26 
The spores kept in various percentages of glycerin in the incubator 
have apparentl}^ died out, viz, having failed to produce symptoms when 
inoculated directly into mice. Tests were now made (May 9) to see 
whether these spores were really dead or whether their virulence was 
simpl}^ attenuated. Small quantities from each of the test dilutions 
were therefore inoculated into freshly prepared bouillon and grown 
anaerobically in a Novy jar in an atmosphere of hydrogen plus pyro- 
gallic acid and caustic potash, with the following results: 
Planted May 9. 
Mixture. 
Result. 
Aqueous solution 
Growth, no spores, contaminated. 
Mixed, no spores, contaminated. 
No growth. 
Many spores. 
Many spores and rods. 
Many spores and rods. 
Many spores. 
Many spores. 
Many spores. 
Spores and many rods. 
Many spores. 
10 per cent glycerin 
20 per cent glycerin 
30 per cent glycerin 
40 per cent glycerin 
50 per cent glycerin 
60 per cent glycerin 
70 per cent glycerin 
80 per cent glycerin .* 
90 per cent glycerin 
100 per cent glycerin 
A few drops of the growths thus obtained were on May 21 inocu- 
lated into mice, with the result that all died within a few hours without 
showing characteristic S 3 unptoms. The inoculations were repeated 
June 3 (i. e., 25 da}^s’ growth in bouillon), 0.0005 c. c. was inoculated 
subcutaneously into the flank of each mouse, with the following results: 
[+ = typical symptoms of tetanus; — = no symptoms of tetanus; k = killed; d = died.] 
Quantity inoculated into mice, per- 
centage of glycerin, and tempera- 
ture. 
1st 
day. 
2d 
day. 
3d 
day. 
4th 
day. 
5th 
|day. 
6th 
day. 
7th 
day. 
8th 
day. 
9th 
day. 
10th 
day. 
11th 
day. 
12th 
day. 
Incubator: 
0.0005 e. c. distilled water 
-fk 
+k 
—a 
+k 
+k 
+k 
+k 
+k 
+d 
+d 
+d 
10 per cent glycerin. . . 
20 per cent glycerin. . . 
30 per cent glycerin. . . 
40 per cent glycerin. . . 
50 per cent glycerin. . . 
60 per cent glycerin. . . 
70 per cent glycerin. . . 
80 per cent glycerin. . . 
90 per cent glycerin. . . 
100 per cent glycerin. . . 
• 
a. This showed no growth. Therefore about 0.25 c. c. of the dilution was injected into a mouse. 
No symptoms. 
The dilutions, etc., for the others were as follows: 10 c. c. aq. + 0.1 c. c. spores = 1:100. 0.05 c. c. of 
this solution would contain 1/2000 of the original, or .0005. 
Showing that although the spores had lost their power of producing 
tetanus when inoclated directl}^ into mice, they were not dead, as they 
regained their original activity and virulence when reactivated by 
growing under favorable conditions. 
