152 PRACTICAL METHODS IN IMMUNITY 



man or the heart of the immunized rabbit, the most convenient method is with the 

 use of an Erlenmeyer flask with a rubber stopper having two perforations in the stop- 

 per. To one of the projecting pieces of glass tubing a stout hypodermic needle is 

 attached through the medium of about 8 inches of rubber tubing, and the second 

 piece of glass tubing is bent at an angle as it leaves the stopper to provide a suction 

 tube. With a man, constrict the upper arm sufficiently to stop venous return with 

 an Esmarck rubber bandage or a towel. Paint tincture of iodine over a prominent 

 vein at the bend of the elbow. Gentle suction will cause the blood to flow into the 

 needle tube and thence into the flask. 



The blood as it is taken from the arm should be received in about 50 c.c. of normal 

 salt solution containing i% of sodium citrate. About 28 to 30 c.c. are usually suffi- 

 cient. Now throw down this red-cell suspension in three or four centrifuge tubes. 

 The resulting sediment should be washed and rewashed with salt solution. Two 

 to three washings with salt solution suffice. 



Now take a large healthy rabbit, shave the lower abdomen and paint the surface 

 with tincture of iodine. The easiest way to inject the rabbit is to hold the animal 

 head down and plunge the needle of a large glass hypodermic syringe containing the 

 washed red-cell sediment into the abdominal cavity in the median line. The intes- 

 tines gravitate downward and by entering the needle below the limits of the bladder 

 we avoid injuring any vital part. 



Make the injections at intervals of five days and give increasing amounts at each 

 successive injection. Thus, first injection, 5 c.c.; second injection, 8 c.c.; third 

 injection, 10 c.c.; fourth injection 12 c.c.; and at the fifth injection give about 15 to 

 20 c.c. of washed red-cell sediment. It is well to dilute the cell sediment with an 

 equal amount of salt solution. About ten days after the last injection, we take some 

 blood in a Wright's tube from a vein of the ear and dilute the serum to make a i to 

 100 dilution. To i c.c. of a i% emulsion of red cells we add o.i c.c. of a 20% dilu- 

 tion of guinea-pig's fresh serum similar combinations being made in a series of 

 10 tubes. To each of these tubes we add varying amounts of the i to 100 dilution, 

 o.i c.c. in the first, 0.2 c.c. in the second, 0.3 c.c. in the third, and so on. If we 

 obtain haemolysis in the tube containing 0.2 c.c. of i to 100 dilution of serum but not 

 in that containing o.i c.c. we note that the serum has a titre of about i to 500. If 

 the o.i c.c. gave haemolysis, the serum would have a titer of i to 1000. 



Having ascertained that the haemolytic serum is sufficiently strong we shave the 

 left side of the thorax of the rabbit and enter the needle of the apparatus similar to 

 that used for taking the blood from a man's vein in one of the intercostal spaces of the 

 left side. 



Having introduced the needle, feel for the heart beat and then plunge the needle 

 into the heart. We can withdraw about 30 c.c. of blood without injury to the rabbit. 

 This blood should be received in a clean empty flask and set, over night, in the re- 

 frigerator. The following morning pour off the clear serum into a clean Petri dish 

 and saturate, one by one, squares of filter paper with the serum. Allow the filter 

 paper to dry on a piece of unbleached muslin. Noguchi recommends Schleich and 

 SchulPs paper No. 597. When thoroughly dry cut strips 10 mm. wide. This makes 

 the amboceptor paper. To standardize, take a series of tubes containing i c.c. of 

 a i% emulsion of red cells and add o.i c.c. of 20% dilution of guinea-pig serum for 

 complement. Next cut across the amboceptor paper strip pieces of varying width, 



