Carmine staining methods
WOLF D. KUHLMANN, M.D.
Division of Radiooncology, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany
Before the era of haematoxylin and synthesized aniline dyes as histological stains, the method
of choice to stain tissue sections was one of the natural dyes such as carmine and saffron. G M
SMITH (1915) reported that in the early days of histological staining H R GOEPPERT and F
COHN (1849) and A CORTI (1851) have already used carmine for the study of cell contents.
Yet, T HARTIG (1854) was given credit for discovering the process of tissue staining with
carmine. A very valuable overview of the history of histological staining is given by G M
SMITH (1915) which includes also some further developments of modern microtechnique.
Carmine is obtained from the bodies (female) of the insect Dactylopius coccus cacti. The
active coloring agent in carmine is carminic acid (C.I. 75470, Natural red 4). For nuclear
staining it is invariably used in conjunction with a mordant such as alum solution.
Today, carmine is quite rarely used for histological staining. Carmine staining, however, can
be an alternative to haematoxylin counterstaining of immunostained tissue sections. A variety
of carmine formulations exist for very clean and sharp nuclear staining. The most popular
formulations are given here.
Carmine, chrome alum (Fyg)
Chemicals Chemical solution
Carminic acid (C.I. 75470) FYG carmine dye solution:
Chromium potassium sulfate (chrome alum) 6.0 g chrome alum dissolved in
100 mL distilled water (heated), add
HCl (25%) 1.0 g carmine
Ethanol
the mixture is boiled for 15 min, then
Distilled water cooled down and filtered
HCl-ethanol solution:
97.5 mL 70% ethanol plus
2.5 mL HCl (25%)
Staining procedure
Immuno-stained sections are passed through distilled water and stained:
FYG carmine dye solution 1-6 hours
distilled water 5 x 1 min
differentiate in HCl-ethanol solution under microscopic control
Dyes and other chemicals in histological staining can be toxic. They must be handled with care
(only when section is overstained)
distilled water 5 x 1 min
Slides are dehydrated in ascending series of ethanol and mounted under coverglass
Carmine, aluminium sulfate (Fyg)
Chemicals Chemical solution
Carminic acid (C.I. 75470) FYG carmine dye solution:
Aluminum sulfate 6.0 g aluminium sulfate dissolved in
100 mL distilled water (heated), add
HCl (25%) 1.0 g carmine
Ethanol
the mixture is boiled for 15 min, then
Distilled water cooled down and filtered
HCl-ethanol solution:
97.5 mL 70% ethanol plus
2.5 mL HCl (25%)
Staining procedure
Immuno-stained sections are passed through distilled water and stained:
FYG carmine dye solution 1-6 hours
distilled water 5 x 1 min
differentiate in HCl-ethanol solution under microscopic control
(only when section is overstained)
distilled water 5 x 1 min
Slides are dehydrated in ascending series of ethanol and mounted under coverglass
Carmine, ammonium alum (Grenacher)
Chemicals Chemical solution
Carminic acid (C.I. 75470) GRENACHER carmine dye solution:
Aluminium ammonium disulfate (ammonium 3.0-5.0 g ammonium alum (or potassium
alum) alum) dissolved in
alternatively aluminium potassium sulfate 100 mL distilled water (heated), add
(potassium alum) 2.0 g carmine
Formaldehyde (37%) the mixture is boiled for 60 min, then
Ethanol cooled down and filtered
Distilled water add 1.0 mL formaldehyde
Staining procedure
Immuno-stained sections are passed through distilled water and stained:
GRENACHER carmine dye solution 1-6 hours
distilled water several rinses
as long as dye veils are seen
differentiate in HCl-ethanol solution under microscopic control
