them with absolute accuracy, and since the resistance of erythro- cytes to haemolysis, even when taken from the same animal on different days, may vary. No two fresh sera, moreover, are entirely alike in alexic activity. The experiments given below will demonstrate the method of determining by titration the minimal haemolytic dose of complement and amboceptor ; they will also demonstrate that this dose varies reciprocally, that is, that with a small amount of complement a relatively larger amount of amboceptor will be necessary to cause complete haemolysis, whereas with a large amount of complement only a small amount of amboceptor is necessary. There is, however, no simple proportionality. Experiment 1 TITRATION OF H^MOLYTIC SENSITIZER OR AMBOCEPTOR. DETERMINA- TION OF UNIT The unit of sensitizer, or amboceptor, is the smallest amount which will cause complete laking of a unit of cells (1.0 c.c. of a 5 per cent emulsion) 1 in one hour, in the presence of an excess of complement. As the degree of dilution is an important factor it is necessary that the volume of all the tubes be brought to a uniform standard before the reaction actually begins. For this reason the reagents are added in the following order : 1. Sensitizer [amboceptor] 2. Alexin [complement] 3. Saline to make all volumes equal 4. Cells A control tube must be set up to prove that the amount of alexin or complement used will not of itself lake the cells. 1 For convenience or economy 0.5, 0.25 or 0.1 c.c. of 5 per cent red cells are sometimes taken as the unit, and in such cases the unit of amboceptor and com- plement must be proportionately reduced. For the sake of uniformity results so obtained can easily be transposed to terms of the 1 c.c. unit, as is often done in reporting Wassermann tests. C6 LABORATORY COURSE IN SERUM STUDY Add saline to 2 c.c. and then 0.5 c.c. of the red cell emulsion. 1 Set up the following tests : Series 1 Immune Serum (Sensitizer) Complement Tube 1. (diluted 1-100) 0.5 c.c. 0.05 c.c. 2. (diluted 1-100) 0.2 c.c. 0.05 c.c. 3. (diluted 1-1000) 1.0 c.c. 0.05 c.c. 4. (diluted 1-1000) 0.5 c.c. 0.05 c.c. 5. (diluted 1-1000) 0.4 c.c. 0.05 c.c. 6. (diluted 1-1000) 0.3 c.c. 0.05 c.c. 7. (diluted 1-1000) 0.2 c.c. 0.05 c.c. 8. (diluted 1-1000) 0.1 c.c. 0.05 c.c. 9. (diluted 1-1000) 0.0 c.c. 0.05 c.c. (Complement Control) Series 2 Immune Serum (Sensitizer) Complement Tube 1. (diluted 1-100) 0.5 c.c. 0.02 c.c. 2. (diluted 1-100) 0.2 c.c. 0.02 c.c. 3. (diluted 1-1000) 1.0 c.c. 0.02 c.c. Add saline 4. (diluted 1-1000) 0.5 c.c. 0.02 c.c. to 2 c.c. 5. (diluted 1-1000) 0.4 c.c. 0.02 c.c. and then 6. (diluted 1-1000) 0.3 c.c. 0.02 c.c. 0.5 c.c. of 7. (diluted 1-1000) 0.2 c.c. 0.02 c.c. the red cell 8. (diluted 1-1000) 0.1 c.c. 0.02 c.c. emulsion. 9. (diluted 1-100) 0.5 c.c. 0.00 c.c. ( (Sensitizer Control) Series 3 Immune Serum (Sensitizer) Complement Tubel. (diluted -100) 0.5 c.c. 0.01 c.c. 2. (diluted -100) 0.2 c.c. 0.01 c.c. 3. (diluted -1000) 1.0 c.c. 0.01 c.c. Add saline 4. (diluted -1000) 0.5 c.c. 0.01 c.c. to 2 c.c. 5. (diluted -1000) 0.4 c.c. 0.01 c.c. and then 6. (diluted -1000) 0.3 c.c. 0.01 c.c. 0.5 c.c. of 7. (diluted 1-1000) 0.2 c.c. 0.01 c.c. the red cell 8. (diluted 1-1000) 0.1 c.c. 0.01 c.c. emulsion. 9. (diluted 1-1000) 0.0 c.c. 0.00 c.c. J (Salt Solution Control) 1 A half c.c. instead of one c.c. is used in order to save materials. The amounts of complement and amboceptor needed are of course half those that would be needed for 1 c.c. of cells. 