Types Of Stains In Biology

Types Of Stains In Biology – Staining is a technique that is used to enhance the visibility of microorganisms. It is often used in histology which is the microscopic study of biological tissues and also in medical fields that focuses on the study and diagnoses of disease at a microscopic level. It is the process of dyeing microorganisms.

Stains in biology are used to define the biological tissues, cell population and the organelles within individual cells. It is also used to mark cells in flow cytometry and to flag proteins or nucleic acids in gel electrophoresis.


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Staining is not only limited to biological materials as it can also be used to study the structure of other materials like lamellar structures of semi-crystalline polymers.

In this article, we are going to be looking at some types of stains in biology. In a natural state, most of the cells and microorganisms that are observed under the microscope might lack color and this makes it difficult to identify them, detect important cellular structures and their special characteristics.

So with stains, it will be easily to see and identify these microorganisms.

Without further delay, let’s delve into the article proper…

Types Of Stains In Biology

We will be looking at some of the most common stains in biology.

  • Acid fuchsin
  • Acridine orange
  • Bismarck brown
  • Carmine
  • Coomassie blue
  • Cresyl violet
  • Crystal violet
  • Dapi
  • Eosin
  • Ethidium bromide
  • Haematoxylin
  • Hoechst stains
  • Iodine
  • Malachite green
  • Methyl green
  • Methylene blue
  • Neutral red
  • Nile blue
  • Nile red
  • Propidium iodide
  • Rhodamine
  • Safranine

Staining Techniques In Biology

We will be looking at some of the stains in biology used to make microorganisms more visible.

Gram Staining

This is used to determine gram status to classify bacteria based on the composition of their cell wall.  It is more like a differential stain as it allows biologist to gather additional information about the bacteria they are working with.

The Gram stains use more than one stain and the cells will have a different appearance based on their chemical or structural properties.

It was first developed by Danish microbiologist Hans Christian Gram in 1884 as an effective method to distinguish between bacteria with different cell walls and it has grown to become one of the most frequently used staining techniques in biology.

With the knowledge of Gram reaction of a clinical isolate can help health care professional make a diagnosis and choose the appropriate antibiotic for treatment.

Let’s look at the Gram stain procedure:

  • First, crystal violet which is a primary stain is applied to a heat-fixed smear and it gives all the cells a purple colour.
  • Then Gram’s iodine which is a mordant is added. A mordant is a substance that is used to set or stabilize stains or dyes. So the Gram iodine acts like a trapping agent that complex with the primary colour (crystal violet) to make the crystal violet-iodine complex cluster and stay in thick layers of peptidoglycan in the cell walls.
  • At this stage, you add a decolourizing agent with is usually ethanol or an acetone/ethanol solution. The cells that have thick peptidoglycan layers in their cell walls are not always affected by the decolourizing agent as they retain crystal violet dye and remain purple. The decolourizing agent can easily wash the dye out of cells with thinner peptidoglycan layers, making them again colourless.
  • Finally, you add a secondary counterstain like safranine as it stains the decolourized cells pink although it might not be much noticeable in the cells that still contain the crystal violet dye.

The purple, crystal-violet stained cells are called the gram-positive cells and the red, safranine-dyed cells are gram-negative. With the gram staining, clinicians will be able to know gram-positive bacteria and gram-negative bacteria and know how to go about bacteria treatment.

Gram-negative bacteria are more resistant to certain antibiotics than gram-positive bacteria.

Capsule Staining

Some of the bacteria and yeast have a protective outer structure called a capsule and it is very important to determine whether cells in a sample have capsules since the presence of a capsule is related directly to a microbe’s virulence.

Capsules do not absorb most of the basic dyes and so a negative staining technique which is more like staining around the cells is used in capsule staining.

The dye stains the background but does not penetrate the capsules and so it will look like halos around the borders of the cell.

Acid-Fast Staining

This is another differential staining technique in biology that is used to differentiate two types of gram-positive cells: those that have waxy mycolic acids in their cell walls and those that do not have.

There are two different methods of acid-fast staining and they are the Kinyoun technique and the Ziehl-Neelsen technique.

Both of these methods use carbolfuchsin as the primary stain. The waxy, acid-fast cells retain the carbolfuchsin even after adding a decolourizing agent (ethanol solution).

A secondary counterstain like methylene blue is applied which turns non-acid-fast cells to blue.

The difference between the two acid-fast methods is if heat is used during the primary staining process. The Ziehl-Neelsen method uses heat to infuse the carbolfuchsin into the acid-fast cells but the Kenyan method does not use heat.

Both of these methods are important diagnostic tools because acid-fast bacteria are the causes of a large number of diseases. So if acid-fast bacteria are present in a tissue sample, their red or pink colour will be seen clearly against the blue background of the surrounding tissue cells.

Flagella Staining

These are tail-like cellular structures that are used for locomotion by some bacteria, eukaryotes and archaea. The flagella cannot be seen under a light microscope because they are so thin and so there is a need for a specialized flagella staining technique.

In the flagella staining, the flagella is thickened by first applying mordant (tannic acid) which coats the flagella and then the specimen is stained with a pararosani-line or basic fuchsin.

Clinicians do not really use this staining technique but microbiologist use it as the result would help them know the location and number of flagella which is useful in classifying and identifying bacteria in a sample.

Endospore Staining

Endospores are structures that are produced within certain cells that make it very difficult for them to die even in harsh conditions. The bacterial spores are difficult to stain as they are not permeable to aqueous dye reagents.

The endospore staining uses two stains to identify endospores in cells. The Schaeffer-Fulton method which is the most commonly used endospore staining technique uses heat to push the primary stain (malachite green) into the endospore. When you wash with water, the cell is decolourized but the endospore still remains green. Then the cell is counterstained with safranine which is pink and the result shows the shape and location of endospores if they are in the cell.

The green endospores will appear within the pink vegetative cells or separated from the pink cells and if no endospores are present, only the pink vegetative cells will be seen.

Final Note

We have talked about the types of stain in Biology and from all we have said, you would see that staining is an important technique in Biology to help in the study and diagnosis of microorganisms and diseases.

Through staining, scientists and biologists will be able to identify the microorganism, the type of disease and how to come up with treatment of the disease.

So all the types of stains in biology and the technique of staining have their functions and importance.




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