(only when section is overstained)
96% ethanol 2 x 1 min
Slides are dehydrated in ascending series of ethanol and mounted under coverglass
Carmine, acetic acid (Schneider)
Chemicals Chemical solution
Carminic acid (C.I. 75470) SCHNEIDER carmine dye solution:
Glacial acetic acid (carmine acetic acid)
55.0 mL distilled water plus
Ethanol 45.0 mL glacial acetic acid plus
Distilled water 1.0 g carmine
the mixture is heated to boil weakly for
about 30-60 min using a reverse cooling
system
when cooled down the stain is filtered
Staining procedure
Immuno-stained sections are passed through distilled water and stained by covering the section
with drops of the dye solution:
SCHNEIDER carmine dye solution 1-2 min
glass slide is slightly heated by a Bunsen flame
from below and then transferred to the cold
distilled water rinse (2 x)
96% ethanol 2 x 1 min
Slides are dehydrated in absolute ethanol and mounted under coverglass
Carmine, alum (Mayer)
Chemicals Chemical solution
Carminic acid (C.I. 75470) MAYER carmalum dye solution:
Potassium aluminium sulfate (potassium alum) 10.0 g potassium alum dissolved in
200.0 mL distilled water (heated) plus
Formaldehyde (37%) 1.0 g carmine
Ethanol
the mixture is cooled down and filtered
Distilled water
add 1.0 mL 10% formaldehyde solution
potassium alum solution:
0.5 g potassium alum dissolved in
100.0 mL distilled water
Staining procedure
Immuno-stained sections are passed through distilled water and stained:
MAYER carmalum dye solution 15 min
distilled water 2 x 1 min
differentiate in potassium alum solution 1-2 min
distilled water 5 x 1 min
96% ethanol 2 x 1 min
Slides are dehydrated in absolute ethanol and mounted under coverglass
Carmine, aluminium chloride - calcium chloride (Mayer)
Chemicals Chemical solution
Carminic acid (C.I. 75470) MAYER paracarmine dye solution:
Aluminium chloride 0.5 g aluminium chloride dissolved in
100.0 mL 70% ethanol (heated) plus
Calcium chloride 4.0 g calcium chloride plus
Glacial acetic acid 1.0 g carmine
Ethanol the mixture is cooled down and filtered
Distilled water ethanol-acetic acid solution:
98 mL 70% ethanol plus
2.0 mL glacial acetic acid
Staining procedure
Immuno-stained sections are passed through distilled water and stained:
MAYER paracarmine dye solution 15-30 min
differentiate in ethanol-acetic acid under microscopic control
70% ethanol rinse
70% ethanol 3 x 1 min
96% ethanol 3 x 1 min
Slides are dehydrated in absolute ethanol and mounted under coverglass
Carmine, lithium carbonate (Orth)
Chemicals Chemical solution
Carminic acid (C.I. 75470) Lithium carbonate stock solution:
Lithium carbonate add as much lithium carbonate to distilled
water to give an aqueous saturated solution
Ethanol
ORTH lithium carmine dye solution:
HCl (25%) 2.5 g carmine added to
Distilled water 100.0 mL lithium carbonate stock
mixture is heated in a boiling water bath
(for 10-15 min)
solution is then cooled down and filtered
prior to use
HCl-ethanol solution:
97.5 mL 70% ethanol plus
2.5 mL HCl (25%)
Staining procedure
Immuno-stained sections are passed through distilled water and stained:
ORTH lithium carmine dye solution 2-5 min
differentiate in HCl-ethanol solution under microscopic control
distilled water 5 x 1 min
70% ethanol rinse
70% ethanol 3 x 1 min
96% ethanol 3 x 1 min
Slides are dehydrated in absolute ethanol and mounted under coverglass
References for further readings
Goeppert HR and Cohn F (1849)
Corti A (1851)
Hartig T (1854a, 1854b)
Gierke H (1884)
Mayer P (1887)
Mayer P (1892)
Smith GM (1915)
Romeis B (1968)
Eisner A (2007)
Llewellyn BD (2007)
© Prof. Dr. Wolf D. Kuhlmann, Heidelberg 10.09.2006
