DS 21.10 



DYE STAINS OF SPECIAL APPLICATION 



379 



be done but to throw the specimen away. 

 If, however, nothing is wrong save too 

 much residual brownish color, it is only 

 necessary to wash out the glycerol by 

 immersion in running water overnight and 

 then to replace the specimen in the potas- 

 sium hydroxide-ammonia mixture. 



The specimens are difficult to mount as 

 microscope slides unless one uses glycerol 

 jelly (see Chapter 5). The glycerol-impreg- 

 nated specimen should be transferred to 

 molten glycerol jelly in a sealed capsule, 

 and kept in it for about a month or until 

 it is completely impregnated. By this time 

 it is probable that the residual alkaUs will, 

 however, have hydrohzed the glycerol 

 jelly to the point at which it will no longer 

 set; but it is possible, by using a fresh 

 batch of glycerol jelly as a mountant, to 

 secure a fairly permanent preparation. If, 

 of course, one is prepared to take the 

 trouble involved in sealing a glycerol 

 mount (see Chapter 3) the specimen may 

 be mounted in a deep cell in glycerol. 



If the specimen is required as a museum 

 preparation it may be sealed in the 

 customary manner in a museum jar of 

 pure glycerol. 



Preparation of an embryo salamander 



to show the cartilagenous skeleton 



by the method of van Wijhe 1902 



Unhke the preparation of a wholemount 

 stained for bone, which has just been de- 

 scribed, the present method lends itself 

 better to microscope-shde preparation 

 than it does to museum jar preparation. 

 A salamander larva has been selected for 

 demonstration purposes for the reason 

 that it is convenient to secure, but the 

 same technique may be used with any 

 embryonic material, and it is surprising 

 that so few of these preparations, by which 

 the development, for example, of a 

 chrondrocranium can be so perfectly 

 shown, find their way into the hands of 

 classes. 



It does not very much matter what size 

 salamander larva is selected, but a 20-mm. 

 larva of Triturus is not only easy to secure, 

 but is also very generally useful for 

 demonstration purposes. The chief diffi- 

 culty is in the selection of the fixative, 



and though it is stated that alcohol- 

 hardened specimens arc satisfactory, the 

 author much prefei's to use a mercuric- 

 acetic fixative of the type of Woltereck 

 (Chapter 18, F 3000.0010 Wolterok (1910)). 

 A specimen of the size described sliould 

 be left in fixative for about 24 hours and 

 should then be washed in a dozen changes 

 of 70% alcohol. It is very important that 

 all the mercuric chloride be removed from 

 the specimen; therefore, it should be trans- 

 ferred from the last wash alcohol to a 

 solution of iodine i)rei)ared by adding 

 about 2 millihters of Lugol's iodine (Chap- 

 ter 22, ADS 12.2 Lugol 1905) to 100 milli- 

 liters of 70% alcohol. The specimen re- 

 mains in this iodine wash overnight and 

 is then again washed in 70% alcohol 

 until the last trace of color is removed 

 from it. It cannot be too strongly empha- 

 sized that a specimen which has been 

 fixed in a picric mixture can never under 

 any circumstances be used for a van Wijhe 

 preparation. 



The staining solution (DS 21.12 van 

 Wijhe 1902) contains 0.1% each of 

 toluidine blue and hydrochloric acid in 

 70% alcohol. The specimen should remain 

 in this solution until it is completely satu- 

 rated with the color. For the specimen 

 under discussion a period of 24 hours is 

 probably sufficient, but as overstaining 

 cannot occur, a period longer than this 

 may be employed. The subsequent process 

 of differentiation and dehydration is a 

 long one, and it is better to start with a 

 thoroughly stained specimen than to com- 

 plete a lengthy preparation with the dis- 

 covery that an additional day's time in 

 the beginning would have saved wasting 

 the whole period. Differentiation consists 

 merely in washing with 0.1 % hydrochloric 

 acid in 70% alcohol until no more color 

 comes away. Repeated changes of 70% 

 alcohol can be avoided by taking about a 

 liter for a specimen of the size under dis- 

 cussion, and placing this in a tall, narrow, 

 cyhndrical museum jar fitted with a cork 

 from which is suspended a loosely woven 

 cloth bag containing the small embryo. 

 Streams of color will at once start falling 

 from the stained specimen, and when 

 these color streams have ceased, it will 

 be necessary to place the specimen in fresh 



