Types of Staining Techniques With Examples and Uses

Staining techniques is the process used to introduce colour into biological specimens so that different structures can be seen clearly under the microscope.

It is used because many cells and tissues are almost transparent, and without staining it is difficult to observe their internal parts. It is the process in which dye molecules bind with specific components of the cell and increase the contrast of the image. The binding usually occur due to chemical interactions between the dye and the cellular substances, and this is referred to as the staining reaction.

Basic dyes mostly attach to acidic cell parts like nucleic acids, while acidic dyes attach to basic components like cytoplasmic proteins. It is also divided into vital staining done in living cells, and non-vital staining which is performed in fixed or dead tissues. This is one of the important methods used in histology and microbiology for identifying cellular structures, tissue arrangement and different types of microorganisms.

A. Fundamental Physicochemical Staining

• It is the process where dyes bind with tissues due to physicochemical interaction mainly depending on electrical charges.
• The staining is controlled by attraction between dye ions and charged cellular components.
• These techniques do not need antigen–antibody reaction.
• Two major types of staining is observed based on the charge of the dye.

1. Basophilic staining (basic dyes)

Basophilic staining is the process in which the cellular structures is stained by using basic dyes. It is the process where the basic dyes carry a positive charge and these bind with the negatively charged components present in the cell. The affinity between the dye and the cellular structures is due to electrostatic attraction.

It is based on the fact that acidic or negatively charged structures in the cell shows attraction towards the basic dyes. The basic dye molecules are cationic in nature and these interact with the anionic groups like phosphate groups of nucleic acids. This interaction produces a deep blue or purple appearance on these structures.

Basophilic substances are those components which shows strong attraction for basic dyes. It is mainly due to the presence of DNA and RNA in the nucleus and nucleolus. These are rich in phosphate groups. Rough endoplasmic reticulum (RER) also shows basophilia due to abundance of ribosomes (RNA). Some acidic mucins and cartilage having sulphated polysaccharides also shows basophilia

Some of the main basophilic components are–

• Nucleus (contains DNA)
• Nucleolus (rich in rRNA)
• Rough endoplasmic reticulum
• Acidic mucins
• Cartilage matrix

Examples of Basic Dyes

These are some important basic dyes used for staining–

1. Hematoxylin– It is used as the major nuclear stain in histology. The reaction is carried out after oxidation of hematoxylin to hematein. It forms a dye-mordant complex and stains the nucleus blue or purple.
2. Crystal Violet– It is used as the primary stain in Gram staining. It is the dye that binds with the negatively charged components in Gram-positive bacterial cell wall and forms a crystal violet–iodine complex.
3. Methylene Blue– It is used in simple staining and also as a counterstain in acid-fast staining. It stains the acidic structures like nucleus and provides the general shape of bacteria.
4. Safranin– It is used mainly as a counterstain in Gram staining and endospore staining. It stains Gram-negative bacteria pink or red after decolorization.
5. Malachite Green– It is used as the primary stain in endospore staining. The stain penetrates the tough spore coat when heat is applied and stains the spores green.
6. Acridine Orange– It is a fluorescent basic dye. It binds with DNA and RNA by electrostatic attraction. Under fluorescence microscope it produces orange or green fluorescence and used in detecting bacteria in blood samples.

2. Acidophilic staining (acidic dyes)

Acidophilic staining is the process in which the tissue components is stained by using acidic dyes. These dyes are anionic in nature and carry a negative charge. It is the process where the dye ions bind with the positively charged structures present in the cell. This affinity occurs due to electrostatic attraction between opposite charges.

It is based on the fact that the acidic dyes dissociate into negatively charged ions and these anions are attracted towards the basic or positively charged cellular components. Most of the cytoplasmic proteins have a net positive charge and thus they show strong acidophilia when stained with acidic dyes. The structures usually appear pink, orange or red depending on the dye used.

Acidophilic structures are those components which shows attraction for acidic dyes. These include cytoplasm, muscle fibres having actin and myosin proteins, mitochondria containing respiratory enzymes and extracellular collagen fibres. Red blood cells also stain strongly due to presence of hemoglobin.

