OUTGROWTH OF THE NERVE FIBER 
801 
Although the first definite results were obtained by the above 
methods, it was found that bacteria quickly invaded the prepara- 
tions, often destroying them as soon as the second day after im- 
plantation. Continued observation over a long period was there- 
fore impossible, and many otherwise good specimens were spoiled 
before they had yielded anything but negative results. After 
experimenting a little with antiseptics such as thymol and ace- 
tone-chloroform, it became apparent that satisfactory prepara- 
tions could not be obtained except by working aseptically. The 
procedure necessary for this involved much tedious detail, though 
it offered no insuperable diff culties. ^ A 11 glassware, such as slides, 
covers, pipettes and dishes, was sterilized by dry heat, either in a 
hot air sterilizer or by passing them through a flame. For cloths 
and filter paper an Arnold sterilizer or an autoclave was used, and 
the needles, scissors and forceps were sterilized by boiling. The 
sterilization of the embryos and the frogs from which the lymph 
was to be taken offered greater difficulties, and in fact was 
accomplished only approximately, though the number of organ- 
isms seems to have been so reduced as not to interfere with the 
purpose of the experiments. The embryos were simply cut oat of 
their jelly in water which had been boiled or passed through a 
Pasteur-Chamberland filter. They were then washed in about 
six successive changes of this water. The salt solution in which 
the operations were performed was sterilized in the same way. 
The frogs were chloroformed and then washed thoroughly in ster- 
ile water, laid out upon moist filter paper and kept in a covered 
dish. In some cases they were first washed in mercuric chloride 
(0.1 per cent) and in others they were kept for 24 hours before 
chloroforming in a solution of copper sulphate one part to 500,000, 
but I am not prepared to say whether these means were sufficiently 
effective to be of material advantage. 
The results of these manipulations are altogether satisfac- 
tory as regards asepsis, although the making ready of the appara- 
tus consumes so much time, and the constant attention to the 
5 I am greatly indebted to Prof. Leo F. Rettger for valuable suggestions as to 
this procedure, and for his generosity in putting at my disposal the apparatus 
in his laboratory. 
JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 9, NO. 4. 
