540 Major W. J. Tulloch. Serological Types of B. tetani in 



pai'affin, sufficient to cover completely the bottom of a flask to a depth of 

 roughly 1/8 inch, together with 3 grm. of sodium formate, dissolved in 

 10 cub. centim. of distilled water per litre of medium. 



The medium is then filtered, first through a Berkfeld and then through a 

 Doulton candle, into the sterilised flask. The flask should be provided with 

 a hooded delivery tube, so that the medium may be distributed as required. 

 The medium can be kept in cold storage in the sterile flask, and keeps fairly 

 well — about three weeks. It should never be sterilised by heat. Before 

 use, the sterility of the medium should be tested by inoculating quantities 

 from 5 cub. centim. to 0*1 cub. centim. into tubes of meat-water medium and 

 incubated anaerobically for at least seven days. 



If the medium is required for cultures that may ultimately be used for 

 animal inoculation, it should be tested by the in vivo method, to ensure that 

 the medium is in itself non-toxic. 



Before use, fresh sterile kidney is added to the medium in the proportion 

 of roughly 1/16 part of a kidney to 5 cub. centim. of the medium. The 

 tubes to which the kidney has been added should be used within three days. 



Prepared thus, the medium inhibits the growth of B. sporogenes, but allows 

 of the growth of B. tetani, other atoxic round end-sporing, and certain oval 

 end-sporing bacilli. 



Latterly, in place of using meat- water tubes for cultures A and B, the 

 following medium has been employed : — 



Chop up finely the flesh of one rabbit, add 1 litre of tap-water and 5 grm. 

 of sodium carbonate, and allow the mixture to decompose at 37° C. for 

 roughly 16 hours. Then render the mixture slightly alkaline to litmus, and 

 add 2 per cent, of trypsin. Incubate for a further period of 16-24 hours 

 at 37° C. The material is then filtered through filter paper, rendered 

 slightly acid, boiled to coagulate proteins, filtered again and neutralised. 



Before neutralising at room-temperature, the samples of medium to be 

 tested should be boiled and rapidly cooled. 



The mean of two titrations with (1) phenophthalein and (2) a-naphthol- 

 phthalein is taken, and the requisite amount of sodium hydrate added to 

 the bulk of the medium. 



Boil again and finallj' filtei'. Occasionally it may be necessary to pass the 

 product through a Doulton candle to remove any dead organisms that might 

 ■be present. The medium may then be tubed and autoclaved, or autoclaved 

 in bulk and kept in cold storage. Before use, 1/16 part of a fresh sterile 

 rabbit kidney should be added to each tube of 5 cub. centim. 



Tubes which have been autoclaved and stored should be boiled for 30 minutes 

 and rapidly cooled before the kidney is added, and should be used as soon 