Some of the main acidophilic components are–

• Cytoplasm
• Muscle fibres
• Mitochondria
• Collagen fibres
• Red blood cells

Examples of Acidic Dyes

1. Eosin (Eosin Y)– It is the most common acidic dye and used mainly as counterstain in H&E staining. It stains the cytoplasm, collagen and muscle fibres in pink or red shades and also highlights protein aggregates in the tissues.
2. Acid Fuchsin– It is a strong acidic dye used in different staining procedures. In Masson’s trichrome it stains muscle and cytoplasm red. In Van Gieson’s method it stains collagen fibres red. It is also used for staining mitochondria in Altmann’s method.
3. Nigrosin / India Ink– These are acidic dyes used for negative staining in microbiology. Because bacterial cells usually carry negative surface charge, the dye does not bind to them and stains only the background. It is used to visualise capsules like those of Cryptococcus neoformans.
4. Biebrich Scarlet– It is an acidic dye used in trichrome staining. It stains cytoplasm and muscle fibres red.
5. Orange G– It is used as an acidic counterstain in Pap staining. It stains keratin and helps in differentiating the cell components in cytological smears.

B. Simple Staining

• It is the process of using a single dye to colour the bacterial cells.
• It is mainly done to increase the contrast between the cells and the background.
• The stain helps in observing the size, shape and arrangement of the bacteria.
• It is referred to as monochrome staining because only one reagent is used.
• It does not differentiate between types of bacteria, it only shows the presence of the cells.
• Two general methods are used in simple staining– positive staining and negative staining.
• In positive staining, basic dyes (methylene blue, crystal violet, safranin) is used which carry positive charge, and these dye molecules are attracted to the negatively charged bacterial surface.
• The cells take up the colour of the stain and appear dark against a clear background.
• In this method, a smear is prepared and heat-fixed before applying the stain.
• In negative staining, acidic dyes (nigrosin, India ink) is used which carry negative charge, so the dye is repelled by the negatively charged bacterial cell surface.
• The background becomes coloured and the bacteria remain colourless, showing a clear outline.
• This process occurs without heat fixation, so it helps in observing delicate structures.

1. Positive Staining (Direct)

Positive staining is the process in which the specimen itself is stained and appears colored against a clear background. It is the process where basic dyes are used because these dyes carry a positive charge. The dye ions bind directly with the negatively charged components present on the microbial cell surface. This produces a visible image of the organism under the microscope.

It is based on the fact that the bacterial cell wall and nucleic acids have negatively charged groups. The basic dyes dissociate into positively charged ions and these ions show attraction towards the cell surface. The staining occurs without the use of a mordant. This is referred to as direct staining because the stain is applied directly to the specimen and the color is taken up by the cell.

Some Features

• It stains the microbial cell directly.
• The background remains clear.
• No mordant is used in this technique.
• It is mainly used to study shape, size, and arrangement of the cells.

Examples of Positive (Direct) Stains

1. Methylene Blue– It is a basic dye that stains the nucleic acids and the bacterial cell. The cells appear blue in color and it is used in simple staining.
2. Crystal Violet– It is a widely used basic dye. It stains the cells purple and used as the primary stain in Gram staining but also in simple staining.
3. Safranin– It is a basic dye that stains the cells pink or red. It is used as a counterstain in Gram staining and also used as a simple stain.
4. Malachite Green– It is a basic dye mainly used in endospore staining. The spores appear green when heat is applied in the staining process.
5. Basic Fuchsin– It stains the cells red. It is used in acid-fast staining and simple staining techniques.

Vital Stains (Live Cell Staining)

Some stains are used for living cells. These stains are taken up by the cell without immediate killing.

• Janus Green B – It stains mitochondria in living cells and appears blue-green.
• Neutral Red – It accumulates in lysosomes or vacuoles and stains them red.

2. Negative Staining (Indirect)

Positive staining is the process in which the specimen itself is stained and appears colored against a clear background. It is the process where basic dyes are used because these dyes carry a positive charge. The dye ions bind directly with the negatively charged components present on the microbial cell surface. This produces a visible image of the organism under the microscope.

It is based on the fact that the bacterial cell wall and nucleic acids have negatively charged groups. The basic dyes dissociate into positively charged ions and these ions show attraction towards the cell surface. The staining occurs without the use of a mordant. This is referred to as direct staining because the stain is applied directly to the specimen and the color is taken up by the cell.

Some Features

• It stains the microbial cell directly.
• The background remains clear.
• No mordant is used in this technique.
• It is mainly used to study shape, size, and arrangement of the cells.

Examples of Positive (Direct) Stains

1. Methylene Blue– It is a basic dye that stains the nucleic acids and the bacterial cell. The cells appear blue in color and it is used in simple staining.
2. Crystal Violet-It is a widely used basic dye. It stains the cells purple and used as the primary stain in Gram staining but also in simple staining.
3. Safranin– It is a basic dye that stains the cells pink or red. It is used as a counterstain in Gram staining and also used as a simple stain.
4. Malachite Green– It is a basic dye mainly used in endospore staining. The spores appear green when heat is applied in the staining process.
5. Basic Fuchsin– It stains the cells red. It is used in acid-fast staining and simple staining techniques.

