The history, chemistry and modes of action of carmine and related dyes

Carmine has been used in biological staining to demonstrate selectively nuclei, chromosomes or mucins, depending on the formulation. Throughout its history in science, complaints and frustrations have been expressed about dye quality. Inconsistencies in dye quality or identity have prevented thorough understanding of staining mechanisms and have caused many stain solutions to behave unsatisfactorily. The aim of this review is to (1) detail causes of these problems, which are rooted in history, geography and production, (2) offer ways to minimize problems and (3) provide modern explanations for stain behavior. Carmine is a “semi-synthetic” dye, i.e., a complex of aluminum and the natural dye cochineal (carminic acid). Carmine shows considerable batch-to-batch variability. Geography, politics, history, agricultural practices and iconography all contribute to the variability of cochineal. In addition, widely divergent manufacturing methods are used to produce carmine. Also, confusion in terminology has led to mislabeling. Pressure from the food industry for a more satisfactory colorant for acidic foods led to the introduction of a new dye, aminocarminic acid, which could enter the biological market inadvertantly. Improved methods of analysis should help the certification process by the Biological Stain Commission. Further standardization could be achieved by replacing most of the methods of solubilizing carmine. The majority of these methods use heat, which is likely to damage the dye molecule. Fortunately, carmine is readily dissolved by raising the pH of the aqueous solvent above 12, and a new form of the dye, now available commercially, is soluble in water without the need for heat or pH adjustment. Chemical structures and physical properties of carminic acid, carmine, aminocarminic acid and kermesic acid are reviewed. A new configuration for carmine is proposed, as well as possible changes to carminic acid and carmine molecules as a result of decomposition caused by heating. Each of the major classes of carmine-based stains is described as are possible mechanisms of attachment to specific substrates. Glycogen binds carmine through hydrogen bonding, and it is here that carmine decomposed by heat could have the greatest detrimental impact. Nuclei and chromosomes are stained via coordination bonds, perhaps supplemented by hydrogen bonds. Finally, acidic mucins react ionically with carmine. Specificity in the latter case may be due to unique polymeric carmine molecules that form in the presence of aluminum chloride.     

A method for determining identity and relative purity of carmine, carminic acid and aminocarminic acid

Carmine is one of the few dyes currently certified by the Biological Stain Commission that is not assayed for dye content. Existing assay methods are complex and do not differentiate the three cochineal derivatives carmine, carminic acid and aminocarminic acid. The latter dye is relatively new to the food trade as an acid-stable red colorant and may eventually enter the biological stains market. The assay proposed here is a two-step procedure using quantitative spectrophotometric analysis at high pH (12.5-12.6) followed by a qualitative scan of a low pH (1.90-2.10) solution. Carmine is distinct at high pH, and the remaining dyes are easily distinguished at low pH. Four instances of mislabeling are documented from 18 commercial products, but the mislabeled dyes were not certified dyes. Samples from nearly all lots of carmine certified by the Biological Stain Commission from 1920 to 2004 proved to be carmine, but they varied widely in dye content. Batches from 1920 through the 1940s were significantly richer in dye content. Variability has been extreme since 2000, and most of the poorest lots have been submitted since 1990.     

A method for determining identity and relative purity of carmine,carminic acid and aminocarminic acid

Carmine is one of the few dyes currently certified by the Biological Stain Commission that is not assayed for dye content. Existing assay methods are complex and do not differentiate the three cochineal derivatives carmine, carminic acid and aminocarminic acid. The latter dye is relatively new to the food trade as an acid-stable red colorant and may eventually enter the biological stains market. The assay proposed here is a two-step procedure using quantitative spectrophotometric analysis at high pH (12.5–12.6) followed by a qualitative scan of a low pH (1.90–2.10) solution. Carmine is distinct at high pH, and the remaining dyes are easily distinguished at low pH. Four instances of mislabeling are documented from 18 commercial products, but the mislabeled dyes were not certified dyes. Samples from nearly all lots of carmine certified by the Biological Stain Commission from 1920 to 2004 proved to be carmine, but they varied widely in dye content. Batches from 1920 through the 1940s were significantly richer in dye content. Variability has been extreme since 2000, and most of the poorest lots have been submitted since 1990.     