38 LABORATORY COURSE IN SERUM STUDY In measuring 0.05, 0.02 and 0.01 c.c. of complement it is best to use a half c.c., .2 c.c. and .1 c.c. of a 1-10 dilution. Shake each tube thoroughly when all the reagents have been added. Incubate one hour in water bath at 37 C. and record by -f- or signs haemolysis at 15, 30 and 60 minutes in the following protocol: IMMUNE SERUM (SENSITIZER OR AMBOCEPTOR) Diluted 1-100 Diluted 1-1000- 0.5 0.2 1.0 0.5 0.4 0.3 0.2 0.1 0.0 Complement 0.05 . Complement 0.1 Complement 0.2 Indicate what the unit is in this case. The tabulation given for final record is the manner in which an experiment of this kind is best recorded by experienced workers. We have inserted the simpler protocols of the three series before this in order to make the purpose of this experiment and its execution a little more easy. (On account of the costliness of guinea-pig serum students should divide themselves into groups of 3, of whom each student should set up one series of amboceptor dilutions with one of the complement quan- tities.) Individual guinea pigs vary in the complementary activity of their serum. For accurate hsemolytic experiments, however, it is necessary to use a constant amount of complement activity. For this reason the unit of amboceptor (which if carefully pre- served remains constant over long periods) is taken as a standard to which the strength of each fresh complement serum is adjusted. Strictly speaking, the unit of complement should be that amount which gives complete laking with one unit of cells and one unit of amboceptor. In practice, however, this turns out to be too large an amount, and for this reason complement is usually titrated with two units of amboceptor. The unit of complement then may be defined as the least amount that completely hemo- lyses 1.0 c.c. of a five per cent emulsion of red cells in the presence of two units of amboceptor in thirty minutes in the water bath. If the test is done with smaller quantities of red cells the unit is correspondingly smaller. Set up titrations with both two and one units as follows: 40 LABORATORY COURSE IN SERUM STUDY TUBE COMPLEMENT 1-10 AMBOCEPTOR 1 0.5 c.c. 2 units ' 2 0.4 2 units Add 3 0.3 2 units Saline to 2 c.c. 4 0.2 2 units 5% cells, 0.5 c.c. 5 0.1 2 units t 6 0.5 1 unit 7 0.4 1 unit Add 8 0.3 1 unit Saline to 2 c.c. 9 0.2 1 unit 5% cells, 0.5 c.c. 10 0.1 1 1 unit Incubate. Read results at 15, 30 and 60 minutes. (Indicate unit found.) It should be remembered that even with these two reagents, com- plement and amboceptor, titrated, the amounts determined might vary somewhat if cells from another sheep or taken from this sheep on another day were used since the resistance of the cells is also a variable factor. In many experiments it is expedient to use red cells previously sensitized with a definite number of amboceptor units. In order to do this the following method is employed. The red cells after washing are taken up in salt solution in a definite concentration which for ordinary work is 5 per cent. Inactivated immune serum (amboceptor or sensi- tizer) diluted in salt solution is then added so that the required number of previously determined haemolytic units shall be present for every unit (1.0 c.c. of 5 per cent) of red cells. Therefore, supposing that 0.001 c.c. of the immune serum was found to be one haemolytic amboceptor unit, 1.0 c.c. of the 1-1000 dilution of this serum added to 1.0 c.c. of a 5 per cent emulsion of red cells would sensitize them with one unit. Having made such a mixture and having given it 15 minutes to allow the union to take place, every 2.0 c.c. would represent 1.0 c.c. of 5 per cent red cells and 1 unit of amboceptor united. Or if desired the cells could be centrifugalized, washed, and made up to the original concentration of 5 per cent. On this principle any number of units up to the maximum absorption power can be added to the red cells. As we approach the maximum absorption power of amboceptor by red cells it is always well to centrifugalize and reemulsify the red cells so that our experi- ment may not be confused by the presence of excessive unabsorbed amboceptor. 