BUREAU OF ANIMAL INDUSTRY. 607 



and a sterile cover-glass, upon which a bit of spleen tissue had been drying for five 

 days under a plugged funnel, was dropped into each tube. These were immediately 

 j)laced in ice water. A preliminary experiment had shown that the temperature in 

 these tubes fell below 40' C. in less than a minute. The spleen had been previously 

 found to contain the bacteria in large numbers. Of four tubes treated in this way 

 two became turbid with the specific bacterium; the others remained sterile. 



In a subsequent experiment, four tubes were inoculated near the boiling tempera- 

 ture and one as a check. This latter developed into a pure culture of the bacterium; 

 the heated tubes remained permanently clear. An almost momentary exposure of 

 the dried bacteria is sufficient, therefore, to destroy their vitality. 



Resistance to various chemical substances or disinfectants. — In 

 the following experiments on the effect of various agents on the 

 vitality of the bacterium of hog-cholera the methods used by Koch 

 were not adopted, because liquids are far more sensitive to bacteria 

 than solid media. A single colony upon gelatine, the descendants of 

 a single germ, may escape the eye, but the same microbe in a nutri- 

 ent liquid would cloud it within a few days. There is, to be sure, for 

 this very reason, greater danger in the use of liquid media, since the 

 introduction of a single foreign microbe might lead to the same con- 

 clusions as the introduction of a dozen or a hundred, while a few 

 bacteria accideutly caught on the gelatine would lead to no errors of 

 interpretation. The results obtained by the method given below 

 were so uniform, the absence of contamination was so constant, that 

 we can recommend it in all similar determinations. 



The disinfectant solution was diluted with sterile distilled water 

 in a test-tube or watch-glass previously sterilized by heat. A few 

 drops from a pure culture of the hog-cholera bacterium were mixed 

 with 4*"' or 5*=*' of this dilution, and a minute portion transferred at 

 given intervals, by means of a platinum loop, to culture tubes contain- 

 ing beef broth. These tubes were then pl'aced in a temperature of 95° 

 to 100° F. , where they remained from one to four days. Tubes which 

 remained clear at the end of this period were sterile, as shown by 

 numerous tubes which were watched for several weeks; 



The experiments given below refer to the active vegetative state 

 of the bacterium in nutrient liquids, as experiments had failed to re- 

 veal any other more resistant state. The cultures were, as a rule, 

 but one or two days old. Previous experiments having shown that 

 older cultures areless resistant to heat than recent ones, it was as- 

 sumed that the vitality is gradually reduced as the culture grows 

 older. 



All the tubes about which there seemed the slightest suspicion of 

 impurity were examined microscopically and of ten on gelatine plates. 

 In all cases the last of a series of inoculated tubes which became tur- 

 bid was carefully examined. This served as a check upon tubes ex- 

 posed for a shorter period of time to the action of the disinfectant. 

 The percentage of the solution used indicates the ratio by weight in 

 grams of chemically pure substances to grams of distilled water. 



Mercuric chloride was found destructive to the bacterium when 

 diluted in the proportion of 1 : 75000 (.001 1-3 per cent.). 



Several drops of a culture were mixed with about 1'^"' of a .1 per cent, solution, 

 and tubes moculated from this at the end of 2, 4, 6, 8, and 10 minutes. Tubes re- 

 main sterile. To show tliat the antiseptic effect of the liquid transferred with the 

 jilatinum loop was 7iil, one of these tubes was inoculated again from another cult- 

 ure. This tube was turbid on the following day. 



Five tubes treated in the same way Avith .05 per cent, solution. All remain sterile, 



live tubes moculated from a culture exposed for the same periods of time to a .01 

 per cent, solution. All remain clear. 



Five tubes treated as before, using a .005 per cent, solution. Permanently clear. 



