TOXIN 529 



3. The toxin of anthrax. 



The nature of the toxin secreted by the anthrax bacillus is not yet definitely 

 determined. 



The toxalbumin which Hankin prepared from cultures of the bacillus grown in 

 Liebig's broth containing fibrin and that which Brieger and Frsenkel extracted from 

 carcases dead of anthrax were only obtained by complicated processes hardly com- 

 patible with the isolation of a very delicate chemical substance. It is to be feared 

 that impurities were responsible for many of the results which followed the inocula- 

 tion of these toxins. 



Sidney Martin obtained an albumose in solutions of alkali albumin which in doses 

 of 3 eg. killed mice weighing 22 grams with symptoms very similar to those seen 

 in anthrax septicaemia. 



Marmier's toxin. Marmier obtained an active toxin by growing the organism 

 at a low temperature in a solution of pure peptone containing glycerin. 



Marmier's medium. To prepare Marmier's medium the first step is to purify 

 ordinary commercial peptone, thus : dissolve a certain amount of peptone in water;, 

 add sufficient sulphate of ammonium to saturate the solution at 100 C. then boil 

 for a few minutes and filter. To the filtrate add sufficient barium hydroxide to 

 precipitate all the sulphuric acid present. Heat the mixture for several hours to 

 a temperature near the boiling point to drive off the ammonia : filter to remove 

 the barium sulphate and keep the filtrate boiling while passing a current of air 

 through it to remove all traces of ammonia ; then pass through it a current of carbon- 

 dioxide to precipitate the excess of barium hydroxide, and finally filter. With 

 this purified peptone solution the following medium is prepared : 



Water, ... 1000 c.c. 



Peptone, 

 Common salt, 

 Sodium phosphate, 

 Potassium phosphate, 

 Pure glycerin, 



40 grains. 

 15 



0'5 gram. 



0-2 

 40 grams. 



Filter, distribute in flasks of 250 c.c. capacity and sterilize at 115 C. 



To prepare the toxin sow the medium with a virulent anthrax bacillus 

 and incubate for 48 hours at 37 C. and afterwards at 20 C. for a fortnight. 



Now filter the culture through porcelain and saturate the filtrate with 

 ammonium sulphate at the temperature of the laboratory. After standing 

 for about 15 hours filter through paper and wash the filter with a saturated 

 solution of ammonium sulphate. Wash the precipitate which remains on 

 the filter paper with as small a quantity of glycerin as possible : leave for 

 2 days, decant the glycerin, replace it by fresh glycerin and decant again. 

 Mix the glycerin solutions together and add the mixture to four times its 

 weight of strong alcohol : pour the precipitate on a filter and wash first with 

 absolute alcohol, then with ether and finally dry in vacuo. The product is 

 an amorphous, easily powdered substance dark brown in colour and contains 

 small quantities of ammonium sulphate. It is soluble in distilled water and 

 in 1 per cent, carbolic acid. It has none of the properties of albuminoid 

 substances, peptones, parapep tones nor alkaloids. 



This substance is rather toxic for rabbits and the inoculation of small 

 doses may cause the death of the animal, but the dose varies for each individual 

 within somewhat wide limits. Thus some rabbits succumb to the inoculation 

 of 25 mg. (in aqueous solution) while for others the fatal dose is as much as 

 120 or even 200 mg. 



A few hours after inoculation there is a well-marked rise of temperature : for a 

 few days the temperature oscillates widely, and then if the animal is going to die it 

 falls steadily (perhaps as low as 8 C. below normal), if on the other hand the animal 

 is going to recover the temperature oscillates less and less. The animal is ill, 

 cachectic, and may lose one-third of its weight ; as a rule diarrhoea is a symptom. 



2L 



