SEROLOGICAL METHODS 203 



5-10 ml of blood allowed to drop into a large tube; the bleeding is stopped 

 by pressure from a cotton pledget. The blood is allowed to clot, the clot 

 is separated from the wall of the tube with an applicator stick, and the 

 serum is centrifuged until clear. 



The same technic is used for trial bleedings after a series of injections 

 has been concluded. Six to eight days should elapse between the last 

 injection and the trial bleeding. Serum from the trial bleeding is titrated 

 for antibody content, and if a satisfactory level of antibody has been 

 reached, the rabbit is bled from the heart. 



Cardiac bleeding is not a difficult procedure but should be learned 

 under the guidance of an experienced person. The rabbit should not be 

 fed for 24 hr before bleeding to avoid excess lipid in the serum. The 

 animal is tied securely to an animal board, and the hair shaved from the 

 chest. The ribs are palpated until the area of maximal pulsation is 

 located, and the site is painted with tincture of iodine. For quantities of 

 blood up to 50 ml, one may use a syringe fitted with a 17-, 18-, or 19-gauge 

 needle. If a rabbit is to be exsanguinated, the needle may be attached to 

 a rubber tube leading into a flask fitted for suction. All materials should 

 be sterilized in the autoclave. 



The animal may be anesthetized with ether or nembutal, but after 

 experience is gained, it is better to use no anesthesia. 



The needle is inserted at the site of maximal pulsation, and gentle suc- 

 tion is applied. If the heart is not located on the first attempt, the 

 needle should be completely withdrawn before reinsertion. 



The blood is allowed to clot in centrifuge tubes or an Erlenmeyer flask. 

 Clotting will usually occur within a few minutes at room temperature, 

 and the clot can then be freed from the walls of the vessel with a sterile 

 glass rod or applicator stick. Storage overnight in the refrigerator will 

 cause the clot to contract, and the serum can be decanted off and freed 

 from cells b}^ centrifugation. Merthiolate may be added to a final con- 

 centration of 0.01 per cent as a preservative; however, glycerol added to 

 serum in a 1:1 ratio is more effective in preventing contamination if its 

 inclusion is not objectionable. If no preservative is desired, the serum 

 may be stored in the deep freeze. 



Antibody purification. Many of the reactions between microbial anti- 

 gens and their homologous antibodies can be studied with crude anti- 

 serum. For some operations, however, it may be advantageous to effect 

 at least a partial purification of antibody globulin. 



A degree of purification can be accomplished by precipitating the 

 globulins, including antibody, with ammonium sulfate. Each 100 ml of 

 serum is diluted mth 100 ml of water and mixed with 200 ml of saturated 

 ammonium sulfate. The globulin precipitate is removed by centrifuga- 



