270 EMBRYOLOGICAL METHODS. 



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, 85) ; 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). 



592. Injection and Clearing of Larger Embryos. -A con- 

 siderable 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 Prd- 

 paraten, 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 cannulse 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 per- 

 forated 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 ferro- 

 cyanide, 0-5 grins., 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. HC1. 



