330 The Philippine Journal of Science 1915 
The following steps were undertaken to keep larvz alive in a 
medium containing cholera until emergence of the imagines 
should take place. 
As was noted in series I, a fly emerged on April 8 in the 
large jar. On April 10 there were, in addition to the emerging 
flies, a considerable number of large and well-advanced larve. 
As these larve were due soon to emerge as imagines, they were 
selected because they would have only a short time to be culti- 
vated. They measured from 3 to 4 millimeters in length. 
a. Thirty of these larve were removed from the large jar and 
placed in a sterile glass-covered stender dish containing sterile 
sand. The sand and the larve were drenched with a broth 
culture of cholera vibrios. A fresh hen’s egg was opened asepti- 
cally and the albumen transferred with a sterile pipette into the 
stender dish as food for the larve. 
b. On the following day (April 11) no active larve could 
be seen, as several of them had pupated. 
One of these pupz was removed with a sterile platinum loop 
and placed in 5 per cent lysol for five minutes, in order to kill 
the cholera vibrios adhering to its surface. After three wash- 
ings in sterile salt solution to remove the lysol, a final washing 
was made in a tube containing Dunham’s peptone solution. 
This tube was incubated for twenty-four hours and then 
streaked on Dieudonné’s medium. Examination of the plate 
showed the absence of cholera vibrios. There was present, how- 
ever, a growth consisting of a coccus and a spore-bearing 
bacillus. 
The lysol-sterilized and washed pupa was placed in a tube 
containing Dunham’s peptone solution and macerated. A por- 
tion of the contents of this tube after being incubated for 
twenty-four hours was streaked on Dieudonné’s medium. Plate 
examination revealed the presence of cholera vibrios. There 
was, also, a growth containing a coccus and a spore-bearing 
bacillus. 
c. Three new pup were secured and subjected to the same 
steps asin b. The results proved identical. 
d. A portion of the sand was inoculated into Dunham’s pep- 
tone and later streaked upon Dieudonné’s medium. Examina- 
tion showed typical colonies of cholera vibrios. There were, 
also, present colonies consisting of cocci and variously formed 
bacilli, many of which contained spores. 
e. On April 14 some actively moving larve were noted. 
These had not yet pupated. 
One of these larve was removed, and the extremities were 
