186 G. G. Heslop: 



tion immediately prior to use in the tests. Although the quan— . 

 tity of carbolic acid present in the amount of serum dilution used 

 in the tests was infinitesimal, it was considered that better results 

 might be obtained if the carbolic acid was eliminated altogether- 

 It was therefore decided to inactivate the bovine sera at 56°C 

 for half an hour on two consecutive days as soon as possible 

 after collection, and store them in the ice chest in an undiluted 

 form for use as required. It has been found that sera so treated 

 invariably remain clear and sterile. 



Tests made with two quantities of serum from the one animal, 

 one quantity of which was only once heated at 56°C. for half 

 an hour, the other quantity being heated at 56°C. for half am 

 hour on two consecutive days, showed no difference in their com- 

 plement fixing properties. The double heating is advisable if 

 the sera are to be preserved for any length of time. 



(6) Antigen. — Cultures in Martin's broth plus ox serum or 

 horse serum were used as Antigens, but before use were pre- 

 pared as follows: — The culture was heated in a water bath at 

 56°C. for one hour, then diluted in equal parts with 1 per cent, 

 carbol saline solution. The mixture was then placed in a mechani- 

 cal shaking apparatus, and thoroughly shaken for 5 hours. It 

 was then placed in the ice chest for 24 hours, and before use ira 

 the test was further diluted by using 1 part of culture mixture 

 to 4 parts of 0.9 per cent, saline solution, forming a 1 in 10 dilu- 

 tion of the original culture. 



With the exception of "Antigen A," all the culture prepara- 

 tions used from time to time as antigens in the complement fixa- 

 tion tests were prepared in the above manner. The various cul- 

 tures prepared and tested as antigens are enumerated in the fol- 

 lowing table: — 



Antigen A. — A fourth subculture of the filter-passing organ- 

 ism in Martin's broth ox serum. It was first used on 18/11/19, 

 after 8 days of growth, in a 1 in 10 dilution with saline solu- 

 tion. It was anticomplementary when any quantity in excess of 

 0.1 c.c. of a 1 in 10 dilution was used. On 24/11/19, it was not 

 anticomplementary when used in an amount of 0.25 c.c. of a 

 1 in 10 dilution. On 11/12/19 (after 30 days of growth) it 

 showed anticomplementary action in quantities of 0.1 c.c. of a 

 1 in 10 dilution in saline. 



Antigen B. — A first subculture, 12 days old, in Martin's brothi 

 ox serum. Not anticomplementary in doses of 0.25 c.c. of a i 

 in 10 dilution. 



