56 University of California Fiiblicalions in Zoologi/ [Vol. 16 



The amphibians used in these cultures were frogs of different kinds, 

 Plethodon, Siren lacertina, and Dieniijcfi/lus turosus. The extreme 

 smallness of Plethodon, allowing only two or three drops of blood, 

 forbade its use. Siren lacertina proved too hard to sterilize, due to 

 its slimy skin; frogs were not readil.y obtained, so in the majority of 

 the work Diemijctylus torosus was used, and this proved to be very 

 favorable material. Diemyctijhis adults appear here at the beginning 

 of the rainy season in December, and lay their eggs beginning in 

 January and extending into March. In order to keep embryos in.side 

 of the gelatinous covering, and thus free from infection, until the 

 latter part of April, the refrigerator was used. 



In passing, mention should be made of attempts to cultivate gold- 

 fish tissue. Entirely negative results were obtained, due to the prac- 

 tical impossibility of getting non-infected blood serum, and even if 

 this was obtained the tissues themselves were nearly always infected. 

 Often nematodes were embedded in the muscular tissue. The fins and 

 tail parts could not be successfully disinfected without killing the 

 tissue itself. 



The spherical form of the gelatinous egg capsules of Diemyctylus 

 makes it very easy to wash and disinfect them in corrosive sublimate, 

 without injuring the inner eggs or embryos in the least. Embryos 

 giving best resiilts were those about ten millimetera long, although 

 any stage previous to breaking through the egg-case, gave good results. 

 About eighty per cent of the cultures made from embryonic tissue 

 showed very visible outgrowtlis. 



Nerve outgrowths were more rare than any other type, yet pos- 

 sibly most interesting. One tissue was most vigorous, sending out 

 twenty-six distinct fibers, some of them thirty-two mieromillimeters 

 in length. Harrison, in his first memorable work on nerve outgrowths, 

 was first to demonstrate that the protoplasmic strand theory of nerve 

 origin is entirely incorrect, that instead there is an active outgrowth 

 of the nerve itself. Several observations were made to discover the 

 precise method of nerve outgrowth. Under the oil-immersion lens 

 could always be found an expanded bulb with two or more small 

 amoeboid pseudopods at the tip of each nerve. Nearly always there 

 was a dominant larger pseudopod which would determine the direction 

 of growth. Increase in length of nerve was caused by these pseudopods 

 creeping along, always with the very tips attached to the under sur- 

 face of the cover-glass, producing here apparently a sort of rolling 



