336 VITAL STAINING 



weaker alcohols avoided as much as possible. Bouin's fluid can be used 

 for fixing spread tissues and for cellular exudates. 



752. Staining with Vital New Red. This dye is a good vital 

 stain, and is less toxic than trypan blue, but it has the dis- 

 advantage of being much more difficult to retain in fixed pre- 

 parations. It is a convenient dye to employ in double-staining 

 experiments with trypan blue, when only examination of the 

 living tissues is intended. 



Methods of Staining and Amounts to Inject. Inject either 

 subcutaneously, intraperitoneally or intravenously. Mice (per 

 20 grm. bodyweight) : 0-5 to 1-0 c.c, of a 1 per cent, solution 

 daily. Rabbits (per 1,000 grm. bodyweight) : 5 c.c. of a 5 per 

 cent, solution. Guinea-pigs (per 100 grm. bodyweight) : 2 c.c. of 

 a 2-5 per cent, solution. 



Fixation. The dye dissolves in the lower alcohols, but not in 

 absolute alcohol, which may therefore be employed as a fixative. 

 Cappell {loc. cit., 1929) recommends corrosive sublimate solution, 

 and states that Zenker or Bouin also are satisfactory with some 

 tissues. 



753. Staining with Diaminefast Scarlet. This is another 

 suitable dyestuff to employ for double-staining experiments with 

 trypan blue. It has the advantage of being retained well in fixed 

 preparations. For mice (per 20 grm. bodyweight) inject 0-5 to 

 1-0 c.c. of a 1 per cent, solution, either subcutaneously, or intra- 

 peritoneally, and for rabbits (per 1,000 grm. bodyweight) inject 

 20 c.c. of a 5 per cent, solution. 



According to Cappeli. (loc. cit., 1929) the best fixatives are Bouin 

 and corrosive-sublimate. 



754. Staining with Carmine. Lithium carmine has the advantage 

 of being well retained after fixation, and resists solution by the 

 alcohols used for dehydration. It is, however, rather more 

 poisonous than trypan blue. The best method to administer it is 

 by intravenous injections. 



(i.) Preparation. (1) Dissolve 2-5 grm. of carmine in 100 c.c. 

 of a saturated solution of lithium carbonate. (2) Boil ten to 

 fifteen minutes on a water-bath. (3) Filter immediately before 

 using — filtration through a Berkefeld filter will ensure sterility. 



(ii.) Intravenous Injections. With mice, injections should be 

 made into the tail vein. It is advisable to dilute the stock solution 

 to 20 per cent., and to commence by injecting 0-2 c.c. of this. 



Repeated injections are necessary, and should be made daily 

 if the condition of the animals will allow. The amount of the dye 

 injected may be increased with later injections. A general vital 

 staining is attained after five to eight days. 



Rabbits may be given 10 c.c. of a 10 per cent, solution daily. 



(iii.) Fixation. Tissues can be fixed in 10 per cent, formalin in 



