PREPARATION OF THE TOXIN 



259 



distributed in thin layers (3-4 cm. deep) in large flasks. The organism soon 

 becomes accustomed to the medium, and grows well in it. 



Character of growth. A film forms on the surface during the first 24 hours and 

 increases both in area and in thickness during the next 24 hours (reject all organisms 

 which do not form a film about the end of the third day). If the culture is growing 

 well the film breaks up and falls to the bottom ; a new film then forms and this in 

 turn sinks to the bottom about the sixth day, after which no further film is formed. 

 The medium is never acid to litmus, but on the other hand about the second to the 

 fourth day it is alkaline to phenol-phthalein. 



The culture should be filtered about the end of the first week when its 

 toxicity is at its maximum ; after about a fortnight the toxin content begins 

 to diminish. 



3. [G. Dean's method. Recommended. This is a less complicated method 

 than Martin's and is somewhat similar to that described by Park and Williams. 



[The broth is prepared with " silverside " of beef and the meat may be used 

 either perfectly fresh or after hanging for 7-12 days at a temperature of 8 C. Free 

 the beef from all fat and fascia and then mince very finely. To each pound (about 

 500 grams) of beef add 1 litre of fairly alkaline tap water. Put the meat and water 

 into an enamelled saucepan, cover with the lid, and allow to boil quietly for -2 

 hours. Filter through Swedish filter paper, thoroughly squeezing out all the juice 

 from the beef. To the filtrate add 2 per cent. Witte's peptone and 0'5 per cent, 

 sodium chloride. Steam at 100 C. for 1 hour. Filter. Make the filtrate neutral 

 to litmus and then add 7 c.c. of normal soda per litre while still hot. Steam again 

 for 1 hour. Filter. Distribute in Erlenmeyer flasks. Steam at 100 C. for 20 minutes 

 and then allow the temperature to run up to 120 C. before turning out the gas. 



[The toxicity in the case of 39 toxins prepared by Dean with this medium and the 

 American bacillus (p. 257) is shown in the following table. 

 1 killed within 6 days at 



8 f 



9 



7 

 7 

 1 



5 did not kill at ^iW ] 



4. Other methods. The methods of Spronck, Park and Williams, Nicolle, 

 and Mace which are in use in some laboratories are less reliable than those 

 given above. 



Spronck's first method. This was based upon the absence of glucose in stale meat. 



Prepare 2 per cent, peptone broth in the ordinary way but with meat which has 

 been hanging for some days until it has acquired a slight smell. Make alkaline and 

 then add 0'5 per cent, salt and a little calcium carbonate. Distribute in quantities 

 of 300-400 c.c. in half litre flasks. Sterilize. Sow when cool. Incubate at 37 C. 

 for 3-4 weeks. Filter. 



Spronck's later method. This was based upon a possibly beneficial action of 

 yeast in promoting the production of toxin. 



Revive the bacillus by sowing first on coagulated blood serum and then on peptone 

 yeast water (p. 37). Sow from the latter on to a shallow layer of peptone yeast 

 water contained in a large flat flask. After incubating for 24 hours the growth has 

 formed a continuous pellicle on the surface and at the end of a week the content of 

 toxin is at its maximum : a dose of ^fa c.c. suffices to kill a guinea-pig. Spronck 

 does not use a porcelain filter but adds 3 grams of carbolic acid per litre and filters 

 through paper. 



Massol's method. Proceed as in Spronck's method but use the following medium : 



High veal, 500 grams. 



Peptone (Witte), 20 



Water, - ... 1000 



Neutralize. Add 7 c.c. normal soda solution. Filter through filter paper. Steri- 

 lize by filtration through a Chamberland bougie. 



