526 THE ANTHRAX BACILLUS 



perature of 70 C. for a very long time and a temperature of 85 C. for 5 

 minutes. In the dried state and especially in presence of albuminous matter 

 such as blood their resistance is even greater ; they will then tolerate a tem- 

 perature above 100 C. and resist the action of absolute alcohol, of compressed 

 oxygen, complete absence of oxygen, exposure to sunlight, etc. 



Arloing however has shown that in cultures spores resist the action of sunlight 

 less well than the non- spore- bearing bacillus. This may be explained by assuming 

 that sunlight acts more powerfully on young bacilli developing from spores than on 

 the full-grown bacillus. 



The spore only develops into a bacillus in presence of free oxygen. 



The non-sporing bacillus. Pasteur noted that in old cultures on gelatin the 

 organism sometimes loses its power of forming spores. 



If broth be sown with anthrax blood and incubated at 42 '5 C. the bacillus 

 grows but no spores are formed : in the rods small bright granules are seen 

 the false spores of Chauveau but these have none of the properties charac- 

 teristic of spores. 



Non-sporing bacilli obtained in this way when resown in a medium kept 

 at the optimum temperature soon revert to the sporing form. To obtain a 

 definitely non-sporing variety one of the following methods must be adopted : 



A. The carbolic acid method of Roux. Recommended. 1. Distribute 

 about 10 c.c. of a slightly alkaline peptonized veal broth into each of a 

 number (30 to 50) of tubes. 



2. Divide these tubes into series of ten each. Number the tubes of each 

 series 1, 2, 3, .'. . 10, and to each tube add a 1 per cent, aqueous solution 

 of carbolic acid (without alcohol) in the following quantities : 



to tube No. 1 equivalent to 10 * 00 of carbolic acid = 0'2 c.c. of the carbolic acid solution. 

 <> No. 2 Iir f 0o =0-4 c.c. 



-, No. 3 I | Fo =0'6 c.c. 



No. 10 ^^U =2-0 c.c. 



3. Plug the tubes with wool, seal off the ends in the blow-pipe above the 

 plug to prevent evaporation of the carbolic acid, and then sterilize in the 

 autoclave. 



4. When cool open the tubes and sow with a drop of anthrax blood : care 

 should be taken to drop the blood into the broth and not on to the side of 

 the tube. 



5. Cover the mouths of the tubes with india-rubber caps and incubate 

 at 33-37 C. 



6. After incubating for about 10 days examine the cultures. In some 

 of the tubes, those containing the largest amount of carbolic acid (tubes 

 7-10, for instance) growth will have been altogether inhibited ; the tubes 

 numbered say 1 to 3 will contain both bacilli and spores ; the tubes numbered 

 3, 4, 5, 6 will contain bacilli but no spores. 



To determine whether or not spores are present draw up a little of the culture 

 into a very fine Pasteur pipette constricted below the wool plug (p. 75). Seal the 

 ends of the pipette in the flame and put it in a water bath at 65 C. or 70 C. for 

 15 minutes or so. If the bacillus is non-spore-bearing it is killed at this temperature 

 so that when the heated contents of the pipette are sown in broth the medium 

 remains sterile. If on the other hand the bacteria are spore-bearing growth takes 

 place. 



Sometimes none of the tubes contain asporogenic bacteria, and at other 

 times a large number are obtained which however may revert to the sporing 

 form after a few sub-cultures in broth at 33-37 C. 



Surmont and Arnold have shown that the faculty of becoming asporogenic varies 

 much among bacteria recovered from different sources. A bacterium obtained 



