ORCEIN: FOUNDATION AND PREPARATION - CHEMISTRY - 2025

The orceína is a natural colored purple, extracted from various lichens as orchilla or archill, fruticose lichen Rocella tinctoria and Lecanora parella mainly. The Rocella tinctoria is found in the African and American continent, although it can be replaced by other species located in Europe.

This stain has been widely used in cytogenetic studies since ancient times. In this sense, the orcein preparation that includes acetic acid was introduced by LaCourt in 1941, which is why it was initially called the LaCourt technique.

Chemical structure of orcein and orcein solution. Source: Wikipedia.org/ Oguenther at de.wikipedia (Dr. Guenther)

Likewise, due to its simplicity and low cost, it has been used for the manufacture of litmus (a stain extracted from lichens) and in histological studies (staining of cells and tissues).

Several studies have used this staining combined with other chemicals, such as ammonia picrocarminate and acetic acid, to stain elastic fibers and chromosomes respectively. At present, orcein continues to be used in the cytogenetic studies of animals and plants.

Recently, Silva et al. 2017 successfully used this stain to observe the various changes that occur in the elastic fibers of the skin of the thumb in Chilean older adults. This study revealed how the fibers of the thumb alter with aging.

In this sense, the elastic fibers were observed to be brownish-black in color in individuals older than 80 years; while in the youngest (from 60 years) they are seen as reddish-brown. The cytoplasm and other cell structures turn yellowish-green.

This allowed corroborating the presence and condition of the elastic fibers, in terms of their disposition, quantity and integrity. On the other hand, it has also been indicated that orcein is of great help in identifying aneurysms and diagnosing arteritis, among other conditions.

Basis

Orcein is part of the oxazine group of dyes, and the staining is based on its affinity for DNA. The dye binds to the negative charge of this molecule, represented by the phosphate group. That is why, the chromosomes are stained purple.

However, the rest of the cell's structures take on a different color. Specifically, in the case of elastic fibers, they are colored reddish brown, cell nuclei from blue to dark purple, while collagen does not stain.

For this reason, orcein is used to stain chromatin and differentiate sex in some species of flies. Likewise, nuclei and some liver inclusions produced by hepatitis B antigens can be stained.

Orcein solutions have specific functions in staining. Orcein A has the function of softening the cell membranes, causing their death and paralyzing the process of their division. Meanwhile, orcein B is responsible for completing the staining by its adhesion to the chromosomes.

Preparation

Aceto-orcein solution 1%

To prepare a 1% solution of aceto-orcein, 2 grams of orcein are dissolved in 45 ml of glacial acetic acid, preferably hot, and 55 ml of distilled water are added at room temperature.

Then, it is constantly mixed to homogenize the solution, to later let it rest until it cools. Finally, it is stored at 4ºC in dark jars. This preparation was used by Flores 2012, to observe the maturation phases of cow oocytes.

In this case, for example, the aceto-orcein solution is placed on the tissue previously fixed in acetic acid-ethanol for 24 hours. This staining process is carried out for 30 minutes and the tissue is subsequently discolored.

Likewise, it can be used in the identification of organelles in multicellular tissues. In this case, a smear of the buccal mucosa is stained with a drop of the aceto-orcein solution, the coverslip is placed on the slide and the nucleus of the cell with its substructures can be immediately observed.

Lacto-proponic solution orcein 1%

Additionally, orcein can be prepared with other chemicals such as lactic acid and propionic acid. To do this, one gram of orcein is dissolved in a premixed solution at room temperature of 23 ml of lactic acid and 23 ml of propionic acid; making up the volume with distilled water up to 100 ml.

With this lacto-propionic orcein solution it is possible to observe the different phases of meiotic division of plant cells. In this case, the pre-fixed and hydrolyzed sample is placed for 15 minutes in the lacto-propionic orcein solution and then the tissue is spread on microscope slides.

In Duque's 2016 study on polygenic chromosomes, he used a preparation of 2 grams of orcein diluted in acetic acid and 85% lactic acid, diluting with a 65% acetic acid solution. Finally, he looked at the chromosomes in the salivary glands of Drosophila melanogaster.

Orcein A and Orcein B

It is important to note that depending on the sample and what is to be evaluated, the appropriate orcein solution is prepared. Thus, for example, 1N hydrochloric acid is added to the orcein A solution to observe dividing cells.

While the orcein B solution is dissolved in 45% acetic acid. This technique is an abbreviated staining method and is generally used to supplement the staining of chromosomal structures.

Final thoughts

Since centuries past, lichens such as Lecanora, Parmelia and Umbilicaria can be found in countries of the American continent such as Argentina, which are great sources of orcein. Also European areas such as the Canary Islands, were at the time large producers of the orchilla.

This was exported to other cities of this same continent such as Genoa and countries such as England, while Holland monopolized the manufacture of litmus from the orchilla. Therefore, this lichen was later reduced until it was about to become extinct.

This led to the orchilla being replaced by other plants with dye properties. However, the emergence of synthetic dyes contributed to the termination of the trade in this orcein source lichen.

This was possible thanks to Cocq, who in 1812 described the synthesis of orcein, knowing then that it is synthesized by a phenolic compound called orcinol. However, despite this, natural orcein is still the preferred one today.

This may be because orcinol is a very strong-smelling compound, and its prolonged exposure could carry health risks, such as loss of ability to detect odors. These damages to the pituitary could go from temporary to permanent the greater the exposure.

References

1. Ortega L, García R, Morales C. «Application of laccase produced by fungi for degradation of microbiological dyes (orcein and crystal violet).» Young People in Science 2. 2015; 633-637. Available at: jovenesenlaciencia.ugto.mx.
2. Barcat J. Orceína and Elastic Fibers. Medicine (Buenos Aires) 2003; 63: 453-456. Available at: Medicinabuenosaires.com.
3. Silva J, Rojas M, Araya C, Villagra F. Histological Characteristics of the Flying Face Skin of the Thumb in Chilean Individuals with Dactylogram Fading. J. Morphol. 2017; 35 (3): 1107-1113. Available at: scielo.conicyt.
4. Orrillo M, Merideth B. "Potato reproductive biology and cytogenetics." International Potato Center (CIP). Technical manual. Available at: research.cip.cgiar.org/
5. Flores F. «Collection, culture and in vitro maturation of cow oocytes (bos taurus) in the Bolivian highlands.". National University of the Altiplano, Puno-Peru; 2012.Available at: repositorio.unap.edu.pe.
6. Duque C. Polytenic chromosomes: A look at the phenomenon of endoreduplication. National University of Colombia, Medellín; 2016. Available at: edu.
7. Camarena F. Cellular and Molecular Biology. Practice manual. Autonomous University of Baja California. 2017.Available at: pez.ens.uabc.mx.
8. FAO / IAEA. 2018. Manual to differentiate wild and reared Anastrepha ludens (Loew) flies from normal strain (“bi-sexual”) and genetically sexed strain (Tapachula-7), irradiated and unirradiated. Guillen Aguilar JC, López Muñoz L, López Villalobos EF and Soto García DN Food and Agriculture Organization of the United Nations. Rome, Italy, 95 pp.
9. Orcein. (2018, November 30). Wikipedia, The Free Encyclopedia. Consultation date: 03:38, July 31, 2019 es.wikipedia.org.
10. Merck Millipore. (2018, July 16). Orcein microscopy for Certistain microscopy. Available at: merckmillipore.com