viir47-14 MANUAL OF METHODS FOR PURE CULTURE STUDY 



which will not grow well on agar slants. Organisms requiring a carbohydrate for growth 

 can be grown in sugar broth and then washed free of acid and used as antigen. Before 

 the suspensions are injected, they should be heated for 1 hour at 60°C. On the first 

 injection, use 0.5 ml. of this suspension. Increase the dose by increments of 0.5 ml. 

 at intervals of 5 to 7 days. If the organism is not too virulent and the animal has 

 not lost weight, the last few injections may be made with unheated suspensions of 

 living organisms. About one week after the last injection, bleed the rabbit from the 

 ear vein and obtain sufBcient serum for a preliminary test to determine its potency. If 

 this test shows that the serum contains antibodies in sufficiently high titer, bleed the 

 rabbit from the heart, or in some manner which will provide as large an amount of 

 serum as possible. After the collection of the serum, heat it at 5G° C. for 1 hour to 

 destroy complement, add 0.3% tricresol as a preservative, and store in sealed ampules 

 or bottles. 



It is not possible to lay down an invariable rule as to the total amount of antigen 

 which should be injected to bring about a sufficient production of antibodies or to 

 specify exactly the period of time required for the series of injections. Immune sera 

 obtained after short periods of immunization are usually more specific than those 

 obtained after long periods of immunization. By trial the amounts to be used in the 

 final test can be determined; see p. VIII47-I6-I7. 



(c). Complement. Guinea pig serum furnishes an active and easily fixable comple- 

 ment. It is usually advisable to pool the sera from at least 3 guinea pigs weighing 1 to 2 

 pounds to obtain a sample of complement having average properties. Bleed the 

 guinea pigs from the heart, removing 5 to 10 ml. of blood from each animal. Allow the 

 blood to clot. Pipette ofJ the serum and store in a sterile glass container in the refrig- 

 erator. The most potent complement can be obtained by allowing the clotted blood 

 to stand overnight in a refrigerator before separating the serum. Complement rarely 

 retains its potency longer than 3 days. It is essential to titrate it daily. Very fine 

 work requires titration twice a day, keeping the complement in the refrigerator as much 

 as possible when not actually being used. Complement preserved by the lyophile proc- 

 ess or the cryochem process may be used: see Mudd et al. (1936), Ecker and Pillemer 

 (1938). 



(d). Sheep's Red Blood Corpuscles. With a veterinary needle, or a 19-gauge needle 

 attached to a 50 ml. syringe, withdraw 10 to 50 ml. of Ijlood from the external jugular 

 vein of a sheep. Place the blood at once in a sterile flask containing glass beads. Shake 

 for 15 minutes to defibrinate, and filter through gauze or absorbent cotton to remove 

 the fibrin. Instead of defibrinating in this manner, the blood may be mixed with an 

 equal volume of 0.85% saline containing 2% sodium citrate. This prevents coagula- 

 tion and makes it unnecessary to remove the fibrin. Wash the cells 3 times in 0.85% 

 saline. This is done by centrifuging the cells at about 1500 r. p. m. for 10 to 15 minutes. 

 Pipette off the supernatant fluid and add as much fresh saline as the amount removed. 

 Mix well and repeat the process twice. Final centrifugation should be at 1800 r. p. m. 

 in order to pack the cells. After the final washing, carefully remove the supernatant 

 saline without disturbing the packed sediment of cells. With this sediment make a 

 2.5% suspension of the red cells in saline by adding 2.5 ml. of the packed cells to 97.5 

 ml. of saline. If it is desirable to keep the cells longer than 3 days, 0.1 ml. of a 1-10 

 dilution of 40% formaldehyde may be added to 8 ml. of blood. This mixture as well as 

 any other suspension of blood cells should be kept in the refrigerator until used. Before 

 use, the cells should be washed 3 times in saline (or until supernatant fluid is clear and 

 colorless). For accurate work it is best to use fresh cells. 



For hemolysin production, red cells which have not been treated with formalin 

 should be used. 



(e). Amboceptor (Anti-sheep-red-cell Hemolysin).^ Very strong hemolysin may 

 be obtained by the following method: Two healthy rabbits are given intravenous 

 injections of undiluted and unpreserved washed sheep's corpuscles according to the 

 following schedule: 1st day, 0.5 ml. packed erythrocytes; 3rd day, 1.0 ml.; 5th day, 

 1.0 ml.; 7th day, 1.0 ml.; 11th day, 1.5 ml. 



Eight days after the last injection a trial bleeding is made from the marginal ear 

 vein. If the serum is found sufficiently potent the rabbits are bled to death or enough 

 blood is taken from the ear vein as is desired for stock hemolysin. The latter method 

 should yield all the serum needed, at least if the bleeding is repeated on two or three 

 successive days, and if both ears are used. 



iSee also Beattie, (1934); von Dardnyi, J., (1928); Stafseth (1932); Ulrich and 

 McArthur (1942); Sawyer and Bourke (1946). 



