10 DEVELOPMENT OF THE VEINS IN THE EMBRYO PIG. 



hydrogen peroxide to remove the hemaglobin and other pigments; third, dehydration; and 

 fourth, clearing the specimens in an oil which has the same index of refraction as the tissues. 

 As applied to embryonic tissues, the method, developed under the direction of Professor 

 Spalteholz, to whom I am very much indebted, is as follows: The specimens which have 

 been injected with India ink are fixed for 24 to 48 hours in 5 and 10 per cent formalin. 

 Commercial formalin is slightly acid, which is an advantage for the India-ink injections, 

 since the ink diffuses in an alkaline solution. Specimens which have been injected with 

 silver nitrate are ruined by fixation in formalin, because the silver salt is changed to a white 

 precipitate which obscures the vessels. If injections of bone are desired, the formalin may 

 be made slightly alkaline and the diffusion of the ink prevented as much as possible by tying 

 off all the vessels before fixation. For large fetuses, which are to be cleared in toto, Dr. P. G. 

 Shipley has found that the subsequent bleaching is made easier by washing the specimen in 

 running water before fixation, thus removing much of the hemaglobin. After fixation, the 

 specimens are washed in running tap-water from 12 to 24 hours, followed by distilled water 

 to remove the formalin. The bleaching is done in hydrogen peroxide. Spalteholz adds a 

 few drops of ammonia to precipitate the barium salts. This is not necessary with barium- 

 free peroxide. For adult tissue, Spalteholz uses undiluted peroxide; for the embryonic 

 tissues about 2 to 3 per cent is the best strength. The small embryos with ink injections 

 take about 20 minutes to bleach ; for the silver specimens, 2 to 3 minutes suffice and they 

 must be watched constantly and the bleaching stopped before the silver is affected. Fol- 

 lowing the bleaching, the specimen must be washed thoroughly in running water and in 

 distilled water. The dehydration may be begun with 50 per cent alcohol and the percentage 

 increased successively by five points or less. After two changes of a good grade of absolute 

 alcohol, the specimens are passed through two changes of benzene into the synthetic oil of 

 wintergreen. The small amount of benzene which is carried over evaporates quickly, and 

 the few bubbles which develop in the bleaching process can be removed with needles. The 



011 of wintergreen should be entirely colorless, but both the specimens and the oil will grad- 

 ually become brown with age. This is especially true of the silver-nitrate specimens, but 

 they will keep for six months or a year in oil. They can be returned to alcohol for storage 

 and recleared when desired, or they may be made permanent in balsam. The advantage of 

 keeping the total specimens in oil rather than in balsam is that they can be dissected. On 

 the other hand, they are much more permanent in the balsam. The oil of wintergreen makes 

 the tissues tough, so that it is possible to obtain minute dissections of the injected specimens. 



The Spalteholz method as applied to embryos can be very much simplified by changing 

 the fixative. For mammalian embryos the best fixative is Carney's mixture. This is 

 absolute alcohol 60 parts, chloroform 30 parts, and glacial acetic acid 10 parts. In this 

 mixture the acid is sufficiently strong to bleach the hemaglobin so that the peroxide is 

 unnecessary. The penetrating power of the fixative is very great, which is of importance, 

 since no injected specimen can be cut into until it is thoroughly fixed. The relations of 

 the tissues are well maintained and the swelling due to the acetic acid tends to counteract 

 the shrinkage that always takes place in the oil of wintergreen. The fixative does not 

 affect any of the injection fluids. The process after fixation in the Carnoy's mixture is 

 simple; the specimens remain in the fixative from 2 to 12 hours and are then placed direct ly 

 into 70 per cent alcohol, dehydrated in graded alcohols, and cleared as before. The speci- 

 mens can then be studied in toto, or dissected or embedded in paraffin and sectioned. They 

 should be embedded through a mixture of the oil of wintergreen and paraffin. They do not 

 become brittle in the oil, so that they may be sectioned after staying in the oil for many 

 weeks. The shrinkage in the oil, however, seems to increase on long standing. 