42 LABORATORY COURSE IN SERUM STUDY Each student needs: 10 c.c. of 5% sheep cells 2 c.c. amboceptor (diluted 1-100) 2 c.c. complement undiluted, for each three students 1 test tube rack 20 half-inch test tubes 4 1-c.c. pipettes, and flask salt solution 4 f -inch test tubes LESSON V QUANTITATIVE RELATIONS OF AMBOCEPTOR AND COMPLE- MENT (Continued) Experiment 1 THE ACTIVITY OF COMPLEMENT DEPENDS TO SOME EXTENT ON ITS CONCENTRATION AND NOT ONLY ON THE TOTAL AMOUNT PRESENT A 25 per cent suspension of washed sheep cells is made. A series of tubes is set up (if necessary by instructor) in which the minimal amount of amboceptor necessary to lake 0.1 c.c. of these cells in the presence of 0.05 c.c. of undiluted complement and in a total volume of 1.0 c.c. is determined. The hsemolytic serum (amboceptor) is then diluted so that this minimal amount is contained in 0.1 c.c. The student then mixes 2.0 c.c. of this amboceptor dilution with 2.0 c.c. of the 25 per cent red cell suspension. He then determines, in the following preliminary titration, the minimal amount (unit) of com- plement which will lake 0.1 c.c. of these cells in one hour, in a total volume of 1 c.c. GUINEA-PIG SERUM 1-10 SENSITIZED CELLS (.05 C.C. CELLS + .05 C.C. OF AMBOCEPTOR) SALINE UP TO 1.0 C.C. Tube 1 Tube 2 TubeS Tube 4 TubeS Tube 6 Tube? TubeS 0.15 C.C. 0.2 0.25 0.3 0.4 0.5 0.0 .5 0.1 c.c. 0.1 c.c. 0.1 c.c. 0.1 c.c. 0.1 c.c. 0.1 c.c. 0.1 .05 unsensi- tized cells .75 C.C. .7 .65 .6 .5 .4 .9 .45 Then with 1, 5, and 10 times this minimal amount in the tubes as indi- cated in the protocol the experiment is set up. The protocol given below is constructed on the supposition that, in the above titration, 0.2 c.c. of a 1 to 10 dilution of complement was the smallest amount of the complement which gave complete laking. 44 LABORATORY COURSE IN SERUM STUDY GUINEA Pio SERUM 1 IN 10 SALT SOLUTION UP TO RESULTING DILUTION OF GUINEA PIG SERUM Tube 1 0.2 C.C. 1.0 c.c. in 50 Tube 2 0.2 2.0 in 100 Tube 3 0.2 5.0 in 250 Tube 4 0.2 10.0 in 500 Tube 5 1.0 1.0 in 10 Tube 6 1.0 5.0 in 50 Tube 7 1.0 10.0 in 100 Tube 8 1.0 20.0 in 200 Tube 9 2.0 5.0 in 25 Tube 10 2.0 10.0 lin 50 Tube 11 2.0 20.0 1 in 100 To each tube add 1 unit (0.1 c.c.) of cells previously sensitized with 1 unit of amboceptor. Calculate the concentration of complement in the tubes used in the preliminary complement titration, and compare the results. If the activity of complement does depend upon the concentration, complete laking should occur in one hour in those tubes of the second series which contain a dilution of guinea pig serum equal to that in the first tube of the first series which showed complete haemolysis. Experiment 2 THE ACTIVITY OF SENSITIZER OR AMBOCEPTOR DEPENDS ON ITS TOTAL AMOUNT AND NOT ON ITS CONCENTRATION Suspend 1 unit (0.05 of 25%) of cells in varying amounts (1, 5, 10, 20 c.c.) of salt solution. To each tube add 1 unit of amboceptor. Mix and keep at 37 C. for one half hour. Centrifugalize. To the sediment in each tube add 1 unit of complement, as previously determined, and sufficient salt solution to bring the volume to 1 c.c. Incubate. All tubes should show complete haemolysis in 1 hour if the cells have absorbed the entire unit of amboceptor. Each student needs: 2 c.c. of 25% sheep cells 1 ten-c.c. pipette (about) 2 c.c. of undiluted com- 2 test tube racks for 12 J-inch plement test tubes 25 units of amboceptor 20 J-inch test tubes Flask of saline 1 fifty-c.c. centrifuge tube 4 one-c.c. pipettes 3 fifteen-c.c. centrifuge tubes 6 -inch test tubes 46 LABORATORY COURSE IN SERUM STUDY LESSON VI THE UNION OF IMMUNE-BODY (AMBOCEPTOR OR SENSI- TIZER) AND CELLS \ 1. DISSOCIATION OF AMBOCEPTOR (Mum) RED cells have a very great affinity for their specific immune- body. They can