378 



METHODS AND FORMULAS 



DS 21.10 



be found to be so hardened that they 

 may be stood in a vertical position with- 

 out becoming distorted. They should now 

 (still in the dark) be placed in 95 % alcohol 

 for about 24 hours, the alcohol being 

 changed at least once in this period. It is 

 not necessary to remove all of the iodine 

 from the specimen, but this additional 

 hardening in alcohol makes staining 

 easier, and prevents the break-up of the 

 specimen. 



Considerable quantities of 5% potas- 

 sium hydroxide should now be prepared, 

 and about 100 milliliters of this taken for 

 each specimen. To each 100 milliliters add 

 1 millihter of a saturated solution of 

 aUzarin red S in absolute alcohol. Place 

 the specimens directly from alcohol into 

 this potassium hydroxide-dye mixture to 

 allow the rapid diffusion currents to carry 

 the stain into the bones. The time of 

 exposure to the stain is not very impor- 

 tant, though at least 24 hours should be 

 allowed to elapse in the case of Triturus; 

 but the time should be greatly increased 

 if only slightly larger specimens are at- 

 tempted. Rana tigrina, for example, 

 should be left for at least a week in the 

 solution. It must be remembered that the 

 stain will be deposited nowhere save on 

 the bones, but that, unless a thorough 

 impregnation takes place, some of the 

 deeper bones may not become sufficiently 

 pigmented. As the entire process takes 

 some months, it is a pity to find at the 

 conclusion of the period that an addi- 

 tional day in the stain would have avoided 

 wasting the whole lengthy period of 

 preparation. 



When the specimens are removed from 

 the staining solution, they will be found 

 to be a dull red all over, and, on exami- 

 nation by transmitted light, the small 

 bones of the limbs will be clearly visible 

 as a darker shadow within the reddish 

 flesh. Now place the specimens in fresh 

 5% potassium hydroxide, changed as 

 often as it becomes colored pink or brown, 

 until the pink color has been removed 

 from the flesh. This is a dual process, for 

 the hydroxide both hydrolizes the skin 

 and muscle, and removes at the same time 

 the absorbed stain. The muscle under this 

 treatment does not become white but 



remains a yellowish brown. Care must be 

 taken to distinguish between this inevi- 

 table residual yellowish brown color, which 

 is removed in the next treatment, and the 

 pinkish color which results from incom- 

 plete removal of the stain. The time for 

 this removal of the excess stain varies 

 with specimens, and it is only a rough 

 estimate to say that about two months 

 will be required for a Triturus and from 

 three to four months for a frog. No harm 

 will result to the specimen should it re- 

 main as long as six months in this solution 

 and, if a number are being prepared, they 

 may well be forgotten for this time rather 

 than watched closely. 



The next stage is the removal of the 

 residual brownish color from the muscles. 

 No further translucency is imparted dur- 

 ing this process of decolorization, which 

 is conducted in equal parts of 5% potas- 

 sium hydroxide and 1% ammonia. The 

 solution needs to be changed as often as 

 it becomes discolored, and this changing 

 must be continued until the muscles and 

 skin are bleached to a pure white color. 

 The time for this is again variable, but 

 rarely takes more than about two or three 

 months for a frog or five or six weeks for a 

 Triturus. 



When the flesh has been bleached, the 

 specimen is placed in 5% potassium hy- 

 droxide, which must be changed daily 

 until the smell of ammonia is no longer 

 apparent, and then transferred to 5% 

 potassium hydroxide containing 5 % glyc- 

 erol. After the animal has become thor- 

 oughly penetrated by this solution, which 

 may be determined by the fact that no 

 further diffusion currents rise from the 

 specimen, it may be placed in 10% 

 glycerol until diffusion currents cease, and 

 so on through increasing concentrations of 

 glycerol, 10% apart from each other, until 

 it is finally in pure glycerol, of which at 

 least two changes should be used. The 

 specimen is now almost glass-clear and 

 shows bright red bones in a glassUke flesh. 

 It is all too frequently discovered at this 

 stage that one or another of the processes 

 has not been continued long enough. If 

 the bones finally disclosed in the thickest 

 portions of the flesh are found to be 

 insufficiently stained, there is nothing to 



