336 HARRIET LESLIE WILCOX 



to filter on the fifteenth day, the cultures may be kept in the 

 incubator until the twentieth day without a loss of toxicity 

 but from the twentieth day to the twenty-fifth day, the toxin 

 loses about 20 per cent in potency. 



FILTRATION 



All glassware, filters, etc., should be neutral to phenolphthalein 

 and the greatest care should be taken to exclude light, either 

 direct or indirect, by darkening the room and by covering the 

 filtering apparatus with dark cloths, ordinary black cambric 

 being used at the Research Laboratory. 



The broth cultures are first passed through Buchner filters 

 about 8 inches in diameter, which have been packed with a 

 layer of finely shredded paper pulp 0.25 inch in thickness. It 

 is of importance that the pulp should be so well packed that the 

 filtrate is absolutely clear, otherwise it will clog the Berkefeld 

 filter. The first filtrate, about 200 cc, which passes through 

 the Buchner is discarded, as it contains a considerable amount 

 of water from the pulp, and then the filtering of the toxin may 

 proceed. If, after passing 8 to 14 litres through the pulp, the 

 filtrate begins to appear cloudy, the pulp must be discarded 

 and the Buchner repacked. The clear filtrate is then passed 

 through a sterile Berkefeld filter, and 10 per cent of a 5 per cent 

 solution of carbolic acid solution is added to the toxin which is 

 now placed in the ice-chest, ready for testing its potency. 



POTENCY TEST 



Two 350 gram guinea pigs are inoculated subcutaneously 

 over the abdomen with 1 cc. of a dilution of 15;^ and 25:000 

 of the toxin respectively. If the toxin has a potency of 25:000 

 the pig receiving the rs^o dilution will die on the second to 

 third day and the pig receiving the 257000 dilution should die 

 on the fourth day. If both pigs die with symptoms of tetanus 

 before the fourth day, the toxin is stronger than 25;^ and a 

 higher dilution should be tested. 