absorb a great number of units. Nevertheless at 37 C. corpuscles containing multiple doses of amboceptor give off a certain amount to the surrounding fluid when it is free of it. There appears to be an equilibrium between combined and free im- mune-body. This can be illustrated by the following experiment : To 1.5 c.c. of a" 5 per cent emulsion of sheep corpuscles is added inactivated haemolytic serum so that there shall be 20 units of ambo- ceptor x to every 0.5 c.c. of cells. (The student should calculate and make dilutions for this purpose after being told the unit of the hsemolytic serum given him.) This mixture is allowed to stand at room temperature for 30 minutes. Centrifugalize and set aside the supernatant fluid. This is Tube (a). Wash the cells obtained in the sediment of the preceding centrif uga- tion three times and set aside the salt solution remaining as supernatant fluid of the last washing. This is Tube (6). Make the suspension of red cells up to the original volume by the addition of saline and incubate this at 42 C. for one hour. Then centrif- ugalize and set aside the supernatant fluid of this, which constitutes Tube (c). Make the sediment of the cells obtained in the preceding up to 1.5 c.c. Shake. Remove 0.5 c.c. and add to it 1.0 c.c. of 10 per cent fresh guinea pig complement. This is Tube (d). To (a), (6) and (c), each, add 0.5 c.c. of 5 per cent sheep corpuscles and .5 c.c. of complement (10 per cent fresh guinea pig serum in salt solution). All four tubes are incubated at 37 C. for one hour. Record the degree of laking in each tube. Tube (a) will show how much of the 20 units of amboceptor failed to be absorbed. The exact amount could be determined by titration, which, however, would needlessly prolong the experiment and add nothing to the illustration of the principle. 1 The unit here is the minimal amount which lakes 0.5 c.c., not 1 c.c., of cells. If Experiments 1 and 3 of this lesson are to be done on the same afternoon it is well to sensitize a considerable amount of cells at one time. 48 LABORATORY COURSE IN SERUM STUDY Tube (6) will show whether the three washings were sufficient to remove all amboceptor from the fluid bathing the cells. Any excess of amboceptor remaining unattached to the cells after the washing would show in the supernatant fluid by laking. Tube (c) will show how much, if any, amboceptor was dissociated after one hour at 37 C. 2. VELOCITY OF AMBOCEPTOR ABSORPTION Red cells absorb their homologous immune-body very rapidly. A knowledge of this is of great importance as the following ex- periments show. A Take 3 c.c. of 5 per cent sheep corpuscles in a wide test tube and add 3 c.c. of diluted amboceptor drop by drop, shaking constantly (the dilution of the amboceptor is such that 1 unit is contained in 0.5 c.c. ; the unit here is taken as the minimal amount which lakes 0.5 c.c. of ceUs). B Take 3 c.c. of diluted amboceptor and add 3 c.c. of 5 per cent sheep cells drop by drop, shaking constantly. Set up two parallel series of five tubes each containing varying amounts of guinea pig serum (1-10). Series 1 GUINEA Pro SERUM SALT SOLUTION (TO TUBE DILUTED 1-10 MAKE ALL VOLUMES MIXTURE A (COMPLEMENT) EQUAL) 1 0.75 c.c. 0.0 C.C. 1.0 c.c. 2 0.5 c.c. 0.25 c.c. . 1.0 c.c. 3 0.4 c.c. 0.35 c.c. 1.0 c.c. 4 0.3 c.c. 0.45 c.c. 1.0 c.c. 5 0.2 c.c. 0.55 c.c. 1.0 c.c. Series 2 Duplicate above with mixture B. Incubate one hour and compare results. The observed differences are probably explained by the fact that the first cells which are added to B absorb nearly all the amboceptor, leaving insufficient to sensitize the last cells added. 