AMPHIBIA. 311 



Another way of hardening is to place the egg, after the action of the 

 osmic acid, in a solution of chromic acid which is then raised to boiling- 

 point on a water-bath ; after cooling, the blackened region is cut out, and 

 the hardening completed in the usual way with chromic acid and alcohol. 



593. KOLLEB'S Method (Arch.f. mik. Anat., xx, 1881, p. 182). Chromic 

 acid, O'l per cent., twenty-four hours ; idem, 0*2 per cent., twenty-four 

 hours ; and so forth, with daily increments of O'l per cent, up to 0'5 per 

 cent. When hard, remove the blastoderm together with a segment of the 

 yolk. Water, twenty-four hours. Stain and imbed. 



594. VIALLETON'S Method (Anat. Anzeig., vii, 1892, pp. 624 627 5 

 Journ. Roy. Mic. Soc., 1892, p. 889). Egg opened in salt solution, blasto- 

 derm excised and removed to a glass plate, then treated with 1 per cent, 

 nitrate of silver solution, washed with water, and put into 70 per cent, alco- 

 hol for six to twelve hours in the dark. Borax-carmine, alcohol, dammar. 



Eeptilia. 



595. General Directions. The methods described above for 

 the embryology of birds are applicable to the embryology of 

 reptiles. During the early stages the blastoderm should be 

 hardened in situ on the yolk ; later the embryo can be iso- 

 lated, and treated separately with Kleinenberg's solution and 

 alcohol (STKAHL, Arch.f. Anat. u. Phys., 1881, p. 123). 



596. KUPFFER'S Method (ibid., 1882, p. 4). The ova are opened and 

 the albumen removed under osmic acid of -^ per cent. The yolk is put for 

 twenty-four hours into an ample quantity of ^ per cent, chromic acid solu- 

 tion ; the blastoderm is removed, washed out in water, and put for three 

 hours into Calberla's liquid (aa glycerin, water, and alcohol), and finally 

 hardened in 90 per cent, alcohol. 



597. SAEASIN'S Method (SEMPEE'S Arbeiten, 1883, p. 159). Fix with 

 chromic acid or hot water, and harden with alcohol. Stain with Bismarck 

 brown, alum-carmine, or haematoxylin, or picro-carmine. Imbed in collodion, 

 and collodionise the sections as cut. 



Amphibia. 



598. Preliminary. In order to prepare the ova of Amphibia 

 for section cutting, it is essential to begin by removing their 

 thick coats of albumen. This may be done by putting them 

 for two or three days into 1 per cent, solution of chromic acid, 

 and shaking well ; but ova thus treated are very brittle, and 

 do not afford good sections. A better method is that described 

 by WHITMAN (Amer. Natural, xxii, 1888, p. 857), and by 

 BLOCHMANN (Zool. Anz., 1889, p. 269). Whitman puts the 

 fixed eggs into a 10 per cent, solution of sodium hypochlorite 



