Carmine, also known as cochineal red, carmine red, or magenta, is a water-soluble synthetic pigment. It presents a bright yellowish red color and belongs to the monochromatic pigment category. It can be used for coloring food, beverages, pharmaceuticals, cosmetics, feed, tobacco, toys, food packaging materials, and other products. In water, it appears as a deep red color; in acidic solutions, it turns from yellow to purple. It is soluble in water, glycerol, and concentrated sulfuric acid, slightly soluble in ether and ethanol, and almost insoluble in petroleum ether, benzene, chloroform, and oils.
Natural carmine is derived from dried female cochineal insects native to tropical regions. The insects are ground into powder, from which carmine is extracted, and then treated with alum to remove impurities, yielding the final carmine product. Pure carmine cannot act as a stain on its own; it must first be dissolved in an acidic or alkaline solution to become functional. Commonly used acidic solutions include glacial acetic acid or picric acid, while common alkaline solutions include ammonia water and borax. Carmine is an excellent nuclear stain, and specimens stained with it are resistant to fading. It can be used for staining sections or tissue blocks, and is particularly suitable for whole-mount staining of small specimens.
Carmine Hydrochloride Staining Solution (4%), also called carmine hydrochloride stain or hydrochloric acid carmine stain, is composed of carmine, hydrochloric acid, alcohol, and deionized water, with an acidic pH. This staining solution is mainly used for the staining and observation of parasitic samples excluding large specimens, and can also be applied to the study of plant chromosomes. This reagent is intended for research use only and is not suitable for clinical diagnosis or any other purposes.
Materials to Be Prepared by User:
1. Glass slides, cover slips, incubator, optical microscope
2. Carnoy's fixative, 45% acetic acid, 1% hydrochloric acid-ethanol differentiation solution, graded ethanol series, xylene, neutral balsam, etc.
Operating Procedures (For Reference Only):
(I) For Plant Chromosome Staining
1. Sample Fixation: Plant root tips, young leaves, buds, shoot tips, tender ovaries, and anthers can be used as samples. Anthers require no pre-treatment. For materials intended for karyotype analysis of metaphase chromosomes during mitosis, routine pre-treatment is necessary. The treatment duration for ovaries should be 1–2 hours longer than that for root tips; alternatively, the concentrations of colchicine and 8-hydroxyquinoline solutions used can be doubled while keeping the treatment duration unchanged. Pre-treated materials should be fixed in Carnoy's fixative for more than 24 hours.
2. Washing: Rinse the materials 2–3 times with 70% ethanol, with each rinse lasting over 1 hour.
3. Staining: Blot the surface of the materials dry quickly with filter paper and immerse them in carmine hydrochloride. The volume of the staining solution should generally be more than 3 times that of the materials, and the staining duration depends on the material type. The staining time for anthers is 12–24 hours, while for other materials such as ovaries and young leaves, it is 2–6 days. To shorten the staining time, the samples can be placed in an incubator at 60°C for 1–3 days of staining.
4. Post-staining Washing: Well-stained materials appear purplish-red on the surface. Rinse the stained materials 2–3 times with 70% ethanol, 10 minutes each time. The washed materials can be preserved in 70% ethanol. The staining solution can be reused several times.
5. Softening and Differentiation: Soften the materials in 45% acetic acid at 60°C for 10–30 minutes, with the exact duration depending on the material. After softening, differentiate the materials in fresh 45% acetic acid over an alcohol lamp for 5–20 seconds.
6. Squashing: Place the softened and differentiated materials on a glass slide, add one drop of 45% acetic acid, perform squashing following standard procedures, and examine under a microscope.
7. Permanent Slide Preparation: Carry out freeze peeling, air drying, or low-temperature drying in an incubator, then mount the slide with neutral balsam for permanent preservation.
(II) For Parasite Sample Staining
1. Sample Washing: Parasite samples excluding large specimens can be used. Remove the parasites from the preservation solution and rinse them 2–3 times with 70% ethanol, with each rinse lasting over 1 hour.
2. Staining: Blot the surface of the parasites dry quickly with filter paper and immerse them in carmine hydrochloride. The volume of the staining solution should generally be more than 3 times that of the samples. The staining duration depends on the sample type, usually 6–12 hours. To shorten the staining time, the samples can be incubated at 60°C.
3. Differentiation: Take out the parasites, differentiate them rapidly in 1% hydrochloric acid-ethanol for 10–30 seconds, then continue differentiation in 70% ethanol for 2–3 times, 10 minutes each time. The staining solution can be reused several times.
4. Dehydration and Clearing: Dehydrate the samples sequentially in 80% ethanol for 2–12 hours and 95% ethanol for 2–12 hours (duration varies with sample size). Then transfer the samples successively to fresh 95% ethanol for 1 hour, absolute ethanol twice for 1 hour each time, and xylene twice for 10 minutes each time. Finally, mount the slide with neutral balsam for microscopic observation.
Precautions:
1. For materials intended for metaphase mitosis karyotype analysis, young leaves, shoot tips, anther walls, and ovaries are all applicable, with ovaries being the optimal choice.
2. After the materials are fixed, it is essential to thoroughly rinse off the fixative with 70% ethanol.
3. This reagent contains a high concentration of ethanol, which is highly volatile. In addition, the staining process requires a relatively long time, so it is advisable to perform sealed staining using slide boxes or covered staining jars.
4. Store the reagent in an airtight container; otherwise, the staining efficiency will decrease.
5. For your safety and health, wear a lab coat and disposable gloves during the operation.
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