50 LABORATORY COURSE IN SERUM STUDY 3. LIBERATION OF AMBOCEPTOR IN HAEMOLYSIS When red cells containing several hsemolytic doses of ambo- ceptor are laked by a not excessive amount of complement, some of the amboceptor is liberated in the solution. To 2.0 c.c. of 5 per cent sheep cells add sufficient amboceptor so that there will be 20 units for every unit (0.5 c.c.) of 5 per cent sheep cells. Allow to stand for one half hour. Centrifuge and wash with saline three times and after the third washing make the volume again up to 2.0 c.c. To 1.0 c.c. of cells so sensitized add 1.0 c.c. of 10 per cent guinea-pig complement. Incubate one half hour. Then divide into two equal portions of 1.0 c.c. each. To the first portion add 0.5 c.c. of the sen- sitized cells and incubate again for one hour. Note whether haemolysis of the added cells occurs. To the second portion add 0.5 c.c. of un- treated corpuscle suspension and 1.0 c.c. of 10 per cent complement. Haemolysis, after incubation at 37J degrees centigrade, will indicate that amboceptor was liberated by the laking of the sensitized cells dur- ing the first incubation and was taken up by the added cells in the second incubation. How does the amount of amboceptor liberated compare with the amount dissociated by simple incubation without haemolysis in Experi- ment 1 ? Each student needs : 12 c.c. of 5% sheep cells 160 units of amboceptor 10 c.c. of complement (1-10) Flask saline 4 one-c.c. pipettes 1 test tube rack for half-inch test tubes 1 graduated centrifuge tube 20 half-inch test tubes 3 three-quarter-inch test tubes 1 five-c.c. pipette 1 nipple pipette 1 1 If experiments 1 and 3 of this lesson are to be done on the same afternoon, it is well to sensitize a considerable amount of cells at one time. 52 LABORATORY COURSE IN SERUM STUDY LESSON VII PFEIFFER PHENOMENON VIRULENT cholera spirilla injected into the peritoneal cavity of a normal guinea pig proceed to multiply rapidly after a pe- riod of about half an hour, and the animal dies usually within 24 hours with symptoms of profound intoxication. The same spirilla injected into an animal which has been immunized by previous injections of killed spirilla or of sub-lethal doses of the living microorganisms become granular and in some cases swell, take on globular and vacuolated forms and gradually undergo lysis. The guinea pig in such cases recovers completely from the in- jection. This destruction of invading bacteria by the immune animal was termed by Pfeiffer " bacteriolysis." It has been ob- served with certain of the Gram-negative bacilli such as the typhoid and the dysentery bacillus, but is best observed in cholera. Similar lysis is observed if the spirilla are injected into a normal pig together with serum of an immunized guinea pig or rabbit. For this experiment it is necessary to employ a virulent 1 strain of cholera spirilla, since old laboratory cultures do not show the phenomenon sharply and rarely kill the guinea pigs in the con- trols. The lethal dose of the particular strain of cholera used for the guinea pigs should be determined roughly before carrying out the experiment. It is inadvisable to allow students, except in very small groups, to handle virulent cholera cultures, so that in carrying out this experiment, the preparation of the cholera suspension, the in- jection of the pig and the removal of specimens from the peri- toneum, as described below, should be carried out by the instruc- tor. The smears should be fixed immediately in 10 per cent foimalin, after which they may be distributed to the members of the class for study. Two pigs one normal, the other previously immunized with cholera spirilla are intraperitoneally injected with the determined dose, say a 1 The strain must be recently isolated or one that has been passed through guinea Digs until it will kill in quantities of one loopful of an agar slant. 