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Heligmosomum muris Yokogawa 
2. Development of the Larvae in the Free-living Stage. 
(a) Method of Culture. 
In my studies of the formation of the embryo and the free development of 
the larvae I made many cultures of the faeces of wild rats infested with 
Heligmosomum muris. I found that cultures made in liquid media are best 
for the study of the formation of the embryo and the earlier stages of larval 
developnient, while those made in solid media are most suitable for the study 
of the development of the later larval stages. 
The best results with cultures in liquid media were obtained by using a 
filtered decoction of faeces prepared in the following manner. One teaspoonful 
of normal faeces from white rats was boiled in 100 c.c. of tap water. The 
mixture was then filtered, and the decoction poured into a glass dish in a 
very thin layer. To this were added many eggs fresh from the females. The 
glass dish was then kept at room temperature, since this work was carried 
on in the summer time and was covered to prevent evaporation. For the 
purpose of examining the progress of development the dish can be placed 
from time to time under the low power of a microscope. In this way the 
hatching of the egg or any of the developmental stages can easily be observed 
on a number of larvae at the same time. By means of a pipette the larvae 
can be picked out, transferred to a drop of water on a glass slide and examined 
under high power. This method of cultivation is very convenient for the 
studying of the early stages of larval development. It is not suited, however, 
for the study of the development of the infective larvae, since the majority 
of the larvae cannot complete the first moult in liquid media. 
The method of making the cultures of solid media was very simple. Fresh 
faeces from rats infected with H. muris was smeared on moist filter paper in 
a covered dish. This culture must be kept moist and the faeces must be spread 
out well on the filter paper to give sufficient air. It is not necessary, however, 
to add charcoal to avoid fermentation as in the cultures of human faeces 
containing hookworm eggs. The mature infective larvae appear on the filter 
paper or on the walls of the dish four to five days after the making of the 
culture. They are later found in great numbers along the edge of the filter 
paper giving the appearance of minute threads. 
(b) A General Description of the Stages of the Post-embryonal Development. 
In the literature on the development of nematodes the statement is made 
that four moults are necessary for the complete development. Looss (1911) 
makes this definite statement: “the number of the moults in the case of the 
nematodes is apparently always four. It remains to be seen whether further 
experience will confirm or overthrow this apparent rule." In the case of 
H. muris only three moults were observed. Early in my work I was struck 
with the fact that I could find only one moult in the free-living stage and J 
suspected that I had overlooked the first moult. After repeated examina- 
