146 IOWA ACADEMY OF SCIENCE 



by Carno}^ and Lebrun."'' This method will, be described somewhat in 

 detail. 



Ova and embryos which are still enclosed in their albumenous enve- 

 lopes are fixed in Gilson's mercuro-nitric fluid* 1.5 hrs. or longer. The 

 albumen is now sufficiently hardened that it can be incised to permit 

 the egg or embryo to be squeezed out by gently applying pressure at 

 the opposite pole. This operation must be made with care, as any 

 distortion of the egg or embryo after fixation may •separate or break 

 the germ-layers ; consequently, perfect sections cannot be obtained. 

 Embryos which are free from their albumenous envelopes are fixed in 

 Gilson's fluid 45 min. to 1 hr. If large enough to swim freely, they 

 may to advantage be placed in water to which a few drops of chloro- 

 form have been added until they become quiescent, before being placed 

 in the fixing fluid. This treatment prevents tearing of the delicate 

 tissues along the neural tube and the notochord by muscular exertion 

 after they have been partly penetrated by the fixing agent. 



After fixation the embryos are washed thoroughly in water and de- 

 hydrated in the usual manner, care being taken not to leave them in 

 95% alcohol longer than 15 min. or in absolute alcohol longer than 5 

 or 10 min. From absolute alcohol they are brought into a mixture of 

 absolute alcohol and chloroform in equal parts. After the embryos 

 sink in this mixture they are brought into pure chloroform for an hour 

 or longer. Paraffin is now added to about double the volume and the 

 whole is placed in a thermostat at about 35° C. for 3 hrs. The embryos 

 are now placed in pure paraffin in the paraffin bath for 15 to 30 min. 

 After imbedding sections may be cut to any desired thickness and 

 stained by any of the common methods. 



Amphibian eggs or embryos successfully carried through the pro- 

 cesses above outlined afford preparations which stain well and show 

 no evidence of shrinking. 



='Lee. Microtomist's Vade-Mecum (sixth edition), p. 331. 



'Gilsos's fluid: Nitric acid, 15 c. c; Glacial acetic acid, 4 c. c; Corrosive su- 

 blimate, 20 grs.; 60% alcohol, 100 c. c; Distilled water, 880 c. c. 