54 LABORATORY COURSE IN SERUM STUDY twentieth of the emulsion obtained from an agar slant, and after ten minutes a specimen of the peritoneal fluid is withdrawn for observation. This is done by shaving the left side of the abdomen of the pig, holding the pig, back down, on a tray. This can be done satisfactorily with the operator's left hand. The shaved surface of the skin is sterilized by wiping with 5 per cent carbolic, a small cut made through the skin, but not through the muscles, with a sharp pair of scissors. Then the sharp tip of a capillary pipette is inserted through the muscles and the peritoneum. If the pipette is held horizontally or with the open end depressed, a small amount of peritoneal fluid will run up into the capillary portion even if no suction is used. When sufficient amount of fluid [0.1 or 0.2 c.c.] is obtained, the pipette is withdrawn and the puncture covered with cotton soaked in carbolic solution. A hang-drop preparation is made with a drop of this fluid and the remainder blown out into a watch glass. With a platinum loop a series of thin smears are made of this fluid on glass slides, allowing one slide for each member of the class, and as soon as the films are dried they are placed in Coplin jars containing a 10 per cent solution of formalin (4 per cent formaldehyde) for five minutes. They are then withdrawn, allowed to dry, and are stained by the students with methylene blue and studied under the oil immersion lens. The hang-drop preparation should be observed under a high-power dry lens and one preparation may be observed by the whole class. Preparations are made in this way from the normal and immune pig ten minutes, thirty minutes, one hour and two hours respectively after injection. By the end of this period if a proper dose of the organisms has been injected, the spirilla will be found to have multiplied enor- mously in the peritoneal fluid of the normal animal, whereas in the smears made from the immune animal after thirty minutes to an hour the spirilla will appear granular or swollen, and after two hours, or, if the observation is followed that long, after three or four hours, will be found to have practically disappeared. The two animals should be kept for observation after the experiment, and the unprotected animal will probably be found dead the next day. The passive transference of bacteriolytic immunity to another animal by simultaneous injection of cholera spirilla and anti-cholera serum obtained from the rabbit immunized to cholera may be demon- strated in a similar way, and by this technic the strength of a bacteriolytic serum may be titrated by injecting series of guinea pigs with varying amounts of the serum. 56 LABORATORY COURSE IN SERUM STUDY A type protocol of such an experiment is given below: WEIGHT or GUINEA Pio DOSE OF BACTERIA 1 CHOLEBA SPIRILLA AMOUNT OP INACTIVATED IMMUNE SERUM RESULT (1) 215 gm. 2 mg. 0.1 c.c. in 1 c.c. Complete dissolution in less salt solution than one hour. Lives. (2) 230 gm. 2 ing. 0.05 c.c. About the same as first. (3) 200 gm. 2 ing. 0.01 c.c. Somewhat slower than in other two ; a few unchanged spirilla after 1 hour. Final dissolution. Pig lives. (4) 245 gm. 2 mg. 0.005 c.c. Similar to (3) but complete dissolution in 2 hours. Pig lives. (5) 220 gm. 2 mg. 0.001 c.c. After 30 minutes the spirilla seem to have begun to multiply. Dies with innumerable active spirilla in peritoneum. Normal Con- trol (6) 210 gm. 2 mg. 0.1 c.c. normal Very slight lysis at the begin- inactive rab- ning. Soon rapid multiplication. bit serum Dies. LESSON VIII BACTERICIDAL TEST IN VITRO STERN KORTE THE destruction of bacteria which takes place in the peri- toneum of immune animals may also be demonstrated for certain species of bacteria in the test tube. Small quantities of typhoid bacilli, as shown in a previous experiment, are killed by exposure to certain normal sera