f 
DESTRUCTION OF FLY LARVA IN HORSE MANURE. 3 
pupe in each pile was counted about eight days after the last treat- 
ment. -A sample of 200 pupe from each pile was kept in the labora- 
tory, and the percentage of emergence determined. From these data 
the apparent larvicidal effect was calculated. For some of the New 
Orleans experiments, cages (PI. I, fig.1,p. 16) were constructed to cover 
the piles, and instead of counting the number of pupz the flies were 
allowed to emerge and were caught in traps attached to the tops of 
the cages. Temperatures and samples for analysis were taken 
through the armholes in the sides of the cages. Soil was banked 
against the base of the cages to prevent the escape of maggots and flies. 
Chemical and bacteriological analyses were made of samples of 
manure from most of the cage and open-pile experiments. 
SAMPLING FOR QGHEMICAL AND BACTERIOLOGICAL ANALYSES. 
It is very evident that a manure pile with the unequal distribution 
and great variation of its physical and chemical constituents will 
necessarily be exceedingly difficult to sample, especially to secure 
from it a few hundred grams which will be thoroughly representative. 
An attempt was made to secure representative samples by taking 
equal portions of manure from three different parts of the pile, 
spreading them on a clean sheet of paper, and finely dividing and 
thoroughly mixing them. When the material appeared quite uni- 
form the sample was quartered. One quarter was then cut into 
half-centimeter lengths with clean shears. The straw or shavings 
were cut with the other material. When this operation was com- 
pleted the sample was again thoroughly mixed. For chemical 
analysis the material was twice passed through a grinder. Both 
bacteriological and chemical examinations were made on the same 
sample. As the bacterial content of manure is very high, no attempt 
was made to work under absolutely sterile conditions, because the 
contamination arising from ordinary handling of the material was 
of no importance when compared with the great number of organisms 
present. However, precautions were taken to prevent excessive 
contamination by using clean paper, shears, etc., for each sample. 
BACTERIOLOGICAL EXAMINATION. 
Two 10-gram samples of the manure, prepared as just described, 
were taken for each bacteriological determination. One of the 
10-gram samples was dried at 100° C. for one hour to determine the 
percentage of solids. The other sample was brushed into a 2-liter 
flask containing 1 liter of sterile water. The flask was then vigorously 
shaken for five minutes and again, after a five minute interval, for 
three minutes. A 1-c.c. sample was then withdrawn and run into 
100 c. c. of sterile water. Five dilutions were prepared, ranging from 
1 part in 10,000 to 1 part in 100,000,000. A duplicate series of Petri 
dishes was then prepared from these dilutions and standard beef agar. 