A method for determining identity and relative purity of carmine, carminic acid and aminocarminic acid.

Carmine is one of the few dyes currently certified by the Biological Stain Commission that is not assayed for dye content. Existing assay methods are complex and do not differentiate the three cochineal derivatives carmine, carminic acid and aminocarminic acid. The latter dye is relatively new to the food trade as an acid-stable red colorant and may eventually enter the biological stains market. The assay proposed here is a two-step procedure using quantitative spectrophotometric analysis at high pH (12.5-12.6) followed by a qualitative scan of a low pH (1.90-2.10) solution. Carmine is distinct at high pH, and the remaining dyes are easily distinguished at low pH. Four instances of mislabeling are documented from 18 commercial products, but the mislabeled dyes were not certified dyes. Samples from nearly all lots of carmine certified by the Biological Stain Commission from 1920 to 2004 proved to be carmine, but they varied widely in dye content. Batches from 1920 through the 1940s were significantly richer in dye content. Variability has been extreme since 2000, and most of the poorest lots have been submitted since 1990.     

The red insect dyes: carminic,kermesic and laccaic acids and their derivatives

Three groups of insect dyes are described: three cochineal dyes, the kermes dye and the lac dye. The major color components are carminic acid, kermesic acid and laccaic acids, respectively. These dyes are red anthraquinone derivatives. The chemical structures are described. All of these dyes have extensive histories that are related briefly; however, only American cochineal is of commercial importance today. Two manufactured derivatives of cochineal, carmine and acid-stable carmine (4-aminocarminic acid) are described in some detail including the chemical identity, toxicity, stability, and staining and non-staining applications.     

Revised procedures for the certification of carmine (C.I. 75470, Natural red 4) as a biological stain

Carmine is one of the original dyes certified by the Biological Stain Commission (BSC). Until now it has lacked both an assay procedure for dye content and a means to positively identify the dye. The methods for testing carmine in the laboratory of the BSC have been revised to include spectrophotometric examination at pH 12.5-12.6 to determine that the dye is carmine (λ_max=530-335 nm). The maximum absorbance of a solution containing 100 mg of dye per liter of water, adjusted to pH 12.5-12.6, which provides a relative measure of dye content, should lie in the range 1.2 to 1.8. If the dye is not carmine, spectrophotometry at pH 1.9-2.1 shows whether it is carminic acid (λ_max=490-500 nm) or 4-aminocarminic acid (λ_max=525-530 nm). The latter two dyes, which are also called carmine when sold as food colorants, have physical properties different from those of true carmine. The functional tests for carmine as a biological stain are Orth's lithium-carmine method for nuclei, Southgate's mucicarmine method for mucus, and Best's carmine method for glycogen.     

The staining of Brunner's gland and other neutral mucins by carmine, hematoxylin and orcein in alkaline solutions.

Brunner's glands and other neutral mucins may be stained red, brownish red, and violet, respectively, by carmine, hematoxylin, and orcein from appropriate alkaline solutions. Carmine and hematoxylin in concentrations of 0.2-1% are dissolved in 60-70% alcohol containing 1% potassium carbonate; orcein is used in a 0.2% alcoholic solution of sodium hydroxide. Staining times are 15 to 30 minutes. The stained sections are rinsed in 95% or absolute alcohol prior to xylene and mounting. The staining of these mucins is blocked by mild bromine oxidation. By using alcian blue 0.1% in 3% acetic acid for 5 minutes prior to the above stains, mucins may be characterized in the same preparation as acid, neutral or mixed.     

