STAINS, ETC. 



1375 



ExMBRYOLOGIC METHODS 



EMBRYOLOGIC METHODS. 

 Artificial Fecundation. This practice can be easily 

 carried out with the Amphibia anura, Teleostea, 

 Cyclostomata, Echinodermata and many Vermes 

 and Ccelenterata. In the Amphibia, the ova should 

 be extracted from the uterus, placed in a watch- 

 glass, and treated with water in which the testes or 

 vasa deferentia of the male have been teased. The 

 spermatozoa of fish rapidly lose their vitality in water, 

 hence, the milt must be added immediately to the 

 spawned ova, then a little water added, and the whole 

 placed in a suitable hatching apparatus with running 

 water. Artificial fecundation of Invertebrates is per- 

 formed in a like manner, and can sometimes be ac- 

 complished under the microscope. The penetration 

 of the spermatozoon and some of the subsequent 

 changes can thus be observed. Amphibia. Ova of 

 amphibia are covered with thick coats of albumin, 

 which must be removed in their preparation for section- 

 cutting. Whitman places the fixed eggs in a 10 per 

 cent, solution of sodium hypochlorite, diluted with 5 

 or 6 volumes of water, until they can be shaken free. 

 Blochmann recommends eau de Javelle (see Bleach- 

 Fluids), diluted three or four times with water, 

 and agitates the eggs, previously fixed in Flemming's 

 solution ^see Fixing Fluids) , for from 15 to 30 minutes. 

 Preserve the ova in alcohol. Axolotl. These ova 

 have an albuminous layer separated from the yolk by 

 a liquid which is not coagulated by reagents. . Place 

 them for a few hours in picrosulphuric acid, then 

 pierce the inner chorion, and gently press out the ova. 

 Harden in alcohol. Stain in the mass with borax - 

 carmin or Henneguy's acetic acid alum-carmin, and 

 embed in paraffin or celloidin. Collodionize the sec- 

 tions. Rana. Place the ova in water heated to qo°- 

 96 C. for 5 to 10 minutes. Incise the albuminous 

 coverings, and remove the ova under water. Place them 

 in 0.5 per cent, osmic-acid solution or in alcohols of 

 70, 80, and 90 per cent. Salamandra. Fix in 

 warm, platinum-chlorid solution (0.25 to 0.3 per cent.) 

 for 3 to 24 hours, according to the size of the embryo. 

 Wash in water, and pass through successive alcohols. 

 Stain sections on the slide. Triton. Incise the sev- 

 eral concentric coats of albumin which surround the 

 ovum ; remove, and place it in Kleinenberg's fixing 

 solution (see Fixing Fluids) . Or, put the eggs in a 

 solution of acetic acid 2 per cent. , chromic acid o. 5 

 per cent. , and after ten hours incise the membranes 

 and turn the embryos out. Finally, pass through suc- 

 cessive alcohol's. Aves. Superficial Examination. 

 During the first 48 hours of incubation of the egg 

 (hen's i the blastoderm is always uppermost. To open 

 the egg, place it in a dish and cover it with a 0.75 per 

 cent, sodium-chlorid solution at a temperature of 38 

 C. Break the shell at the broad end over the air- 

 chamber, to keep this end from tilting up. The shell 

 is then filed through at one point, and the opening 

 enlarged with forceps. Remove the upper half of the 

 shell, bit by bit. Then remove the shell-membrane 

 in the long axis of the egg, and the yolk and embryo 

 will come into view. A quicker but less satisfactory 

 method is to break the egg across and pour the yolk 

 and white into the sodium-chlorid solution. Maintain 

 the salt-solution during the period of examination at 38 

 C. over a sand-bath. Duval's Orientation Method. To 

 obtain sections of any desired direction of the ova of 

 Aves, before the development of the primitive streak, 

 Duval proceeds as follows. During incubation the 

 embryo is generally lying on the yolk, so that the large 

 end of the egg is to its left, and the small end to its 

 right; hence, the position of the blastoderm can be 

 marked out. Construct a triangular, bottomless box 



from a strip of paper 5 mm. wide and 50 mm. long ; 

