96 KAllM NARAYAN BAHL 



Since the cocoons of Pheretima have a transparent shell, 

 the embryo inside a cocoon can be easily seen under a binocular 

 microscope by transmitted light. It is therefore easy to know 

 the age and size of the embryo before opening the cocoon. 

 Since in a given lot of earth the cocoons are of all ages, we can 

 at once select an (anbryo of the desired age, provided we have 

 a large number of cocoons. As a rule there is (mly one embryo 

 in each cocoon, but we sometimes meet with two or even 

 three. 



The cocoons are opened in salt solution by means of a pair 

 of sharp needles under the binocular microscope. Very early 

 stages (blastulae and gastrulae) were mounted whole in clove 

 oil after staining with paracarmine. Two pieces of hair were 

 placed below the coverglass, which enabled the rounded embryo 

 to be rolled under the coverslip in order that it could be 

 examined from all sides. 



Embryos of about 4 to G mm. in length were used both for 

 whole mounts and sections for the study of integumentary 

 nephridia. The embryos while in salt solution were always nar- 

 cotized by ether and fixed either with Bouin's fluid or corrosive- 

 acetic or Petrunkewitsch. The latter solutions were found 

 preferable to Bouin, since this fluid hardens the food-yolk very 

 nuich and makes it brittle for section-cutting. Serial longitudinal 

 sections of a few embryos of suitable age enables one to follow 

 the development of integumentary nephridia fairly completely. 

 A series of transverse sections, 5 m in thickness, of an embryo 

 about 300 M in length, was very useful in following the very 

 early stages of development, e.g. the teloblasts and their 

 development. For the later stages of development of primary 

 nephridia, as represented in Text-fig. 4, embryos fixed in 

 Bouin or corrosive are slit open by means of a sharp needle 

 along the mid-dorsal line, the roll of albuminous material 

 fining the gut is removed, and with a little care the endoderm 

 itself is removed. What is left is the body-wall with the 

 nephridia attached to it. This is stained with paracarmine, 

 flattened out, and mounted whole. 



Older embryos, about 10 mm. or more in length, are fixed 



