202 MANUAL OF MICROBIOLOGICAL METHODS 



best to start near the tip of the ear so that if the vein is missed, another insertion can 

 be made nearer the base of the ear. During this process, the rabbit can be held by an 

 assistant or placed in an animal box which allows the neck and head to protrude. 

 After the needle is removed, bleeding is stopped by pressing a piece of cotton against 

 the site of injection. 



For intraperitoneal injections, the animal is held by an assistant. The fore paws 

 are grasped in one hand, and the hind paws in the other. The animal is held with 

 the head down so that the abdominal viscera are pushed against the diaphragm. The 

 area over the lower part of the abdomen is clipped and washed with 70 per cent 

 ethanol. The skin over the abdomen is held between the fingers, and the needle 

 (21- to 23-gauge) inserted through the skin and peritoneum into the peritoneal cavity. 

 Care should be taken not to perforate the intestine. 



Subcutaneous injections are made by pulling up the skin of the back or thighs with 

 one hand and inserting a 21-gauge needle into the raised skin. 



If hypersensitivity is suspected, it may be wise to administer a small 

 dose of antigen intracutaneously before these other routes are used. A 

 portion of the back, sides, or belly is shaved, and the antigen injected 

 through a 24- to 25-gauge needle into the epidermis. The needle should 

 be held nearly parallel to the surface of the skin with beveled edge upward. 

 A volume of 0.1 or 0.2 ml is injected, and if done properly, a bleb should 

 be raised. 



When antigen is administered to the guinea pig, intraperitoneal, sub- 

 cutaneous, and intracutaneous injections are made in a manner similar 

 to that described for the rabbit. Intravenous inoculations can also be 

 given to the guinea pig, using the vein on the dorsal inner region of the 

 hind leg; however, this procedure requires some practice and should be 

 demonstrated by an experienced person. 



Immunization schedules. There are a number of satisfactory sched- 

 ules for immunization. One good method is to give daily injections of 

 antigen for the first 3 days of each week, allowing the animal to rest 

 during the intervening 4-day period. This cycle is repeated for 3 to 6 

 weeks, and a test bleeding is made. If the titer is sufficiently high, the 

 animal is bled from the heart; otherwise, the series is repeated. It is 

 advisable to give the first injection each week by the intraperitoneal or 

 subcutaneous route and the other two by the intravenous route. In the 

 absence of more definite information on dosage, soluble proteins can be 

 injected in amounts of 10-100 mg contained in 1 or 2 ml of saline. With 

 bacteria, 10^ or 10'* cells per injection may be used unless the cells are too 

 toxic. 



Harvesting of antiserum. Rabbits should be bled from the marginal 

 ear vein before immunization is begun in order to secure a few milliliters 

 of reference serum. The animal is placed in a box with the head protrud- 

 ing, and the ear rubbed with xylene and alcohol as described under ''Immu- 

 nization," When the vein is congested, it is nicked with a razor blade and 



