Staining Techniques: Definition, Types, and More

Staining is a fundamental practice in microbiology and histology that involves applying coloured dyes to specimens to enhance their visibility under the microscope. If you have ever wondered, “What is the staining technique?”, it is simply a method by which specific dyes bind to cellular structures, thereby making it easier to distinguish between different components such as the cell wall, nucleus, and cytoplasm.

Staining is crucial in identifying and classifying microorganisms, as well as in diagnosing diseases. It can also be extended beyond the biological realm to study crystalline polymers. In essence, staining techniques form the basis for many diagnostic procedures in laboratories worldwide.

How to Prepare Biological Specimens

The process of preparing specimens can vary depending on the staining techniques microbiology labs employ. Below are some key steps:

1. Wet Mounting

• Fresh, living specimens (like cells or microorganisms) are placed on a glass slide with a drop of water and then stained for immediate observation.

2. Fixation

• This step preserves the specimen’s structure.

• Heat Fixation: Uses gentle heat to kill and fix cells onto the slide.

• Chemical Fixation: Involves chemical agents (e.g. formaldehyde or ethanol) that form cross-links, stabilising the specimen.

3. Mordant Application

• A mordant enhances the binding between the dye and the biological specimen.

• Basic Mordants bind with acidic dyes, while Acidic Mordants bind with basic dyes.

• Staining with a mordant is called “indirect staining”, whereas staining without a mordant is referred to as “direct staining”.

4. Permeabilisation

• A surfactant is used to disrupt the cell membrane, making the specimen more receptive to the dye.

Types of Staining Techniques

Below is a comprehensive overview:

1. Gram Staining

Among all gram staining techniques, this one is used to differentiate bacteria based on their cell wall. Bacteria can be categorised as Gram-positive or Gram-negative.

• Primary Stain: Crystal violet

• Mordant: Iodine solution

• Decolouriser: Alcohol or acetone

• Counterstain: Safranin or fuchsin

2. Endospore Staining

Some bacteria form robust endospores, making them resistant to standard dyes.

• Stain Used: A combination of 5% malachite green (primary stain) and safranin (counterstain)

• Fixation: Typically heat-fixed to enhance dye penetration

3. Ziehl-Neelsen (Acid-Fast) Staining

Certain bacteria like Mycobacterium tuberculosis have waxy cell walls that do not take up the Gram stain.

• Primary Stain: Carbol fuchsin

• Counterstain: Methylene blue

4. Haematoxylin and Eosin (H&E) Staining

Widely applied in histopathology for tissue sections:

• Haematoxylin imparts a blue hue to nuclei.

• Eosin stains the cytoplasm and its components pink or red.

5. Periodic Acid–Schiff (PAS) Staining

Used specifically to detect carbohydrate-rich molecules, such as glycogen:

• Commonly employed in examining tissues from the liver, kidneys, pancreas, and ovaries.

6. Masson’s Trichrome Staining

A tricolour method that helps differentiate cells from connective tissue:

• Muscle fibres and keratin: Red

• Collagen: Blue or green

• Cytoplasm: Red to pink

• Nuclei: Black

Common Biological Stains

Different dyes offer distinct advantages, helping highlight various structures:

1. Acridine Orange

• A cationic, fluorescent dye that targets nucleic acids. Useful in DNA analysis during the cell cycle.

2. Coomassie Blue

• Commonly used in protein gel electrophoresis to identify protein bands.

3. Crystal Violet

• The primary stain in gram staining techniques gives Gram-positive bacteria a purple hue.

4. Eosin

• A counterstain for haematoxylin, colouring the cytoplasm red or pink.

5. Ethidium Bromide

• Fluorescent dye that intercalates with DNA, producing an orange-red glow under UV light.

6. Iodine

• Acts as a mordant in Gram staining by binding with crystal violet.

7. Malachite Green

• Employed as a primary or counterstain (notably in endospore staining) to impart a blue-green colour.

8. Methylene Blue

• Enhances the nucleus in animal cells, turning it a deep blue.

9. Safranin

• A red dye that serves as a counterstain in both Gram and endospore staining procedures.

Additional Insights to Make Learning Fun

Mnemonic for Gram Staining Steps

Try the mnemonic “CIM-S” (pronounced “Kim’s”):

• C – Crystal violet

• I – Iodine (mordant)

• M – Mixture of alcohol/acetone for decolourisation

• S – Safranin counterstain

Quick Quiz

1. What makes endospores resistant to dyes?

2. Name the dye that specifically detects carbohydrates in tissues.

3. How does heat fixation assist in staining?

4. Which staining technique uses carbol fuchsin as the primary stain?

Try answering these to test your understanding of staining techniques.

Additional Resources for Deeper Understanding

• Difference between Gram-positive and Gram-negative Bacteria

These resources will help you explore more about staining, microbial classification, and the specific roles of various dyes.