382 . METHODS. 



Boil vigorously for twenty minutes; allow the fluid to settle; decant the clear 

 fluid; add more water and boil again; filter the entire solution and evaporate the 

 filtrate to 80 c.c.; add a small piece of thymol or camphor to prevent the growth 

 of fungi. Alum cochineal is, on the whole, the best reagent for in toto staining, 

 as it will penetrate quite large objects and color them uniformly throughout, and 

 gives a good differentiation of the tissues. 



For in toto staining place the specimen in water until it sinks; then transfer 

 it to the cochineal for twenty-four hours, or for large specimens longer; the depth 

 of the stain will depend upon the strength of the' solution; transfer to clean water 

 for fifteen to twenty minutes to extract the alum, which otherwise will crystallize 

 in the tissues when the specimen is placed in alcohol; the object must not be 

 left too long in water, because it extracts the color also; put in 50 per cent 

 alcohol for one hour, then successively in 70 per cent, 80 per cent, and 95 per 

 cent, when the specimen will be ready for imbedding. 



2. BORAX CARMINE. 



Formula: Best carmine 3 gm. 



Borax 2 gm. 



Water 50 c.c. 



Boil for twenty minutes; allow the solution to cool; 'add water enough to restore 

 that lost by evaporation, then add 50 c.c. of 70 per cent alcohol, let the solution 

 stand twenty-four hours; filter. Borax carmine gives a good nuclear stain and 

 may be advantageously supplemented by counterstains. 



For in toto staining place the specimen in water until it sinks; transfer to the 

 carmine for twenty-four hours, or longer for large specimens; wash in water for 

 five minutes; then place it in 70 per cent alcohol, to every 100 c.c. of which 2 c.c. 

 of hydrochloric acid have been added; after one hour transfer to fresh 70 per 

 cent alcohol, which must be renewed in an hour or two, and finally transfer to 

 80 per cent and 95 per cent alcohol, and the specimen will be ready to imbed. 



3. ALUM HEMATOXYLIN. 



Formula: i. Dissolve 10 grms. hematoxylin in 60 c.c. absolute alcohol. 



2. Dissolve 15 grms. ammonia alum in 100 c.c. warm water, and allow the solution to cool. 



3. Mix the two solutions and allow the stain to "ripen" in a shallow open dish exposed to 



the light for three days. 



4. Filter, then add 25 c.c. pure glycerin and 25 c.c. methyl alcohol. 



5. After three days filter. 



The solution thus prepared will keep indefinitely. It is one of the best nuclear 

 stains known. Eosin may be used with it as a counterstain giving beautiful 

 preparations. 



i. Place sections from water into a filtered diluted solution of the stain for 

 from fifteen minutes to twenty-four hours, according to the strength of the staining 

 solution. (Slow staining gives the best results.) 