Vital Stains (Live Cell Staining)

Some stains are used for living cells. These stains are taken up by the cell without immediate killing.

• Janus Green B – It stains mitochondria in living cells and appears blue-green.
• Neutral Red – It accumulates in lysosomes or vacuoles and stains them red.

C. Differential Staining

• It is the process in which more than one stain is used to differentiate between two groups of bacteria or two different cell structures.
• A primary stain, a mordant, a decolorizer and a counterstain is used in sequence.
• The main step is the decolorization step, as some cells retain the primary stain while others lose it.
• It is important because cells with different chemical composition show different reactions to the staining reagents.
• This technique help to identify bacterial groups and special structures like spores and acid-fast cells.

Types of Differential Staining

1. Gram Staining

• It is the most common differential stain used to divide bacteria into Gram-positive and Gram-negative types.
• Crystal violet is used as primary stain and iodine is used as mordant.
• Alcohol or acetone is used as decolorizer.
• Safranin is used as counterstain.
• Gram-positive cells appear purple and Gram-negative cells appear pink.

2. Acid-Fast Staining

• It is used to stain bacteria having waxy cell walls containing mycolic acid.
• Carbol fuchsin is used as primary stain, and acid-alcohol is used for decolorization.
• Methylene blue or brilliant green is used as counterstain.
• Acid-fast cells remain red and non-acid-fast cells appear blue or green.

3. Endospore Staining (Schaeffer–Fulton method)

• It is the process used to stain resistant endospores of Bacillus and Clostridium.
• Malachite green is used as primary stain and heat helps in forcing the dye into the spores.
• Water is used as decolorizer.
• Safranin is the counterstain.
• Spores appear green and vegetative cells appear red.

4. Trichrome Staining

• It is used mainly in tissues to differentiate collagen, muscle and cytoplasm.
• Hematoxylin stains nuclei, acid fuchsin stains cytoplasm, and aniline blue or light green stains collagen fibres.
• It helps in identifying connective tissue changes and structural details.

D. Structural Staining

• It is the process used to demonstrate special bacterial structures which do not take up ordinary stains easily.
• These structures have special chemical nature, so simple stains or differential stains cannot show them clearly.
• Structural staining help to visualize spores, capsules, flagella, granules and other inclusion bodies.
• Each structure needs its own specific staining method.

Types of Structural Staining (with examples)

1. Endospore Staining

• It is used to stain resistant spores formed by Bacillus and Clostridium.
• The spore coat is thick and does not allow normal stains to enter.
• Schaeffer–Fulton method uses malachite green with heat and safranin as counterstain.
• Dorner’s method uses carbol fuchsin with heat and nigrosin for background.
• Wirtz–Conklin method is another variation using malachite green and heat.
• Spores appear green or red depending on the method and vegetative cells appear pink.

2. Capsule Staining

• It is done to show the capsule made of polysaccharide or polypeptide.
• Capsule does not take up stain and is destroyed by heat, so negative staining is used with a simple stain.
• Nigrosin or India ink stain the background and crystal violet or safranin stain the cells.
• Capsule appears as a clear halo around the stained cell.

3. Flagella Staining

• Flagella are very thin and cannot be seen directly under microscope.
• Mordants like tannic acid are used to increase the thickness of the flagella.
• Leifson’s method is commonly used, where flagella appear red and cells appear blue.

4. Granule and Inclusion Staining

• It is used to show storage granules like volutin, lipid granules or polysaccharides.
• Albert’s method stains metachromatic granules bluish-black and the cytoplasm green.
• Burdon’s method stains lipid granules deep blue with Sudan black.
• PAS method stains polysaccharide inclusions red.

5. Cell Wall and Nuclear Material Staining

• Dyar’s method stains the cell wall red and the cytoplasm blue.
• Feulgen technique is used for DNA, which stains the nuclear material pinkish purple.

• These staining methods help in identifying the organisms by showing the special structures clearly.

E. Histological (Tissue) Staining

• It is the process used to colour thin sections of tissue so that the cells and their internal parts can be seen clearly under microscope.
• Biological tissues are almost transparent, so staining is needed to give contrast and show the structure.
• It is mainly divided into routine staining and special staining.
• These stains help in identifying tissue organisation, pathological changes, fibres, pigments, mucins and microorganisms present in the tissue.