such as those of the rabbit and the guinea pig. A more powerful action is observed in sera of animals immunized to the typhoid bacillus, and, as in the case of red cells, the immune body or sensitizer can be shown to be relatively thermostable in that it will act after exposure to 56 C. for half an hour provided it is reactivated by the addition of a small amount of normal serum. The bactericidal effect of immune serum shows more clearly than most other antibody phenomena the presence of a prezone, 1 The bacteria may be measured for such an experiment by standard loop- fuls (one loop being equal to 2 milligrams), or by volume in emulsion with salt solution. 58 LABORATORY COURSE IN SERUM STUDY that is, the failure of the antibodies to act if present in excessive concentration. In the sera of artificially immunized animals or of typhoid patients amounts greater than 0.01 c.c. of a strongly im- mune serum are usually ineffective, the best results being obtained with amounts varying from 0.001 to 0.0001 c.c. in a volume of 1 c.c. Since the inactivated immune serum must, of course, be re- activated by fresh normal serum used as complement or alexin, it is important to use an amount of the normal serum too small to be of itself bactericidal ; and the first step to determine is the bactericidal power of the normal serum against the strain of bacteria to be used. On the other hand, to secure striking results it is necessary to use as large an amount of fresh normal serum as possible without entering the zone of normal bactericidal power. Normal rabbit serum is usually definitely bactericidal for typhoid bacilli in amounts ranging from 0.1 to 0.02 c.c. (in a total volume of 1 c.c.). Reagents : 1. Anti-typhoid serum serum of a rabbit immunized against typhoid bacilli obtained in sterile condition and heated at 56 C. for half an hour. 2. Normal rabbit serum, which must also be sterile. 3. 24-hour broth culture of typhoid bacilli. The glassware and salt solution used in the experiment must, of course, be sterile, and the reagents handled with care to avoid contam- ination during the setting up of the experiment. TENTATIVE SCHEDULE l Set up the following tubes: IMMUNE SERUM TUBE Actual Quantity of the SOLUTION SERUM 1-50 Immune Serum 1 0.01 C.C. 0.5 c.c. (1-50) 0.0 c.c. 0.5 c.c. 2 0.002 c.c. 0.1 c.c. (1-50) 0.4 c.c. 0.5 c.c. a 0.001 c.c. 0.5 c.c. (1-500) 0.0 c.c. 0.5 c.c. 4 0.0001 c.c. 0.05 c.c. (1-500) 0.45 c.c. 0.5 c.c. 5 * 0.01 c.c. 0.5 c.c. (1-50) 0.5 c.c. 6 None 0.5 c.c. 0.5 c.c. 7 None 1.0 c.c. 8 None 1.0 c.c. 1 By a previous test of the serum the instructor should adjust the schedule so that one or two tubes lie within the "killing " zone and others above and below it. 2 The serum in this control tube should be that amount which shows maximum bactericidal power when used with active serum. 60 LABORATORY COURSE IN SERUM STUDY Add to each tube about 0.1 c.c. of a 24 hour broth culture of B. typhosus, diluted 1-500. This should be measured by making a mark on a sterile capillary pipette, filling accurately to this mark and dis- charging the contents against the side of the tube, close to the level of the fluid, being careful not to touch the upper portion. The actual amount used is unimportant, but precisely equal amounts must be added to each tube. Shake. Incubate the tubes two hours at 37 C. At the end of this time convey two loopsful from each tube into melted and cooled agar tubes and pour plates in the usual way. Transfer of two loops from tube eight should also be made into agar in this way immediately after mixing without incubating. In careful experiments, two additional controls, the one containing 0.5 c.c. of 1-50 normal serum, the other containing 0.5 c.c. of 1-50 im- mune serum without the addition of typhoid bacilli, should also be