fibrin and blood cells by eentrifuging, these washings invariably 

 give g reaction with the homologous type serum, provided 



tin- pneumococci have grown well in the peritoneal cavity. 



Preparation and Test of Protective Sera. 



The sera prepared against Types I, II and III were made 

 with heated culture followed, in my earlier experiments, by 

 living virulent culture, as I thought that the latter might be 

 necessary for the production of protective substances. It was 

 found, however, in the course of the preparation of immune sera 

 against the strains not conforming to the three main types, that 

 sera of considerable protective potency could be produced in 

 rabbits solely by the inoculation of virulent culture killed by 

 heat. Consequently, the technique ultimately adopted was 

 the same, in all respects, as that described for preparing agglu- 

 tinating sera, and it was found that a serum with a high agglu- 

 tination titre was generally serviceable as a protective 

 serum. 



The protective power of the sera was tested on mice and the 

 method was, in general, that first used by Neufeld. In this a 

 fixed quantity of serum is used and the test is designed to deter- 

 mine the largest amount of broth culture against which this 

 quantity of serum will protect the mouse from death, the period 

 of observation being usually limited to 4 days. 



So long as sufficient serum is used, the quantity is not a 

 matter of importance and the dose of 0*2 c.c. used by Neufeld 

 has been continued by most investigators, including the American. 

 The serum is always tested for its prophylactic and not for its 

 curative potency, but there is some difference of practice in 

 regard to the length of the interval between the injection of the 

 serum and the test culture. The method which I have adopted 

 has proved satisfactory and has certain advantages in point of 

 view of convenience; the serum is administered about 4 o'clock 

 in the afternoon and the test culture between 10 and 1 o'clock 

 the next day. Both serum and culture are injected intraperi- 

 toneally. It may perhaps be useful to describe some of the 

 technical details of a protection experiment on mice. 



Serum. — The serum is diluted 1 in 2-5 with broth so that 0-2 c.c, 

 the prophylactic dose, is contained in 0-5 c.c. 



Culture. — A pneumococcus culture is grown for 18 to 24 hours, in 

 0-5 c.c to 1 c.c of blood broth containing 50 per cent, of fresh whipped 

 rabbit blood. The culture is well shaken before the quantity necessary 

 for the first dilution is withdrawn. Virulence tests give more regular 

 results with this medium than with plain broth; the diplococci remain 

 single and all stain uniformly well with Gram. 



The dilutions of culture are made as follows : — a series of six-inch 

 t. -t -tubes is filled with broth, the first containing 4-9 c.c. and each of the 

 rest 4- 5 c.c. To the first tube • 1 c.c. of the blood broth culture is added and 

 the mixture is well shaken. From the first tube, now containing 1 in 

 50 dilution of culture, 0-5 c.c. is taken over to the second tube to make a 

 dilution of 1 in 500 and so on to the end of the series. A separate pipette 

 is used for each dilution. The test dose is given in a total bulk of 0- 5 c.c. ; 



