370 EMBRYOLOGY 



1887, p. 107 (picro-sulphuric acid for the mouse, and picric acid with 

 j\ per cent, of chromic acid for Didelphys) ; Keibel, Morph. Arb., ii, 

 1893, p. 11 (Sus scrofa) ; Neumayer, Festschr.f. Kupffer, 1899, p. 458 

 (embryos of the sheep best fixed in Carnoy's acetic acid, alcohol, and 

 chloroform, § 90) ; Winiwarter, Arch. Biol., xvii, 1900, p. 39 (mixture 

 of 50 parts saturated sublimate in salt solution, 50 parts alcohol, 20 of 



1 per cent, platinum chloride, and 5 of acetic acid) ; Spee, Encycl. mik. 

 Techn., 1910, p. 353 (cornua of Cavia fixed for twelve to twenty-four 

 hours in sublimate, and put into 0-5 per cent, osmic acid till light 

 brown, then into iodine alcohol, in which the osmium is reduced) ; 

 WiDAKOWiCH, Zeit. wiss. ZooL, xciv, 1909, p. 243 {Mus. rattus, 

 fixation in Zenker's mixture, or 2 parts of alcohol of 80 per cent, with 1 

 of formol ; also instructions for dissection). 



809. Injection and Clearing of Larger Embryos. A considerable 

 amount of useful work has lately been carried out on embryonic 

 blood and lymph vessels, and on the cerebro-spinal cavities, by 

 micro-injection apparatus. A suitable injection medium is blown 

 or forced into the vessels of an embryo, the latter is fixed and then 

 dehydrated, and cleared by the Spalteholz method {Uber das 

 Durchsichtigmachen von menschlichen und tierschen Prdparaten, 

 und seine theoretischen Bedingungen, Leipzic, S. Herzel, 1911; 



2 Aufl., 1914). 



In an early stage in the formation of embryonic vessels and 

 cavities the walls are thin and often ill-marked, and care must 

 be taken not to burst through boundaries by excessive pressure. 

 Very fine metal needles, or, better, finely drawn out glass cannulfe 

 are used for injecting the specimens ; the tube leading to the 

 cannula is filled with the injection medium, which, by means of a 

 rubber tube leading to the operator's mouth, is blown carefully 

 into the perforated vessel or cavity. Or, one may use a rubber 

 bulb either worked by hand, or placed on the floor and compressed 

 by the foot. See E. M. Gregory, Anat. Record, xi, 1917. 



The injection media most commonly used are India ink, a 

 saturated solution of Prussian blue, an aqueous suspension of 

 lamp black, or silver nitrate (5 per cent.). The Prussian blue 

 and India ink give about equal results, the blue clearing better, 

 the ink being more opaque. The ink flows the better. Silver 

 nitrate preparations are very beautiful and easy to analyse, but 

 its caustic action prevents the finer vessels from filling. Lamp 

 black tends to precipitate in fine flakes (Cunningham, vide infra). 

 Evans {vide infra), for cerebro-spinal spaces of pig embryos, 

 injected potassium ferrocyanide, 0-5 grm., iron ammonium citrate, 

 0-5 grm., aq. dest., 100 c.c, and afterwards immersed the embryo 

 for one to ten minutes in a 10 per cent, formaldehyde solution 

 containing 1 per cent. HCl. 



The embryo was then fixed in Bouin's fluid, but the Prussian 

 blue faded after about a year. 



Sabin {vide infra) and Cunningham, after India ink injection, 

 fix in Carnoy's fluid, place in 80 per cent, alcohol, dehydrate in 



