CONNECTIVE TISSUE IN AMPHIBIA 451 



MATERIAL AND METHODS 



In the present work, embryonic amphibian material has been 

 used. It was obtained from a number of species, chiefly Rana 

 sylvatica, R. palustris, R. catesbiana and Amblystoma punc- 

 tatum. From each of these species, five series of nine or ten 

 embryos, each ranging from the medullary plate stage by regular 

 gradations to well developed free swimming larvae were obtained 

 for preservation. In all about 200 embryos were selected for 

 permanent preservation. In addition to this, through the kind- 

 ness of Professor R. G. Harrison, access was had to several series 

 of transverse, sagittal and frontal serial sections of R. palustris, 

 cut 7.5/x and stained in haematoxylin and Congo red. Figures 

 1 and 2 are taken from this series. 



The following preserving fluids were used: a) Zenker with 

 5 per cent glacial acetic acid; b) Zenker without acetic acid; 

 c) saturated solution of mercuric chloride with 5 per cent 

 glacial acetic acid; d) saturated solution of mercuric chloride 

 without acetic acid; e) 80 per cent alcohol. Several series of 

 embryos were preserved in these fixing fluids. They were later 

 imbedded in paraffin, and transverse, sagittal and frontal serial 

 sections were made ranging from 7 to 10 micra in thickness. 



The following stains have been used: a) Delafield's haema- 

 toxylin; h) iron haematoxylin; c) picro-fuchsin connective tissue 

 stain; d) Mallory's anihn blue connective tissue stain, and e) 

 Mall's modification of the Mallory stain. With the large amount 

 of material that was available, preserved by various methods, 

 and with the various methods of staining which were used, it 

 was possible to test a large nmuber of combinations and to 

 determine the combination which was best adapted for the pres- 

 ent work. Considerable time was spent in making this compara- 

 tive study of the preserving fluids and stains and their reactions 

 with the tissues, and it is believed that the combination, con- 

 sisting of Zenker with acetic for the killing fluid and Mallory's 

 aniline blue connective tissue stain which was finally adopted 

 and used for most of the material is the best possible one for 

 the problem in hand. 



