584 PROTOZOA 



used as simple counterstains after nuclear stains such as the 

 above. Sections and films are placed in them for a minute or 

 two before final dehydration in absolute alcohol. They may be 

 used dissolved in weaker alcohols, if desired, and eosin, fre- 

 quently used after hematoxylin, may also be used in 1 per cent, 

 aqueous solution. Eosin may be removed from tissues to some 

 extent by prolonged washing in tap-water owing to its solubility 

 in alkalies. 



For staining protozoa in tissues, double or triple stains are 

 specially useful. 



Light green and picric acid is recommended by Minchin as a 

 double counterstain. (1 grm. of " licht gri'm " and 0-5 grm. picric 

 acid are dissolved in 100 c.c. 90 per cent, alcohol.) This stain or 

 light green alone gives especially good results after safranin, 

 magenta or carmine. 



Eosin and light green is recommended by Chatton {Arch. 

 Zool. Exper., lix, 1920, p. 21). This is a simplification of Prenant's 

 method {Arch. d'Anat. niicr., vii, 1905, p. 430), as follows : Alcohol 

 (95 per cent.) is saturated with eosin W.G. and light green F.S. 

 This solution keeps indefinitely. 



Sections are stained for five minutes. They are then rose, 

 and are differentiated in absolute alcohol containing 5 per cent, 

 of acetic acid until the connective tissue of vertebrates or the 

 chitin of arthropods is green. The preparations are then washed 

 in xylol and mounted in neutral balsam. The method is said to 

 be particularly useful for the study of parasites in Copepods. 



Picro-nigrosin, a mixture of 1 part of saturated aqueous solution 

 of nigrosin with 9 parts of saturated aqueous solution of picric 

 acid, is used as follows : Transfer films or sections from distilled 

 water into the stain for five to seven minutes, wash in tap-water, 

 then rinse rapidly in 70 and 90 per cent, alcohol. Complete 

 dehydration and inount. Connective tissue and chitin should 

 be blue and muscles yellow. Very pretty preparations may be 

 obtained after carmine. The method works especially well after 

 sublimate fixation, it is not so successful after Bouin's fluid. 



Alcoholic solutions of this stain are sometimes recommended. 



Indigo-carmine solutions may be made up in many ways. 

 A saturated solution in 70 or 90 per cent, alcohol is satisfactory 

 but a much stronger solution may be made in water. 



Picro-indigo-carmine mixtures may also be made in a variety 

 of ways. Three parts of a saturated solution of indigo-carmine 

 to 1 part of a saturated solution of picric acid, both in 70 per 

 cent, alcohol, makes a good counterstain. 



Ramon y Cajal's uses a watery solution (§ 426) after carmine or 

 magenta. 



BoRREL {Annal. Inst. Past., xv, 1901. p. 57) recommends a mixture 

 of 2 volumes of a saturated watery solution of indigo-carmine with 1 



