What is Staining?

Staining in biology is a technique to apply chemical substances or dyes to a biological specimen to enhance the visibility and contrast of its cellular constituents under the microscope. Staining is classified into two types. They are vital and non-vital. Vital staining involves the application of non-toxic water-soluble stains to the living specimen to study them live. Whereas, non-vital staining is typical for dead tissues. Some common examples of stains include methylene blue, orcein, carmine, etc.

What is Staining?

Staining is a technique in biology that enhances the visibility of specific components of a biological specimen under microscopes. This technique involves chemical substances called stains that usually interact with specific cellular components to impart colors. Staining can be broadly grouped as vital staining and non-vital staining. Vital staining is used to study living cells, whereas non-vital staining is used to study non-living cells. One of the most common staining techniques is Gram’s staining which is used to identify gram-negative and gram-positive bacteria.

Types of Staining

Based on the process, staining is grouped into three types: Simple staining, Differential staining, and Special staining.

Simple Staining

Simple staining is quick and is generally used for studying the morphology of cells using a single stain. In simple staining only single stain is used. Depending on the targeted component, simple staining can be classified into two types:

• Positive staining: This technique uses simple basic stains like safranin, crystal violet, etc. In this method the cells are stained and hence this technique is particularly useful in highlighting cell morphology, cellular arrangements, and specific organelles to understand the internal structures of microorganisms or cells.
• Negative Staining: This technique mostly uses acidic stains like India ink, nigrosin, etc. Acidic stains bear a negative charge and hence do not stain the cells due to repulsion from the cell wall. These stain color the background and create a contrast between the cell and the background.

Differential Staining

Differential staining involves multiple stains to distinguish between different types of microorganisms or structures within a specimen. Some examples include gram staining, acid fast staining, etc.

• Gram Staining: Gram staining categorizes bacteria into two groups based on their cell wall composition. This method involves staining with crystal violet followed by iodine treatment, alcohol wash, and counterstaining with safranin. Gram-positive bacteria retain the violet stain, appearing purple under a microscope, while Gram-negative bacteria take on the safranin counterstain and appear red.
• Acid-Fast Staining: Acid fast staining is a staining technique that is used to stain bacteria with waxy coating on cell walls. This method involves staining with carbol fuchsin, heat treatment, acid-alcohol wash, and counterstaining with methylene blue.

Special Staining

Some staining techniques utilize specific stains or procedures to highlight particular cellular structures, substances, or microorganisms. These techniques are used for detailed study of particular cellular component. Some of these staining methods are discussed below:

• Capsule Staining: Capsules are protective structure formed by bacteria. These are made up of polysaccaride chain and do not get stained easily. To detect them alternatively acidic and basic stains are applied. Acidic staining stains the background and basic stain stains the cell. The capsule being resistant to stains remain colourless and distinctly appear under microscope.
• Endospore Staining: This technique is applied to study the bacterial endospores. In this technique the bacterial smear is treated with malachite green followed by heat treatment that results in the penetration and binding of the stains to the endospore.
• Flagella Staining: Flagella are extremely thin structure and do not take up stain easily. They are treated with basic stains like carbol fuschin or fuschin. These stain color the thin flagella and help to determine their divisions and numbers on the cell.

Steps of Preparing a Stained Biological Specimen for Microscopic Study

Steps of staining biological specimens vary greatly depending upon the type of staining techniques being applied. Some common steps in most of the staining techniques are given below:

• Wet Mounting: Wet mounting is the first step in simple vital staining technique where living cells are to be studied. It this step the specimen under study is placed in a drop of water on a glass slide.
• Fixation: Fixation involves treating the specimen with fixatives like formaldehyde and ethanol. This kills the specimen and maintains its structure by preventing decay.
• Embedding: Some cells and tissues needs embedding in suitable medium like paraffin wax to make sectioning easier.
• Deparaffinization: If embedding medium is used, deparaffinization is done using suitable solvent to remove the embedding medium.
• Staining: This step involves application of the stain to the specimen. Sometimes modarnts are added with stains in order to stain the unstainable specimen. Based on the staining techniques the further steps vary. It may involve washing, heating etc, followed by application of counterstain or other substances.
• Mounting: After staining is completed the specimen is mounted onto the slide with a suitable medium. Then a cover slip is placed on it. After mounting the specimen is ready for microscopic studies.

Common Example of Biological Stains

The following are some of the common examples of biological stains:

• Acridine Orange: Acridine Orange is used in fluorescence microscopy for vital staining of live cells. It is a fluorescent dye composed of acridine and phenazine moieties. It emits orange fluoroscence when bound to DNA, making it useful for nucleic acid staining. Acridine Orange is used in fluorescence microscopy for vital staining of live cells.
• Eosin: Eosin is an acidic dye composed of brominated fluorescein. Eosin stains cytoplasm and extracellular structures in tissues, imparting a pink or red color. It is often used as a counterstain in combination with hematoxylin in H & E staining.
• Hematoxylin: Hematoxylin is a basic dye derived from the heartwood of certain trees and undergoes oxidation to form hematein. Hematoxylin stains nuclei blue or purple.
• Malachite Green: Malachite Green is a synthetic dye belonging to the triarylmethane class. Malachite green is used in staining bacterial endospore.
• Methylene Blue: Methylene Blue is a basic thiazine dye. It stains acidic structures in cells, such as nucleic acids.

FAQs on Staining

1. What do you mean by Staining?

Staining is a laboratory method that involves applying colored dyes to biological specimens to enhance their visibility under a microscope. This technique helps in studying and distinguishing cellular structures for research and diagnostic purposes.

2. What are the 3 types of Staining?

Three types of staining are: simple staining., differential staining, and special staining.

3. What is the Principle of Staining?

The principle of staining is the difference in charges of the dye and the cellular component. Opposite charges attract each other and like charges repel each other.

4. What is an Example of Stain?

Crystal voilel is an example of stain. Stains are chemical compound which bind to specific cellular constituents and make them visible under microscope. Some more examples of stains include fuschin, orcein, etc.

5. Why is Staining Important?

Staining is important because it enhances the visibility of complex microsopic structures under microscope. This helps to study the morphology and architecture of different cells and tissues.