196 THE PRINCIPLES OF IMMUNOLOGY 



Reimann found that the cells can be preserved for use in the Wassermann 

 test for 3 to 4 weeks by formalization and for 21 to 25 days by the Rous and 

 Turner method. " The readings obtained differ from those obtained with fresh 

 cells only in so far as some sera produce slightly different results when used 

 with cells from the same specimen of sheep blood." An excellent control for the 

 usefulness of preserved blood is suggested by Kolmer, who maintains that there 

 should be no discoloration of supernatant fluid after the second washing and 

 that the blood should become brighter in color than the dark color it possesses 

 after standing. 



When extreme accuracy is desired cell emulsions are made to 

 contain 1,000,000,000 cells per cubic centimeter. Such emulsions are 

 being more widely adopted, but many laboratories still use 5 per 

 cent, or 10 per cent, emulsions calculated either from the original 

 blood volume or the bulk of the centrifuged cells. The cells should 

 always be most carefully washed, so as to avoid precipitin reactions 

 which may appear if the serum is not entirely removed and to wash 

 out antilytic substances which may appear if the blood is old. 



Influence of Temperature upon the Reaction. This influence may 

 be determined as regards the velocity of the reaction and the amount 

 of complement fixed. The earlier work with complement fixation was 

 based on the general assumption of immunologists that a temperature 

 of 37 C. represents the optimum. In 1912, however, A. McNeil pointed 

 out that ice-chest temperature favors the completeness of complement 

 fixation in the Wassermann test, provided the time of exposure is from 

 eight to twelve hours. This was confirmed by Coca and 1'Esperance, 

 Smith and W. J. McNeil, Berghausen and others, and the ice-chest 

 method has now been adopted by a large number of laboratories as a 

 standard method. The time, however, has been reduced to from three 

 to four hours and the results appear to be entirely satisfactory. The 

 antigen, serum to be tested and complement are mixed and placed in 

 the ice-chest for the required time ; the mixture is then brought to about 

 37 C. in a water bath, the sensitized erythrocytes added and the whole 

 incubated at 37 C. for one hour. 



Dean has investigated the influence of temperature and finds that 

 fixation proceeds most rapidly at 37 C. Noguchi confirms this but 

 finds that at the lower temperature of 23 C., fixation will reach a maxi- 

 mum but proceeds more slowly. He states that with the acetone insol- 

 uble antigen " a serum containing one unit of fixing substance will 

 complete the reaction within thirty minutes at 37 C., sixty minutes at 

 30 C., and two hours at 23 C., irrespective of whether human or 

 guinea-pig complement is used." Dean, however, finds that at o C. 

 the amount of complement fixed is much greater than at 37 C., and this 

 accords with experience in the use of the ice-chest method. Certain 

 unknown factors may delay the action of the complement, as has been 

 pointed out by McConnell, and a second incubation may accordingly 

 have to be prolonged beyond the usual time. 



The Technic of the Wassermann Test. For the demonstration of the 

 method we may use an alcoholic extract of ox heart as the syphilitic antigen, 

 inactivated human serum from a normal individual and from a known victim 

 of syphilis, guinea-pig complement and a sheep hemolytic system. 



