DS 21. IDS 21.10 



DYE STAINS OF SPECIAL APPLICATION 



377 



from the more generally employed blood 

 stains. These three divisions have, how- 

 ever, left a necessity for a fourth division, 



known here as "special stains for other 

 tissues" (DS 21.4), in which are grouped 

 some highly specialized techniques. 



21.1 Special Stains for Skeletal Tissues 



The term skeletal tissue is here taken in 

 its broadest sense to cover the supporting 

 structures of both plants and animals. 

 The supporting structures of vertebrates 

 are covered in the first three sections, 

 those of invertebrates in the fourth sec- 

 tion, and those of plants in the fifth sec- 

 tion. This leaves a small division at the 

 end for certain formulas which do not 

 easily fall into any division. 



21.10 TYPICAL EXAMPLES 



Preparation of a wholemount of a 



small salamander with the skeleton 



stained by the alizarin technique 



of Gray 1929 



Though this technique lies on the 

 borderhne l^etween the making of micro- 

 scope slides and the making of museum 

 preparations, the results are sufficiently 

 interesting to warrant inclusion. The tech- 

 nique about to be described deals with the 

 entire skeleton of a small salamander — say 

 Triturus — which is, of course, far too large 

 to mount as a microscope specimen. An 

 exactly similar method of operation, how- 

 ever, applies to the preparation of the 

 skeleton of the hand or arm of a sala- 

 mander, which can be justifiably regarded 

 as an object for microscopic mounting. 

 The method consists essentially in the 

 deposition of a calcium-alizarin lake on 

 the surface of the bones of the specimen, 

 the muscles and skin of which are subse- 

 quently rendered translucent by alkaline 

 hydrolysis, and the replacement of the 

 alkah with glycerol, both to increase the 

 transparency and to render the prepa- 

 ration permanent. 



Any small salamander may be used. 

 This is an excellent method of turning 

 experimental animals which die into useful 

 class-demonstration specimens. Each sala- 

 mander should be permitted to lie un- 

 touched after death until it is entirely 

 limp. It must then be mounted on a glass 

 shde to hold it in position through subse- 



quent operations. An ordinary 3" XI" 

 microscope slide serves excellently for 

 Triturus, but the operator will naturally 

 have little difficulty in adapting this tech- 

 nique to any other nmphiliian. The speci- 

 men is placed flat on the slide and the legs 

 and tail maneuvered into a natural posi- 

 tion. Soft silk thread, of the type once 

 used for surgical ligatures, is used to tie 

 the specimen to the glass slide in as many 

 places as possible, so that it will not shift 

 during the hardening operation. These 

 ligatures need not be made too tight, 

 since the object should always be hardened 

 in a horizontal position. 



The specimen is now hardened in an 

 iodine solution in alcohol, the exact con- 

 centration of which is of Uttle importance. 

 It should be considerably weaker than the 

 solution specified for microscopical prepa- 

 ration, and the suggestion given in the 

 technique below (DS 21.11 Gray 1929) of 

 a 10% dilution of Lugol's iodine in 95% 

 alcohol is approximately correct. The 

 concentration is not critical, and the 

 degree of dilution may be comfortably 

 made b^^ eye rather than by measurement. 

 At least 500 milliliters of this solution 

 should be used for a single salamander, 

 and the author has found it convenient 

 to lay the salamander, or as many sala- 

 manders on slides as will fit the dish, in 

 the bottom of a square glass dish of the 

 type commonly known as a refrigerator 

 jar. This jar is then filled with the iodine- 

 alcohol and placed in a dark spot over- 

 night, or until ne.xt required. The author 

 has invariably found that if the iodine- 

 alcohol is not used in the dark, the speci- 

 men will fall to pieces during the course 

 of subsequent hydrolysis. Though there is 

 no rational basis at present for this ()l)ser- 

 vation, he has made it often enough to 

 recommend that it be followed; and he is 

 sure that objections wliich have been 

 raised to his technique are based on failure 

 to follow this specific instruction, .\fter 

 the specimens have remained in the 

 iodine-alcohol at least overnight they will 