set up and transfers should be made from them into agar to prove the sterility of the reagents used. A successful experiment should show an enormous number of colonies, over 10,000 to the plate, in plates Nos. 5, 6 and 7. Plate 8 should also show a large number of colonies, though perhaps less than the other controls. In plates 2 and 3 there should be very few col- onies or none at all; in plates 1 and 4 some reduction in the number, though in some cases there may be no observable difference between these plates and the controls. Each student needs: Five f-inch test tubes, twelve petri dishes, twelve tubes of agar, four one-c.c. pipettes. LESSON IX I. TITRATION OF AGGLUTININS BY MACROSCOPIC METHOD THE clumping of bacteria by immune serum causes a homo- geneous suspension to form flocculi easily visible to the naked eye, which, on standing, settle to the bottom of the tube, making the supernatant fluid clear. The macroscopic test is considered more accurate than the microscopic since one is less often de- ceived by small clumps of bacteria which may form spontaneously in salt solution or broth for various reasons and are readily visible under the microscope but do not come together in large enough masses to cause a precipitate visible to the naked eye. It is much simpler to carry out a quantitative titration of the aggluti- native power of the serum by setting it up in test tubes than by preparing hang-drops of each dilution. Furthermore, since the quantities used in these tests are relatively large, slight inac- curacies in measurement do not lead to such gross errors as would 62 LABORATORY COURSE IN SERUM STUDY similar inaccuracies in the microscopic method. For these reasons the macroscopic method is the one usually employed in experimental investigations. On the other hand, in testing patient's sera against the typhoid bacillus (the Widal reaction) considerable information is given by the cessation of motility of the bacilli in the presence of immune serum. It is desirable to use small amounts of serum and unnecessary to set up more than three or four dilutions in one test. The macroscopic test may be carried out with living bacteria, with bacteria killed either by exposure to heat (56 C.) or by ex- posure to weak antiseptics such as phenol, 1 per cent solution, or formaldehyde 0.8 per cent. Killed suspensions of bacteria may be kept a long time in the ice box and still remain serviceable. The flocculation is most easily observed in narrow test tubes, f of an inch in diameter, but larger tubes can also be used with- out difficulty. Some workers prefer to use tubes pointed at the bottom, similar to the centrifuge tubes, since the sediments which settle out can be readily observed in the tips of these tubes. The period of incubation varies. A marked reaction is visible after an hour at 37, but with many organisms it is necessary to incubate for two hours to obtain clear-cut results, and the floc- culation often becomes more distinct when the tubes are removed from the water bath and placed in the ice box for from half an hour to 12 hours. Such organisms as the streptococcus 1 and the staphylococcus usually require the longer periods of incuba- tion, and agglutination is favored by a temperature of 56. On the other hand, for organisms of the typhoid-colon group long in- cubation is unnecessary. Reagents : 1. Serum of a rabbit immunized against typhoid. For this test the serum of animals immunized by the class should be used, the members in charge of the rabbits bleeding them from the carotid at the previous exercises and distributing to each other member of the class 2 c.c. of a 1-10 dilution of the serum. 2. B. typhosus 24 hour culture on agar slant. 3. Normal salt solution. 1 Streptococcus agglutination requires a special and complic.it c