23 



still translucent, usually from 6 to 8 hours after a rich inoculation 

 of a previously warmed broth. As a result of comparative 

 tests I selected whole broth cultures as the most satis- 

 factory suspension, and they have been used as a routine for 

 some time. The broth in which the pneumococci are grown is 

 Douglas's trypsinised broth* ; the method of preparation differs 

 from the original formula summarised below in that no glucose 

 or calcium chloride are added and ordinary ox muscle is used in 

 place of heart. 



Method of Preparation of Douglas's Trypsin Broth. 



Cut off the fat and large vessels from an average sized fresh bullock's 

 heart : mince, add 4 litres of water, make fa*intly alkaline to litmus and 

 heat to between 70° and 80° C. After cooling to 45° C, 1 per cent, of 

 liquor trypsinse co. (Allen & Hanbury) is added, i.e., 40 c.c. to the four 

 litres. Incubate at 37° C. for about 3 hours, then boil for i hour after 

 slightly acidifying with acetic acid. Strain through muslin and adjust 

 the reaction by the addition of dekanormal caustic soda solution to -f- 17 

 per litre (Eyre's scale). Add • 13 per cent, calcium chloride and • 25 per 

 cent, sodium chloride : heat to boiling point and filter. Steam for 30 

 minutes on each of three consecutive days. 



The reactionf of the broth should be carefully standardised, 

 Ph 7 - 8 is a favourable point, and the cultures should be incubated 

 for 18 to 24 hours. Different strains produce very uniform 

 growths, so that there is no necessity to estimate density, and 

 there is rarely any tendency to spontaneous precipitation. The 

 precipitate produced by the interaction of serum and whole 

 broth culture is less bulky than with saline suspensions, but it is 

 limited to the homologous type serum and does not occur with 

 heterologous type sera. (After standing overnight the latter 

 sera may produce at the most fine granules just visible to the 

 naked eye). Thus the separation of pneumococci into sero- 

 logical types is emphasised, while the evidence of common re- 

 lationship which can be obtained with more sensitive suspensions 

 is not brought into undue prominence. In testing the type of a 

 pneumococcus culture in the above way, it has never been 

 necessary to titrate the agglutinating sera, the result being 

 either positive or negative, and the sera have been used through- 

 out in a dilution of 1 in 10, which is mixed with equal parts of 

 broth culture. 



When a type reaction occurs it is quite definite. Almost 

 immediately after addition of culture to serum there is produced 

 slight turbidity of the translucent broth culture. After in- 

 cubation at 50° C. for one hour clumps form which increase 

 rapidly in size with gentle shaking. The clumps aggregate 

 into a compact mass after standing overnight and cannot be 

 broken up by shaking. To obtain confirmation of a type reaction 

 when the reaction with culture is not strong, it is useful to infect 

 a mouse intraperitoneally with the culture and after death to 

 wash out the peritoneal cavity with salt solution. Freed from 



* Lancet, 10th October, 1914, p. 891. 



t Medical Research Committee. Special Report Series, No. 35, 1919. 



