482 EEPOKT OF THE COMMISSIONER OF AGKICULTUllE. 



the same time. A pipette was filled with the peritoneal effusion and one 

 with blood. On the following day two tubes, one of meat broth, the 

 other of meat broth with pei)tone, received eaeh several drops of the 

 X>eritoneal exudate from a pipette. Two additional tubes were inocu- 

 lated with a platinum loop from the pipette containing blood from the 

 heart. 



All of the above cultures remained sterile excepting the two liquid 

 cultures of the blood and the gelatine tube coniaining :i portion of tlie 

 spleen. Both the former became faintly clouded on the second day 

 after inoculation, and weni found pnie cultures of the bacillus found in 

 the vaccine. It is ])robable that they had multiplied in the i>ipette over 

 night and made infection possible, as their number in the blood must 

 have been very small. 



In about two weeks after inoculation a faint, cloudlike growth from 

 the bit of spleen, which was about the size of a s])lit pea, downward 

 into the gelatine could be easily seen. AYhen the bit of spleen was re- 

 moved a WL'ek later and rubbed on cover-glasses, exquisite preparations 

 of the rouget bacillus were obtained. Masses of filaments could be 

 seen interlacing with one another in all directions. Most of the fila- 

 ments were of considerable length (20 to 30 micro millimeters). The in- 

 jected bacilli had thus penetrated the system (piite thoroughly, though 

 lirc.sent at any place in but small numbers. 



The three remaining pigs which had been vaccinated a])peared uudiS' 

 turbed by the operation. The temperature chart is given on ])age 484 in 

 connection with the second vaccination. 



A tube of the second vacciue was received at the laboratory on Octo- 

 ber 23. It remained unopened in a cool box at atemperaturoof 00° to 05"^ 

 F., until October 28, when a portion was used for the second vaccina- 

 tion of pigs, ajiother for the inoculation of mice, and a third for micro- 

 scopical examination and cultivation. In general appearance, color, and 

 consistency the liquid resembled the first vaccine closely. On removing 

 the rubber stopper there was a sudden outrush of air, which scattered 

 a portion of the liquid as a fine spray. At the same time minute air- 

 bubbles rose to the surface of the liquid forming a delicate, white foamy 

 'layer. 



Portions were imniediately dried on covers and stained in an aqueous 

 solution of methyl- violet. Examined with a Zeiss y^ homog. Oc. 2, two 

 kinds of bacilli were found. (Plate II, Fig. 4.) Large ones with rounded 

 extremities from 2.5 to 4 micromillimeters long and about .7 micromil- 

 limeters broad, a few in chains of two to four, the majority isolated. 

 Some failed to stain deeply, probably because dead. The other bacilli 

 resembled closely those found in the first vaccine. They occurred in 

 slender filaments, either curved or angled. Some of the filaments were 

 plainly Jointed, the segments measuring not more than from 1 to 1.2 

 micromillimeters in length ; a few isolated bacilli were of the same 

 length as the segments of the longer filaments. 



Plate cultures were at the same time prepared by adding two drops 

 of the vaccine liquid to 10'^'^ of beef broth jicptone and mixing one 

 drop of this dilution with 10'^'= of nutritive gelatine, which was si)read 

 on two plates. At the same time two additional i)lates were prepared 

 by adding merely a platinum loop of the dilution to 10*=" of the gelatine. 

 Though examined from time to time for nearly a week no colonies of the 

 bacillus could be detected. But this was easily explained when the 

 tube, which formed the dilution for the x)late cidtures, was found sterile 

 after four or five days. It seemed as if the microbes were dead ; the 

 large as well as the small bacillus. On October 31 two additional cult- 



