Carmine

CARMINE, a coloring substance of animal origin, red in color, obtained from cochineal (Coccus cacti) by precipitating its aqueous infusion with alum; it is a compound of carminic acid with aluminum, calcium, and protein substances. The best grade of C.-Carmin nacarate (Merck, Griibler and others) is sold in the form of light, dry pieces of a dull red color. C. is almost insoluble even in boiling water; completely insoluble in alcohol, ether; easily soluble in HCl and H₂SO₄, as well as in solutions of ammonia, caustic and carbonic alkalis, and in solutions of borax. In histological technique, both C. and carminic acid are used. -Application of C. Carmine was introduced by Corti (Corti; 1854) and Gerlach (Gerlach; 1858); it is used in animal and botanical histology and in protistology. An excellent nuclear and protoplasmic stain; it also stains glycogen and mucus. It is used on sections, but it also excellently stains pieces. The best results are obtained after mercuric chloride fixatives; after formalin and osmic acid it is not applicable. It is also used for injection masses, as well as for vital staining of the reticulo-endothelium (blockade). Methods of application. I. Carmine. A. Acidic aqueous solutions. 1. Alum Carmine of Grenacher (Grenadier; 1879). Dissolve 3.0-5.0 of potassium and ammonium alum in 100.0 distilled water, add 1.0 C., boil for 15-20 min., cool, filter, add 1.0 formalin. The solution is unstable. Unsuitable for staining pieces. Alum C. is widely used for total staining of small animals, for example flatworms (trematodes, tapeworms), insect larvae, etc. Sections are transferred from distilled water into the stain for 1/24-24 hours, differentiated in distilled water. Result-sharp nuclear staining. Modification of Mayer (Mayer; 1897)-2.0 C., 5.0 alum, 100.0 water; boil for 1 hour. - 2. Acetic carmine of Schneider (Schneider; 1880). Dissolve dry powder of C. in boiling 45% acetic acid to saturation (carefully! in a fume hood!), cool, filter. Used for simultaneous fixation and staining and direct observation without subsequent mounting in balsam. Stain in a weak solution (1:100.0 distilled water) or in undiluted stain; the latter serves simultaneously for fixation. Especially suitable for studying teased tissues, small animal objects (worms, etc.) and some protozoa. In case of over-staining, differentiate in a mixture of 100 cm³ glycerol with 100 cm³ 70° alcohol and 0.5 cm³ HCl (strong); can be transferred from the stain into pure glycerol. -3. Picricarmine (P. Mayer)- an unstable mixture of ammonium picrate, ammonium C. and free ammonia (Ranvier); the mixture gives inconsistent results; it is recommended to replace it with double staining, staining with a saturated solution of picric acid after preliminary staining with carmine. B. Basic solutions. Ammonium C. Ranvier (Ranvier, Gerlach). 1.0 C. is triturated in 100.0 distilled water, add ammonia drop by drop until C. dissolves and leave the solution to mature until the smell of NH₃ disappears. After 14-day fixation in Müller's fluid, it stains the processes of ganglion cells and nerve fibers in the central nervous system after 24 hours. 1. Lithium C. of Orth. 2.5-5.0 C. are boiled for 10-15 minutes in a water bath in 100.0 of a saturated at room temperature (1%) aqueous solution of Lith. carbonicum; filter; stain for 2-5 min.; differentiate in hydrochloric alcohol (Alk. 70°- 100.0; HCl strong-0.5-1.0); wash in water. Excellent nuclear staining. Used for vital staining by injecting under the skin to mice 0.3 for 5-8 days in a row, to rats 2.5-3.0, and to rabbits 10.0 intravenously. -2. Ammonium carmine of Best (Best; 1901-06). Preparation of the basic solution: C.-1.0, ammonium chloride-2.0; lithium carbonate-0.5; boil in 50 cm³ distilled water. After cooling add 25% ammonia 20 cm³. After 2-3 days the solution is ready for use and retains its staining ability for several weeks. Staining method - see Glycogen. - 3. Boric alcohol C. of Grenacher. 2.0-3.0 C. are dissolved by boiling in 100.0 of a 4% solution of borax; after cooling add 100.0 of 70° alcohol and leave to mature for several weeks with frequent shaking. Used for staining pieces, for which they are transferred from 50° alcohol into the stain solution for a period from 24 hours to several days depending on the size of the piece. Differentiate in the piece with hydrochloric alcohol until the latter stops extracting the stain. - 4. Mucicarmine of Mayer. 1.0 C., 0.5 aluminum chloride and 2.0 distilled water are heated for 2 min. on a weak flame until the mixture darkens, after which 100.0 of 50° alcohol are gradually added and after 24 hours it is filtered. For staining, the basic solution is diluted with 10 parts of tap water. Stain progressively (5-10 min.). When properly prepared, only mucus is stained, not nuclei; it is also used for staining hyaline cartilage and elastic tissue. II. Carminic acid is used in histological technique in combination with aluminum and calcium. The following solutions are most commonly used. 1. Carmalum (Mayer; 1891). 1.0 of carminic acid and 10.0 of potassium alum are dissolved in 200.0 distilled water; the resulting red-violet solution is filtered and 1.0 formalin and 1.0 Natr. salicylici are added to it. Sections are stained for 10-15 min., then differentiated in a 1% solution of alum and washed in water. Pieces are stained for 24-48 hours and washed in water. Staining also occurs after osmic fixation. For double staining, a 0.5% solution of Lichtgrun in 70° alcohol is used. - 2. Paracarmine (Mayer; 1892). 1.0 of carminic acid, 0.5 of aluminum chloride and 4.0 of calcium chloride are dissolved in 100.0 of 70° alcohol, allowed to stand and filtered. Sections are stained for 10-15 min., differentiated in 70° alcohol.