70 
Experiments with Flies 
The remaining two pupae were shaken up in a tube of broth and allowed to 
remain in it 25 minutes, then transferred to an agar slope upon which they were 
broken up with sterile needles. Tubes were incubated at 29° C. The broth remained 
sterile, the agar show'ed growth in 11 hours of a pime culture of B. pyocyaneiis. 
The inference is that the interior of the pupa is certainly infected. 
The results of this experiment have been forwarded to Dr J. G. 
Ledingliam, who has kindly verified them; he has also performed 
similar experiments with pupae supplied by me, the results of which 
are appended. 
2. Eight pupae were placed in 3 % lysol, shaken up and allowed to remain in 
the solution for five minutes ; they were then washed in sterilized distilled water. 
Two were placed in clean silver sand in a clean tube to test if sterilization had 
killed them. One fly subsequently emerged. 
Three were placed in a tube of broth, torn up with sterilized needles and 
incubated at 24° C. 
The broth was timbid in 13 hours and the growth again proved to be B. 
pyocyaneus b 
The remaining pupae were placed in a tube of broth and allowed to remain 
at 24° C. for 13 hours. They were then removed to a fresh tube of broth in 
which they were broken up with sterilized needles ; both tubes were incubated at 
29° C. The first tube remained clear for 12 hours after the time the pupae were 
removed from it but showed slight turbidity in 20 hours. 
The second tube became turbid in nine hours. 
These results suggest either that external sterilization was not 
complete or that during the lengthy period of soaking in broth some 
passage between interior and exterior was effected. That the interior 
was infected seems clear from the greater rapidity of growth that 
followed the breaking up of the pupae. 
3. Four pupae were placed in 10 ®/q lysol for seven minutes, washed in 
sterilized distilled water, placed in a tube of broth and allowed to remain in it 
13 hours at 24° C. They were removed to a second tube and broken up with 
sterilized needles. Both tubes were then incubated at 29° C. The first tube 
remained clear for 12 hours but showed .slight signs of growth in 24 hours. The 
.second tube showed strong growth in 12 hours. 
The same remarks apply to this experiment as to Exp. 2. 
4. Seven pupae were passed five or six times through a Bunsen flame on 
a platinum loop. 
Three of these pupae were then placed in broth at 24° C. for 13 hours, after 
which they were transferred to a similar fresh tube and broken up with sterile 
needles. Both tubes were incubated at 29° C. 
^ In all cases the growth obtained appeared both culturally and microscopically to be 
pure B. pyocyaneus except that some nine slides showed slight contamination with 
B. prodigiosus. 