Examples of Histological Staining

• Hematoxylin and Eosin (H&E) staining
  • It is the most common routine stain used in tissue examination.
  • Hematoxylin stains nucleic acids and nuclei blue or purple.
  • Eosin stains cytoplasm, muscle fibres and collagen pink or red.
  • It gives a general idea of the tissue structure.
• Connective tissue stains
  • Masson’s trichrome: Nuclei become black, cytoplasm and muscle become red, collagen becomes blue or green.
  • Verhoff’s stain: Elastic fibres appear black and is useful in arteries.
  • Reticulin stain: Reticular fibres become black.
  • Picrosirius red: Collagen appears bright red.
• Carbohydrate and mucin stains
  • PAS stain: Glycogen, mucins and basement membrane appear bright magenta.
  • Alcian blue: Acidic mucins appear blue.
  • Mucicarmine: Epithelial mucins become red.
• Lipid stains
  • Oil Red O: Neutral fats appear orange or red.
  • Sudan black B: Lipid granules appear black or dark blue.
• Mineral and pigment stains
  • Prussian blue: Ferric iron deposits are stained blue.
  • Von Kossa: Calcium deposits appear black.
  • Fontana–Masson: Melanin becomes black.
• Microorganism stains in tissue
  • GMS stain: Fungal cell walls become black.
  • Brown and Hopps: It is a Gram stain for tissues.
  • Ziehl–Neelsen: Acid-fast bacteria appear red.

• These stains are used when normal staining is not enough to show specific chemical or structural features in the tissue.

F. Vital Staining

• It is the process in which living cells or tissues are stained with non-toxic dyes so that their internal structures can be seen without killing them immediately.
• The dye enters the living cell and shows the movement and activity of different organelles.
• It is also referred to as in vivo or supravital staining depending on whether the cells are inside the body or taken out.
• Some dyes are taken up by living organelles (inclusion stains) while some dyes only enter dead cells (exclusion stains).

Examples of Vital Staining

• Janus Green B
  • It is used to stain mitochondria in living cells.
  • The dye becomes blue-green in the oxidised state inside mitochondria.
  • Mitochondria appear as small blue-green bodies.
• Neutral Red
  • It is used to stain vacuoles or lysosomes.
  • The dye enters the vacuoles and gives red colour.
  • It shows the storage and digestion spaces inside the cell.
• Trypan Blue
  • It is an exclusion stain.
  • Living cells do not take the dye, but dead cells absorb it and become blue.
  • It is used for checking cell viability.
• Propidium Iodide (PI)
  • It stains only dead or damaged cells.
  • It is used in fluorescence microscopy to detect non-viable cells.
• Acridine Orange
  • It stains nucleic acids in living cells.
  • DNA gives green fluorescence and RNA gives red or orange.
• Rhodamine 123
  • It is another stain for mitochondria.
  • It is used to observe mitochondrial activity in living cells.
• Nile Red
  • It stains lipid droplets in living cells.
  • Lipid bodies fluoresce red.
• New Methylene Blue
  • It is used as a supravital stain to show reticulocytes in blood.

These stains help to observe living processes like vacuole formation, cytoplasmic streaming, and organelle movements.

G. Advanced Molecular Techniques

• These are techniques that use specific molecular interaction instead of normal chemical dyes.
• Antibodies or nucleic acid probes are used to detect particular proteins or genetic material in the tissue.
• These methods give very high specificity and help in identifying markers which cannot be seen by ordinary stains.
• Two main techniques are Immunohistochemistry and Immunofluorescence.

Examples of Advanced Molecular Techniques

1. Immunohistochemistry (IHC)

• It uses antibodies to detect specific antigens in tissue sections.
• The antibody is linked with an enzyme like HRP which reacts with a chromogen (DAB) to form a brown deposit.
• The stained slide can be viewed under normal light microscope.
• The stain is permanent and can be stored for long time.
• Hematoxylin is often used as counterstain to show the nuclei.
• It is widely used in diagnosis of tumours and other tissue lesions.

2. Immunofluorescence (IF)

• It uses antibodies labelled with fluorescent dyes.
• Under UV or specific wavelength light, the fluorophore gives green, red or other coloured fluorescence.
• Direct IF uses labelled primary antibody, while indirect IF uses labelled secondary antibody.
• Indirect method gives stronger signal because more secondary antibodies bind to one primary antibody.
• It can detect more than one antigen at a time using multiple fluorophores.
• It is used for co-localization studies and sometimes for live-cell observation.

3. Collagen Hybridizing Peptide (CHP) staining

• It binds only to damaged or denatured collagen fibres.
• It helps in studying tissue injury where collagen structure is disturbed.

4. In situ hybridization (ISH)

• It is used to detect DNA or RNA sequences in cells.
• A complementary probe binds to the target nucleic acid and shows its location.

These techniques are important in research and clinical diagnosis where precise molecular identification is required.