DS 12.30 



DYE STAINS OF GENERAL APPLICATION 



335 



AF 20 may be employed. The writer has 

 a strong preference for a simple solution 

 of picric acid, provided that the specimen 

 has, as in this case, been prior-fixed in a 

 dichromate or a mercuric mixture. The 

 specimen may remain in a saturated solu- 

 tion of picric acid for as long as is neces- 

 sary to secure complete decalcification. 

 Decalcification may be conducted in the 

 same jar used for fixation, with precisely 

 the same technique of suspending the 

 object in a small loosely-woven cloth bag 

 about one-third of the way down the 

 bottle. It is only necessary to place an 

 excess of picric acid in the bottom of the 

 jar, to fill it up with water, and shake it a 

 few times to produce saturation before 

 inserting the cork bearing the specimen. 

 In the case of the mouse, at least two 

 months will be required to decalcify the 

 skull, the test object being the inner ear, 

 the bony protection of which is thicker 

 than that of any other part of the cranium. 

 This may be tested at intervals by re- 

 moving the object from the decalcifying 

 reagent and probing dehcately in the 

 region of the inner ear with a fine needle. 

 It is very easy to distinguish between the 

 tough nature of a decalcified bone and the 

 hard, sharp reaction which one obtains 

 from endeavoring to press the point of a 

 pin into an actual calcified structure. It is 

 to be recommended, however, in the 

 interest of safety, that a period of at least 

 one additional week in the decalcifying 

 reagent be allowed between the time when 

 one imagines the head to be perfectly 

 decalcified and the time when one decides 

 to embed and section it. The great advan- 

 tage of picric acid as a decalcifying agent 

 is that one cannot leave the specimen in 

 it for too long a period; no harm whatever 

 can be occasioned to the head of the 

 mouse if it be left for even a year or two 

 in the reagent. 



After removal from the picric acid, the 

 head is washed in running water for two 

 or three days until it ceases to liberate 

 any j-ellow color. It cannot be made en- 

 tirely white, because compounds will be 

 formed between the picric acid and the 

 protoplasm. Three days' washing, for an 

 object of the size described, should be 

 ample. The specimen is then dehydrated. 



embedded, and sectioned. No special pre- 

 cautions are necessary save to remember 

 that the dehydrating agent, the clearing 

 agent, and the wax will penetrate slowly 

 through so large and so tough an object. 

 It is therefore desirable in embedding first 

 to impregnate the object thoroughly with 

 a saturated solution of wax in the selected 

 clearing agent and then to evaporate the 

 clearing agent off slowly, thus gradually 

 increasing the concentration of the wax. 

 A low-melting-point wax is better than a 

 high melting point for preventing the 

 hardening of the tissues, since it may be 

 presumed that a 12- to 15-micron section 

 will be cut. It is unwise to loosen the 

 string holding the skin in place until at 

 least the last change has been made in 

 the clearing reagent. At this point, a sec- 

 tion for final embedding, about a quarter 

 to three-eights of an inch thick may be 

 cut through the object with a fine saw, 

 although the writer prefers to leave the 

 final trimming of the object until it is in 

 the paraffin block. 



When the paraffin block has been pre- 

 pared, it should be trimmed down roughly 

 with a knife until such time as one can 

 clearly see the eyes and thus distinguish 

 the region of which it is desired to cut a 

 section. The whole front end to within a 

 millimeter or two of this can now be cut 

 off with a fine saw. It is almost impossible 

 to trim off these large masses with a knife 

 without either cracking the paraffin block 

 or so dragging the object within the 

 paraffin that the hold of the embedding 

 agent is loosened. 



Section cutting presents no special diffi- 

 culty, the more so as it is not anticipated 

 that one intends to mount ribbons, but 

 only to select individual sections. If these 

 large sections have a tendency to roll up 

 on the knife, wet the blade of the knife 

 with 70% alcohol in order to hold down 

 the beginning of the ribbon, and then cut 

 as many sections as are desired. The 

 closer one trims the block to the region 

 which one desires to section, the less 

 necessity will there be to substitute a 

 freshly sharpened knife before taking off 

 the sections from the actual regions re- 

 quired. Sections are flattened as usual, 

 though it is recommended that they be 



