SEROLOGICAL METHODS viii,,-5 



cally. The macroscopic tube test is probably the most accurate and 

 is a convenient method. The macroscopic slide test has been used 

 extensively for typing enteric bacteria. The phenomenon of agglu- 

 tination is evidenced by the appearance of granulation in the bacterial 

 suspension. Granulation may be extremely fine, or clump size may 

 range to very coarse. Clumps may be compact and dense as in the 

 case of somatic agglutination, may be light and cottony with flagel- 

 lar reactions, or may be stringy and thread-like with some mucoid 

 organisms. Conditions of optimal incubation time and tempera- 

 ture vary considerably depending on the organisms tested. 



PREPARATION OF IMMUNE SERUM 



The rabbit is the most satisfactorj' animal for the production of agglutinating 

 serum. The technics employed for inuuunization are many, and vary widely with dif- 

 ferent workers. The particular properties of the bacteria under examination are the 

 factors determining the method chosen. 



A healthy, well-developed rabbit is selected for immunization. It may conven- 

 iently be held in a squatting position by an attendant or locked in a special box in 

 such a manner that only the head protrudes. The hair is removed from around a 

 marginal ear vein and along the edge of tlie ear by shaving. The ear is then cleansed 

 with 70% ethyl alcohol. Dilation of the vein is promoted Ijy rubbing or patting, by 

 heat, or by applying xylol. Any sharp sterile instrument will serve to open the vein, 

 a clean wound favoring the escape of blood. About 5 ml. of blood are collected in a 

 sterile test tube to provide serum for determining the presence or absence of natural 

 agglutinins in the blood of the rabbit selected. The technic is described in the next 

 section. Having determined the absence of natural agglutinins the immunization 

 procedure may be undertaken. 



Immune Serum: Immunize the rabbit by repeated subcutaneous, intraperitoneal, or 

 intravenous injections of saline suspensions of young (18-24 hour) cultures of the 

 organism to be studied. Growth can be taken from agar or from silica gel medium or 

 in the case of flagellar antigens young broth cultures may be used. A convenient sus- 

 pension is one containing about 500 million organisms per milliliter, although suspen- 

 sions containing more or less than this number of organisms can be used, depending 

 upon the toxicity of the cidture. The number of organisms in the suspension can be 

 c}uickly and roughly determined by various methods outlined in any treatise on sero- 

 logical methods. If the organism is non-pathogenic for rabbits, suspensions of living 

 organisms can be injected. As a rule, however, the organisms are killed Ijefore injec- 

 tion by heating the suspension in a water bath at (JO°C. for 1 hour, or by mild chemical 

 treatment such as 0.3% formalin. Inject subcutaneously, intrapcritoneally, or intra- 

 venously into a rabbit at intervals of 5-7 days starting with a dose of 0.5 ml. and in- 

 creasing each dose by 0.5 ml. After the third injection a test bleeding m;iy be made to 

 determine titer, and the rabbit bled out or reinjected as necessary. Titrations of the 

 serum should be made following each subsequent injection, and immunization con- 

 tinued until a satisfactory titer is attained or until no further increase occurs. Six to 

 eight injections are usually required to produce agglutinins of sufficient titer. The 

 method of immunization can be varied to meet the needs of special cases. The shorter 

 the period of immunization, the more specific is the imtnune scrum. Long immunization 

 increases the content of group agglutinins. To test the titer of the serum, draw 1-2 ml. 

 of blood from the marginal vein of the ear of the rabbit 5-7 days after the last injection; 

 collect the serum and carry out an agglutination test with it, as described below. If the 

 titer is sufficiently high, bleed the rabbit from the heart or an artery to obtain as much 

 blood as possible. Allow the blood to clot, collect the serum aseptically, and add 0.5% 

 phenol or 0.3% tricresol to the serum as a preservative. Place the serum in ampules 

 or bottles and store in the refrigerator. 



Bleeding of Rabbit: Rabiiits are more easily and readily bled from the heart when 

 large amounts (25-100 ml.) of blood are desired. Etherize the rabbit, clip the hair over 

 the region of the heart, and shave. .\n added precaution to prevent contamination is 

 to wet the hair of the rabbit thoroughly over the entire left side. With the rabbit 

 lying on its right side paint the shaved area with tincture of iodine. Determine the 

 point of maximum pulsation. Using a sterile 50 ml. Luer syringe and a needle of 17 or 

 18 gauge (2-2)^2 inches), insert the needle at the point of maximum pulsation. The 



