144 CARMINE AND COCHINEAL 



from one to ten minutes. The slides are now passed rapidly 

 through the following solutions : 1 part of glacial acetic acid and 

 2 of absolute alcohol ; then 1 part of acetic to 9 of absolute 

 alcohol. Dehydration is completed in pure absolute alcohol and 

 the slide cleared in xylol and mounted. 



For animal tissue the same method which McClintock follows 

 may be used. The coverglass is floated off fresh aceto-carmine 

 preparations in 10 per cent, acetic acid and carried through the 

 steps of dehydration and mounting she uses for plant tissue. 



Buck {Science, Ixxxi, 1935, p. 75) inverts an aceto-carmine 

 slide (with supports) in a Petri dish containing equal parts of 

 glacial acetic acid, absolute alcohol and xylol, until the cover- 

 glass soaks off. Then the slide and coverglass are passed through 

 two changes of equal parts of absolute alcohol and xylol and 

 then placed in pure xylol, from which they may be reunited in 

 damar or balsam. Buck points out that Metz and Gay found 

 that clove oil could be substituted for xylol in the initial steps. 



258. Lee's Iron Carmine. We recommend trial of the following, 

 which Lee has aheady pubhshed in the Traite des Meth. Techniques, 

 Lee et Henneguy, 1902. Sections are mordanted (a few hours will 

 suffice) in sulphate of iron (Benda's liquor f err i, as for iron haematoxylin), 

 washed, and stained for an hour or so in 0-5 per cent, solution of car- 

 minic acid in alcohol of 50 per cent. Wash in alcohol of 50 per cent. ; 

 no differentiation is necessary. When successful, there results an 

 almost pure chromatin stain, quite as sharp as iron haematoxylin, 

 but somewhat weak. 



Iron Carmine. Pfeiffer von Wellheim {Zeit. wiss. Mik., xv, 

 1898, p. 123) mordants for six to twelve hours in a very weak solution 

 of chloride of iron in 50 per cent, alcohol, washes in 50 per cent, alcohol, 

 and stains as above. Overstains may be corrected with 0-1 to 0-5 per 

 cent. HCl alcohol. Lee found this good, but not so good as the last. 



Iron Carmine (Zacharias, Zool. Anz., 1894, p. 62). Stain for several 

 hours in an aceto-carmine (made by boiling 1 grm. of carmine with 

 150 to 200 c.c. of acetic acid of 30 per cent., for twenty minutes, and 

 filtering). Rinse the objects with dilute acetic acid, and bring them 

 (taking care not to touch them with metallic instruments) into a 1 per 

 cent, solution of ammoniated citrate of iron. Leave them, for as much 

 as two or three hours if need be, till thoroughly penetrated and blackened 

 (with sections this happens in a few minutes). Wash for several hours 

 in distilled water. A chromatin and plasma stain. 



259. Hollande's Chlorcarmine Staining Method (C. R. Soc. Biol., 

 1916, Ixxix, p. 662, and Jour. Roy. Micr. Soc, 1920). Place 

 5 c.c. pure hydrochloric acid in a porcelain dish ; add little by 

 little 14 grm. powdered carmine, stirring constantly to make a 

 homogeneous doughy mass. Allow to digest for twenty-four 

 hours ; add 250 c.c. aq. dest., bring to the boil, and keep boiling 

 for half an hour. Filter ; make up to 180 c.c. with aq. dest., 

 and then add enough 75 per cent, alcohol to make a total volume 

 of 200 c.c. Stain sections or pieces of tissue for two to twenty- 