 lay this on the yolk enclosing the cicatricula in such a 

 position that the base corresponds to the anterior, region 

 of the embryo. By means of a pipet fill the paper 

 triangle with 0.3 per cent, osmic-acid solution. When 

 the preparation becomes dark, place the whole egg in 

 a weak chromic-acid solution, remove the white, and 

 place the rest in a fresh chromic-acid solution for sev- 

 eral days ; a black triangle will mark the position of 

 the cicatricula, and may be cut out with scissors and 

 scalpel. Examination of an Opaque Object. Place 

 the blastoderm on a slide, and dry just sufficiently to 

 make its edges adhere to the glass ; immerse in a 

 solution of picric acid for two or three hours, and ex- 

 amine with a simple lens. Examination and Pre- 

 servation in toto. Open the egg in salt-solution, 

 pierce the blastoderm at the outer margin of the vas- 

 cular area with a fine scissors, and carry the incision 

 completely around. Then place the excised blasto- 

 derm in a watch-glass, and remove the vitelline mem- 

 brane by gentle shaking with a needle. The blasto- 

 derm can then be placed on a slide, surrounded by a 

 ring of putty, covered with salt-solution and a cover- 

 glass, and examined under the microscope. Keep 

 the slide at about 38 C. Permanent preparations of 

 embryos in toto, up to about 50 hours, may be made 

 by treatment with osmic acid, I percent. After sepa- 

 ration of the vitelline membrane, hold a drop of the 

 acid, by means, of a pipet in contact with the em- 

 bryo for 15 or 20 minutes. Then mount in a cell in bal- 

 sam. Druelopment of the Blood-vessels. Obtain 

 blastoderms of 30 or 40 hours , immerse in gold chlorid, 

 0.5 percent., for I minute, wash in distilled water, 

 mount in glycerin, and examine. This method renders 

 the nuclei and protoplasmic processes distinct. Or, 

 immerse the blastoderm in I per cent, solution of potas- 

 sium bichromate for 1 day, and mount in glycerin. 

 Or, use a 0.5 per cent, solution of osmic acid for from 

 y^ to I hour, then place in absolute alcohol for I day, 

 and mount in glycerin. Gerlach's Windcnv Method. 

 Remove the shell at the small end of the egg, with- 

 draw a little white with a pipet ; the blastoderm 

 will change its position and appear under the win- 

 dow thus made. Paint the margins of the window 

 with gum-mucilage, and build a small, circular wall 

 of cotton-wool on it, cover with a cover-glass, 

 and ring with gum. The progress of development 

 can be followed thus to the fifth day. Prepara- 

 tion. During the first 24 hours of incubation the blas- 

 toderm can be separated from the yolk only with ex- 

 treme difficulty, so that they must be fixed together. 

 Open the egg in salt-solution, then lift so that the blas- 

 toderm is above the surface of the fluid, and treat it 

 with a fixing solution dropped from a pipet ; then 

 remove it by a circular incision about its margins, free 

 the vitelline membrane, and place the blastoderm in a 

 hardening fluid. Dehydrate in absolute alcohol, clear 

 in chloroform, and embed in paraffin. Segmentation. 

 To observe this process, it is necessary to obtain the 

 eggs from the oviduct of the hen. The yolk must be 

 hardened as a whole, preferably in chromic acid. 

 Fol's Method for Reconstruction of Embryos from 

 Sections. Before cutting sections of the object, 

 make an outline drawing of it, under the magnifica- 

 tion to be employed for the reconstructed drawing, and 

 in a plane perpendicular to that of the intended sec- 

 tions. Then cut the sections, and make drawings of 

 all under the same magnification used for the sagittal 

 drawing. Trace over the sagittal drawing a series of 

 equidistant parallel lines corresponding to the sections 

 cut. (If the sections are ^\^ mm. thick and the draw- 

 ing is magnified 100 times, the lines should be I mm. 