The Staining of Brunner's Gland and Other Neutral Mucins by Carmine,Hematoxylin and Orcein in Alkaline Solutions

Brunner's glands and other neutral mucins may be stained red, brownish red, and violet, respectively, by carmine, hematoxylin, and orcein from appropriate alkaline solutions. Carmine and hematoxylin in concentrations of 0.2-1% are dissolved in 60-70% alcohol containing 1% potassium carbonate; orein is used in a 0.2% alcoholic solution of sodium hydroxide. Staining times are 15 to 30 minutes. The stained sections are rinsed in 95% or absolute alcohol prior to xylene and mounting. The staining of these mucins is blocked by mild bromine oxidation. By using alcian blue 0.1% in 3% acetic acid for 5 minutes prior to the above stains, mucins may be characterized in the same preparation as acid, neutral or mixed.     

The Staining of Brunner's Gland and Other Neutral Mucins by Carmine, Hematoxylin and Orcein in Alkaline Solutions

Brunner's glands and other neutral mucins may be stained red, brownish red, and violet, respectively, by carmine, hematoxylin, and orcein from appropriate alkaline solutions. Carmine and hematoxylin in concentrations of 0.2-1% are dissolved in 60-70% alcohol containing 1% potassium carbonate; orein is used in a 0.2% alcoholic solution of sodium hydroxide. Staining times are 15 to 30 minutes. The stained sections are rinsed in 95% or absolute alcohol prior to xylene and mounting. The staining of these mucins is blocked by mild bromine oxidation. By using alcian blue 0.1% in 3% acetic acid for 5 minutes prior to the above stains, mucins may be characterized in the same preparation as acid, neutral or mixed.     

Iron and aluminum lakes of Gallo blue E as nuclear and metachromatic mucin stains.

Gallo blue E, C. I. No. 51040, Mordant Violet 54, furnishes a blue black nuclear stain when applied to tissue sections in the form of its moderately stable iron lakes. This coloring combined well with such counterstains as orange G and eosin B. The Van Gieson stain tends to decolorize mucins, cartilage, and mast cells previously stained with this dye. Its aluminum lake solutions tend to gel in a few minutes to 24 hours depending on the solvent used and the amount of Al3+ present. Aluminum lake solutions give a moderately good blue to dark blue nuclear stain and a brilliant purplish red to dark purple stain to a variety of epithelial and connective tissue mucins. Acid dye counterstains are poorly tolerated. With either lake, nuclear staining is abolished by deoxyribonuclease digestion or relatively short mineral acid extraction of DNA.     

Iron and Aluminum Lakes of Gallo Blue E As Nuclear and Metachromatic Mucin Stains

Gallo blue E, C. I. No. 51040, Mordant Violet 54, furnishes a blue black nuclear stain when applied to tissue sections in the form of its moderately stable iron lakes. This adoring combined well with such counterstains as orange G and eosin B. The Van Gieson stain tends to decolorize mucins, cartilage, and mast ells previously stained with this dye. Its aluminum lake solutions tend to gel in a few minutes to 24 hours depending on the solvent wed and the amount of Al²⁺ present. Aluminum lake solutions give a moderately good blue to dark blue nuclear stain and a brilliant purplish red to dark purple stain to a variety of epithelial and connective tissue mucins. Acid dye counterstains are poorly tolerated. With either lake, nuclear staining is abolished by deoxyribonuclease digestion or relatively short mineral acid extraction of DNA.     

America's red gold: multiple lineages of cultivated cochineal in Mexico

Cultivated cochineal (Dactylopius coccus) produces carminic acid, a valuable red dye used to color textiles, cosmetics, and food. Extant native D. coccus is largely restricted to two populations in the Mexican and the Andean highlands, although the insect's ultimate center of domestication remains unclear. Moreover, due to Mexican D. coccus cultivation's near demise during the 19th century, the genetic diversity of current cochineal stock is unknown. Through genomic sequencing, we identified two divergent D. coccus populations in highland Mexico: one unique to Mexico and another that was more closely related to extant Andean cochineal. Relic diversity is preserved in the crops of small‐scale Mexican cochineal farmers. Conversely, larger‐scale commercial producers are cultivating the Andean‐like cochineal, which may reflect clandestine 20th century importation.     

