THE ISOLATION OF THE BACILLUS 535 



blood films prepared as described above but there is a minority of cases in 

 which anthrax exists but in which the bacilli cannot be found on microscopical 

 examination because the organisms are present only in very small numbers in 

 the blood. In horses, and especially in pigs, a negative result on micro- 

 scopical examination is of no value. Agar cultures must be sown therefore 

 in every suspected case of anthrax in animals. If bacilli resembling anthrax 

 bacilli were found in the blood films, examination of the cultures will confirm 

 the diagnosis ; the bacillus of malignant oedema does not grow on agar 

 under aerobic conditions. In those cases in which no bacilli were found in the 

 blood films and cultures yield an organism resembling the anthrax bacillus 

 the diagnosis must be confirmed by the inoculation of a guinea-pig.] 



Cina and Stoiesco have shown that it is of great advantage to examine 

 the skin in cases where the carcase has 'begun to decompose. The anthrax 

 bacillus can always be found in the skin even though it may have disappeared 

 from the blood and internal organs. 



Cut off small pieces of the skin and leave them to dry, then scrape them with a 

 scalpel and rub up the scrapings with sterile saline solution (10 volumes of saline to 

 1 volume of tissue). To a series of broth tubes add 15 drops of the emulsion, heat 

 the tubes to 65 C. for thirty minutes, then sow on agar, and examine and identify 

 the colonies which develop. 



Examination of the intestinal contents may also, according to Cin9a and 

 Fenea, afford valuable evidence in diagnosis. 



Isolation of the bacillus from soil. 



Pasteur utilized the resistance of the spores to heat for the purpose of isolating 

 the organism from the soil of infected fields. The technique is as follows : 



Crush a little of the soil in a mortar and make a suspension in sterile water. A 

 granular precipitate is immediately deposited. Decant the supernatant liquid 

 containing only fine light particles with care and let it settle in a sterile test-tube 

 on a foot. The turbid liquid becomes clear and a deposit is formed in the bottom 

 of the tube. Decant the supernatant liquid again. Aspirate the deposit into 

 pipettes and after sealing heat them in a water-bath at 85 C. for 15-20 minutes and 

 use the contents for sowing gelatin plates in Petri dishes. The plates must be 

 carefully watched, and every suspicious colony should be examined microscopically, 

 sown on various media, and inoculated into guinea-pigs and mice. 



The pyogenic micro-organisms are destroyed by heating the material to 85 C. 

 and the method of cultivation eliminates all anaerobic species such as the bacillus 

 of malignant oedema which might lead to error in the investigation. In the method 

 originally used by Pasteur the deposit obtainr by powdering the soil was inoculated 

 immediately after heating and before colonies were isolated, but this technique is 

 not so exact. 



By utilizing a method very similar to that just described, Diatroptoff succeeded 

 in isolating the organism from the mud at the bottom of a well. 



