Why Acetocarmine is Used in Mitotic Chromosome Studies

Different stains are used in the staining of different biological materials. Stains react with a specific substance in a biological sample, giving a specific colour to that substance. Acetocarmine is a stain used for the demonstration of nucleic acids within the chromosome. There are also some other reasons that make acetocarmine more suitable as a nucleic acid stain. They are discussed in this article.

Key Areas Covered

1. What is a Stain
     –  Definition, Types of Stains
2. What is Acetocarmine
     – Definition, Characteristics, Preparation
3. Why Acetocarmine is Used in Mitotic Chromosome Studies
     – Preparation, Use of Acetocarmine

Key Terms: Acetocarmine, Chromosomes, Formaldehyde, Hydrolysis, Mitotic chromosome Studies, Stains

What is Staining

Staining is a technique used in microscopic studies of biological samples to enhance the contrast of the biological sample under the microscope. It highlights the structures of biological tissue such as specific cell populations, organelles, DNA, proteins, carbohydrates, and lipids. The stain can be either synthetic chemicals or natural chemicals originated from plants or animals. The staining methods are described below.

1. Single staining – In single staining, only one stain is used to give a single colour to the whole biological sample.
2. Double staining – In double staining, two stains are used to stain specific organelles or a specific area.
3. Multiple staining – In multiple staining, more than two stains are used for the specific staining of components such as organelles in the sample.

What is Acetocarmine

Carmine is a basic dye prepared from an insect known as Coccus cacti. Acetocarmine is produced by mixing carmine with glacial acetic acid. It is a DNA-specific stain used for the visualization of super-coiled chromosomes during the different stages of mitosis.

Preparation of Acetocarmine Stain

1. Dissolve 10 g carmine in 1 L of 45% glacial acetic acid.
2. Add aluminum granules, and reflux for 24 h.
3. Filter into dark bottles and store at 4°C.
4. The staining can be intensified by adding ferric chloride (add 5 mL of a 10 % ferric chloride solution per 100 mL of % acetocarmine).

Why Acetocarmine Stain is Used in Mitotic Chromosome Studies

Generally, acetocarmine is a dye used in single staining. It stains both nucleus and cytoplasm. In order to stain chromosomes while keeping the cytoplasm colourless, the biological sample should be first treated with formaldehyde, and then, it can be hydrolyzed with HCl at 60 °C with the correct hydrolyzing time. Finally, it can be treated with acetocarmine. Acetocarmine produces large dye aggregates in weakly acidic conditions (pH 4-5). The demonstration of onion root mitosis by staining with acetocarmine is shown in figure 1.

Figure 1: Onion Root Mitosis

However, acetocarmine is lightly-toxic than the other nucleic acid stains such as aceto-orcein. It is also cheaper than other types of stains.

Conclusion

Stains are used in microscopic studies to enhance the contrast of specific biological components in a sample. Acetocarmine is such a stain used to stain nucleic acid inside cells. As acetocarmine specifically-stain chromosomes apart from the cytoplasm, it can be used to visualize chromosomes in mitotic studies.

Reference:

1.“ACETOCARMINE STAINING .” Kansas State University, Available here.
2.J. A. Rattenbury (2009) Specific Staining of Nucleolar Substance with Aceto-Carmine, Stain Technology, Available here.

Image Courtesy:

1. “Onion root mitosis” By staticd – Own work (CC BY-SA 3.0) via Commons Wikimedia