Biological Stains

Posted by Tylor Keller on November 16th, 2018

Since biological samples are mainly composed of elements such as carbon, hydrogen, oxygen, nitrogen and so on. These elements have a relatively low refractive index to light, and their atomic number is low, and their ability to scatter electrons is also weak. Therefore, it is necessary to perform staining to enhance the enhance contrast in the microscopic image. Stains and dyes are frequently used to highlight structures in biological tissues for viewing, often with the aid of different microscopes. 

1 Structure of Biological stains

The dye used in microscopic technology, also known as biological stains, has an ever-changing structure. As a biological stain, two conditions must be met: one is to have color; the other is to have affinity with the dyed tissue. The color of the dye and its affinity with the tissue are determined by the molecular structure of the dye itself, so the dye used in microscopy contains two basic groups no matter how the structure changes: ①chromophore ②auxochrome. The color-forming chromophore group and the auxochrome group that produces affinity with the tissue determine the dyeing properties of the dye. 

1.1 Chromophore

The role of chromophores is to impart color to the dyes because they absorb light of a certain wavelength. Common chromophores include (-NO2), (-N=N-), (=C=C=), (=C=O), (-N=O). Some dyes have only one chromophore, but some dyes have more than one chromophore, and that’s why they have a darker color.

1.2 Auxochrome

The role of auxochrome is to give the dye an affinity for the tissue so that the dye can bind firmly to the tissue. The reason for the affinity is that due to the presence of these groups, the dye can be ionized in solution, giving the dye positive and negative charges, and thus the dye can be combined with the oppositely charged portion of the tissue. Auxochrome groups include acid groups such as -OH, -SO3H -COOH and basic groups such as -NH2, -NHCH3, -N(CH3)2. Colored substances without a auxochrome can not be called dyes.

2 Classification of biological stains

2.1 Artificial dyes and Natural dyes

Biological stains can be divided into artificial dyes and natural dyes according to their different sources. Biological stains are all organic compounds. Artificial dyes are extracted from aniline or coal tar or are artificially synthesized such as gentian violet, eosin, solid green, etc. Natural dyes are extracted from the body of plants and animals. For example, magenta is extracted from the female body of Cochineal; sarranine is extracted from saffron; haematoxylin is extracted from Sappan wood (by using ether).

2.2 Acid dyes and Basic dyes

Dyes having an acidic or basic group in the auxochrome are referred to as acidic or basic dyes, respectively. Chromatin is easily stained dark by basic dyes. In the experiment, an alkaline dye such as gentian violet solution or acetic acid magenta solution is used for dyeing chromatin. These stains are prepared by dissolving gentian violet or magenta in an acetic acid solution, and the prepared gentian violet solution has a pH of less than 7. But why is the gentian violet solution called an alkaline stain? In fact, the definition of the acid dye and the basic dye is not determined by the pH of the dye solution, but is determined by the charge carried by the auxochrome in the dye after ionization. In general, if the auxochrome in the dye has a positive charge, the dye is a basic dye, and if the auxochrome group has a negative charge, the dye is an acid dye. For example, Eosin Y contains a auxochrome -COOH that emits hydrogen ions when ionized in water, so Eosin Y has negative charge. When it is formulated into an Eosin Y dye, it reacts with a strong base NaOH to form a salt (-COONa).