CHAPTER XII. 139 



A similar stain has been prepared with formic acid by PIANESE (see 

 Zeit. wiss. Mik., x, 4, 1894, p. 502). 



For BURCHARDT'S pyroligneous-acid carmines see Arch. mik. Anat., 

 liii, 1898, p. 232 ; and Jena Zeit. Naturw., xxxiv, 1900, p. 720. 



222. Iron Carmine. I recommend trial of the following, which I 

 have already published in the Traite des MetJi. Techniques, LEE et 

 HENNEGUY, 1902. Sections (I have not tried material in bulk) are 

 mordanted (a few hours will suffice) in sulphate of iron (Benda's 

 liquor ferri, as for iron hsematoxylin), washed, and stained for an 

 hour or so in 0-5 per cent, solution of carminic acid in alcohol of 50 

 per cent. Wash in alcohol of 50 per cent. ; no differentiation is 

 necessary. When successful, an almost pure chromatin stain, quite 

 as sharp as iron hsematoxylin, 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. HC1 alcohol. I find 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. 



Hollande's Chlorcarmin Staining Method (G. 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 grms. 

 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-four hours. Rinse in aq. dest. or 30 per cent, alcohol ; 

 immerse in 3 percent, iron alum solution, in which the sections become 

 black, and are then slowly decolourised; when differentiation is com- 

 plete, rinse in a 1 per cent, pyridin solution, and wash under the tap for 

 ten to fifteen minutes. Counterstain and mount as desired. This is a 

 very intense stain suitable for mitochondria and cell granules. 



223. Iron Carmalum (DE G-KOOT, Zeit. wiss. Mik., xx, 1903, p. 21).- 

 Dissolve 0-1 grm. of ferric alum in 20 c.c. distilled water and add 1 grm. 

 carminic acid. Dissolve, add 180 c.c. of water, warm, add 5 grms. 

 potash alum, dissolve, cool, filter, and add 2 drops of hydrochloric 

 acid. To be used as carmalum, and said to give a stronger stain. 