On the structure of carminic acid and carmine

Summary The chemical mechanism and histochemical significance of carmine stains are not yet understood. To determine possible effects of dye configuration on staining patterns we built models of dye molecules with the Stuart-Briegleb-type of atomic models. However, steric hindrance prevented construction of carmine according to the formula suggested by Harms. A review of recent chemical literature showed that the widely accepted formula of carminic acid is incorrect; the carboxyl group is not in the 5 but in the 7-position, and the side-chain is not a methylpentose but a hexose. Models based on the revised structural formula could be combined to 211 carminic acid-Al-Ca complexes. But formation of the central Al-O-Ca-O-Al bridge of the conventional 421 carminic acid-Al-Ca formula of carmine was still impossible. It is suggested that carmine may be a 211 compound analogous to the 211 alizarin-Al-Ca complex established by Kiel and Heertjes. Investigations of carmine were rendered difficult by wide variations in the staining properties of dye samples and the lack of data concerning the composition of various batches of carmine.     

Use of Dactylopius coccus Costa (Hemiptera: Coccoidea) Pigments in the Detection of Larvae and Pupae of Plutella xylostella (L.) (Lepidoptera: Yponomeutidae)

Carmines obtained from the dye of Dactylopius coccus Costa (Hemiptera: Coccoidea) were used for the detection of larvae and pupae of Plutella xylostella (L.) (Lepidoptera: Yponomeutidae) in broccoli inflorescences. Larvae were dyed with carmine II and red cochineal, while the majority of the dyes, with the exception of carmine III and the aqueous extract, were suitable to dye pupae. In the broccoli lumps exposed to the dyes, only the verge of the stems were actually dyed, right in the position where the incision took place, an appropriate characteristic for implementing this technique for commercial use.     

Detection of Indigo Carmine dye in juices via application of photoluminescent europium-doped carbon dots from tannic acid

Indigo Carmine is a hazardous dye and produces an allergic action for humans despite the excessive use of the dye in several industrial fields. A sensitive and simple fluorescent assay for determining Indigo Carmine relying on quenching of the fluorescent europium-doped carbon dots by the action of inner filter effect was developed. This sensing platform involved the preparation of europium-doped carbon dots from the hydrothermal carbonization of tannic acid and europium chloride, which was used as fluorescent reagent with a distinctive excitation/emission wavelength at 307/340 nm. Both excitation and emission fluorescence of prepared carbon dots can be successfully quenched by adding Indigo Carmine dye. The developed spectrofluorimetric method exhibits good linearity with the concentration of Indigo Carmine dye in the range of 1.5 to 10.0 μg/ml and provided a limit of detection (LOD) value of 0.40 μg/ml. Furthermore, the prepared carbon nanoparticles were identified and characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier-transform infrared (FTIR), and ultraviolet (UV)-spectrophotometer techniques. In addition, the developed detecting approach was applied to determine Indigo Carmine in juice samples with acceptable recovery.     

Importance of acidic mucin secretions by foveolar and mucous neck cells of rat fundic mucosa as the defence mechanisms against HCl as revealed by fasting.

