118 Animal Micrology 



paraffin block in order to cut sections in' the proper plane. The immer- 

 sion in the melted paraffin should not be longer than 5 or 10 minutes. 

 The paraffin is best hardened under 95 per cent, alcohol. The sections 

 may be stained by any of the hematoxylin methods; iron-hematoxylin 

 (reagent 51, Appendix B) yields excellent results. 



5. For the Embryology of the Frog and other Amphibia the following 

 are the most useful stages : 



I. Surface views (from side and from animal pole). Unsegmented 

 egg; 2-, 4-, and 16-cell stages; 32 becoming 64 cells; later cleavage; 

 blastopore forming; yolk-plug stage; early medullary folds; late medul- 

 lary folds; later embryo; embryo just before hatching; tadpole shortly 

 after hatching; later stages of tadpole for gross dissection. 



II. Sections. Late cleavage; blastopore forming (sagittal); yolk-plug 

 stage (sagittal); late medullary fold (transverse and sagittal); later 

 embryo (transverse and sagittal); embryo just before hatching (trans- 

 verse and sagittal); tadpole shortly after hatching (sagittal); head and 

 anterior part of the tadpole about the time the hind legs appear 

 (sagittal). 



To study amphibian eggs entire use a hand lens or dissecting micro- 

 scope. Place the eggs on a bit of absorbent cotton under 70 per cent, 

 alcohol in salt cellars. The eggs are fragile, consequently to manipulate 

 them use a soft hair pencil or a current from a pipette. Use the same 

 egg for surface view and for sectioning when possible. 



Amphibian eggs may be fixed (in masses of 15 or 20) in Gilson's 

 mercuro- nitric (reagent 15, Appendix B) or in Worcester's aceto-formol- 

 sublimate mixture (reagent 196, Appendix B). Chromic acid (reagent 10) 

 brings out surface views well but the material becomes very brittle and 

 does not take stains readily. If surface views alone are desired formalin- 

 preserved material will answer. 



Before the egg can be prepared for sectioning the thick albuminous 

 coat which surrounds it must be removed. The fixed eggs may be shelled 

 out by means of needles. Whitman (American Naturalist, Vol. XXII, 

 p. 857) recommends putting the fixed eggs into a 10 per cent, solution of 

 sodium hypochlorite diluted with 5 or 6 volumes of water and leaving 

 them until they can be shaken free. This requires only a few minutes. 

 Rinse the eggs in 35 per cent, alcohol. It is advisable to remove the 

 albuminous coats before hardening in alcohol. 



Child (Zeitschrift fur wissenschaftliche Mikroskopie, Vol. XVII, 

 1900, p. 205) states that the albumen which surrounds many ova becomes 

 transparent and dissolves if after fixation (in any way except with 

 chromic acid) the ova are passed up through the grades of alcohol to 

 80 per cent., hardened, and then passed down again through the alcohols 

 into water which has been slightly acidified with any acid except chromic. 



