142 CARMINE AND COCHINEAL 



and the mixture heated to assist in dissolving. After cooling the 

 glycerine is added, and the mixture filtered. Recommended by 

 Ludford for counterstaining trypan blue material (see § 750). 

 Keeps well, only for sections. 



All solutions prepared with ahim tend to precipitate. Carmalum 

 made up with 500 c.c. of water instead of 200, and with glycerin or 10 

 per cent, of formol or pyroligneous acid added, keeps well. 



255. Mayer's Aqueous Aluminium-Chloride Solution {Mitth. Zool. 

 Stat. Neapel, x, 1902, p. 490). Carminic acid, 1 grm. ; chloride of 

 aluminium, 3 grm. ; water, 200 c.c. Add an antiseptic, as for car- 

 malum . 



Use as carmalum. The stain is of a blue-violet colour, very powerful 

 and elective, but not so purely nuclear as carmalum. It is recom- 

 mended only as a substitute for carmalum in cases in which the latter is 

 counter-indicated on account of the alum in it or the like. 



256. Alum-Carmine and Picric Acid. Alum-carmine objects may be 

 double-stained with picric acid. Legal (Morph. Jahrb., viii, p. 353) 

 combines the two stains by mixing 10 vols, of alum-carmine with 1 of 

 saturated picric acid solution. I find this very recommendable. 



257. Aceto-Carmine (Acetic Acid Carmine) Schneider {Zool. 

 Anz., 1880, p. 254.). To boiling acetic acid of 45 per cent, strength 

 add carmine until no more will dissolve. Cool and filter. With 

 some batches of carmine it is much better to simmer an excess of 

 carmine in 45 per cent, acetic acid, preferably under a reflux 

 condenser, for an hour or two, and after cooling filter. (According 

 to Schneider, the largest proportion of carmine is dissolved in 

 acetic acid of 45 per cent, strength. Usually less than ^ grm. of 

 carmine will dissolve in 100 c.c. of 45 per cent, acetic acid.) 



Belling 's Iron Aceto-Carmine. Belling found that the addition 

 of a trace of iron to Schneider's aceto-carmine makes the chromo- 

 somes, in fresh tissue, stain more deeply {Amer. Nat., vol. 55, 

 1921, p. 573). If the tissue requires teasing in order to separate 

 the elements, this is done on a glass slide in a few drops of aceto- 

 carmine with steel needles and usually enough iron will be dis- 

 solved to serve as a mordant. Or a few drops of ferric hydrate, 

 dissolved in 50 per cent, acetic acid, is added to ordinary aceto- 

 carmine until it becomes a bluish-red, but without a visible 

 precipitate. Then add an equal part of untreated stain. 



The amount of iron needed to give the optimum effect varies 

 with the tissue used and the sample of carmine employed to make 

 aceto-carmine in the first place Too much iron will completely 

 spoil the stain so it is well to go slowly, adding minute traces of iron 

 each time until a satisfactory stain is obtained for the particular 

 material in hand. 



Aceto-carmine, either with or without iron, may be used in a 

 1 per cent, strength as a slow stain, but, for studying chromosomes 

 in fresh tissue, full strength is required. The procedure varies in 

 details but, in general, the method of application is as follows : 

 Fresh material is placed on a glass slide and aceto-carmine is 