The localization of neutral mucin and acidic mucins in both control and fasted rat gastric fundic mucosa were examined by microscopic and electron microscopic histochemical methods. By Carnoy's fixation, the surface mucous coat of the control rat gastric fundic mucosa was found to be composed of alternating layers of acidic mucins and neutral mucin, indicating the synchronous and cyclic secretions of them. In many gastric pits of the fundic glands, the acidic mucins were found to spring out from the deep foveolar regions like volcanoes. This phenomenon may suggest that the acidic mucins play a fundamental role in protecting the pit cells against HCl during its passage, and the layers of neutral mucin and acidic mucins in the surface coat is the safeguard against the HCl and digestive enzymes in the gastric lumen. In the fasting rat gastric fundic mucosa, the acidity and the amount of the gastric juice were markedly decreased, indicating the suppressed secretions of mucins and HCl. The decreased production of sulfomucin was directly demonstrated by 35SO4-autoradiography. Many mucous neck cells existing in close association with the parietal cells were ballooned due to accumulation of alcian blue (AB)-positive but high iron-diamine (HID)-negative sialomucin, which was not demonstrable in the control. The secretory granules of sialomucin contained in the ballooned mucous neck cells were positively stained ultrastructurally with cacodylate-ferric colloid to stain acid mucopolysaccharides.     

A Mordanting Fixation for Intense Staining of Small Chromosomes

A combination iron-mordant fixative in which propionic acid is substituted for acetic acid has been found useful in preparing small plant chromosomes for carmine stained squashes. Propionic acid is better than acetic acid because it holds more iron in stable solution. The fixative is a 3:1 mixture of 95% alcohol and pure propionic acid which contains 400 mg. of Fe(OH)₃ per 100 ml. of propionic acid. The latter is previously prepared by dissolving the dry freshly prepared Fe(OH)₃ in it. To each 10 ml. vial of fixative is added a few drops of carmine stain. Standard aceto-carmine squashes of material fixed in this mixture show quick intense staining and are especially useful for differentiated chromosomes at mitotic prophase.     

Alcoholic Hydrochloric Acid-Carmine as a Stain for Chromosomes in Squash Preparations

A regressive bulk carmine staining schedule was adapted from a formula proposed by P. Mayer. The stain is made by boiling gently 4 gm of certified carmine in 15 ml of distilled water to which 1 ml of concentrated HC1 has been added. After cooling, 95 ml of 85% alcohol is added, and the solution filtered. Fixed tissue is soaked in the stain until thoroughly penetrated; squashes are then prepared as usual, but plain 45% acetic acid is used as the temporary mounting medium instead of aceto-carmine. The advantages of this method are: intense, precise staining of chromosomes coupled with a lightly stained cytoplasm; consistency and uniformity of results; simplicity of use.     

Decolorization and biodegradation of Indigo carmine by a textile soil isolate Paenibacillus larvae

The potential of recently isolated bacteria Paenibacillus larvae for the effective decolorization of Indigo carmine was evaluated. The effects of operational parameters (temperature, pH, dye concentration, shaking/non shaking) were tested. Maximum extent of decolorization was observed when the medium was incorporated with 10 g/l of yeast extract and peptone. Decolorization was strongly inhibited at non-shaken conditions as well as incorporation of inorganic sources (sodium nitrite and ammonium chloride) in the medium. Maximum decolorization was observed at 30°C (100%) and 40°C (92%) at 8 h of incubation. The LC-MS and NMR analysis confirms the oxidation of Indigo carmine .The primary degradation products were found to be Isatin sulfonic acid and anthranilicacid.     

A Mordanting Fixation for Intense Staining of Small Chromosomes

A combination iron-mordant fixative in which propionic acid is substituted for acetic acid has been found useful in preparing small plant chromosomes for carmine stained squashes. Propionic acid is better than acetic acid because it holds more iron in stable solution. The fixative is a 3:1 mixture of 95% alcohol and pure propionic acid which contains 400 mg. of Fe(OH)₃ per 100 ml. of propionic acid. The latter is previously prepared by dissolving the dry freshly prepared Fe(OH)₃ in it. To each 10 ml. vial of fixative is added a few drops of carmine stain. Standard aceto-carmine squashes of material fixed in this mixture show quick intense staining and are especially useful for differentiated chromosomes at mitotic prophase.