STAINING PT.1

Staining

Process of applying dyes on the section to see and study the architectural pattern of the tissue and physical characteristics of cells

Histological, Histochemical, Immunohistochemical

3 Staining Categories

Histological

Tissue components are demonstrated by direct interaction with a dye or staining solution

Histological

Both nucleus and cytoplasm will be stained

Tissue + dye = color

Colors cells and tissues

Histological

Active tissue component is colored

Micro-anatomical stains, bacterial stains, specific tissue stains

Methylene blue, Crystal Violet, Wright’s stain, Geimsa stain, Carbol fuchsin

Examples of Histological Stains

Histochemical

Demonstration of tissue components through chemical reactions

Histochemical

Not by using dyes

To demonstrate fats, certain proteins, enzymes

Perl’s Prussian blue, Periodic acid schiff, Sudan dyes

Example of Histochemical Stains

Hemosiderin

Perl’s Prussian blue reaction

Carbohydrates

Periodic acid Schiff

Fats or lipids

Sudan dyes

Immunohistochemical

Detection of phenotypic markers that are detected by antibodies i.e., monoclonal, polyclonal

Phenotypic markers

Tissue antigens

Immunohistochemical

The trend now

Same principle as immunofluorescence

DIRECT STAINING

Uses aqueous or alcoholic dye solutions to produce a color

Only one dye is used

Uses simple solutions or dyes to produce color

DIRECT STAINING

Only the tissue made to react with dye in order to produce color

To color the tissue is the role of the dye

The color of the dye is also the resulting color

Methylene blue

Example of direct staining

Indirect Staining

Tissue components are demonstrated using a dye but with the help of mordant and accentuator to produce color

Mordant and Accentuator

2 types of Indirect Staining

Mordant

always used

Serves as a link or bridge between the dye and the tissue

2 ways to use mordant

Part of the staining reaction

Can be component of the dye

Part of the staining reaction

Apply the dye and then apply the mordant

Can be component of the dye

Most of the time, mordant is already incorporated into the dye

Potassium alum, Iron (ferric ammonium chloride), Copper, Lead, Phosphotungstic acid, Molybdenum

Examples of Mordant

Potassium alum

Mordant often used

Mayer’s hematoxylin and Ehrlich’s hematoxylin

Stains containing potassium alum

Iron (ferric ammonium chloride)

ferrous not used

Weigert’s hematoxylin

Stains containing ferric ammonium chloride

Accentuator

Substance that heightens color intensity of the dye

Increases the staining power of the dye

Not part of the staining reaction

Progressive Staining

Tissue elements are stained in a definite sequence

Gradual application of dye to the tissue until desired color is achieved

Progressive Staining

Tissue is not overstained

Decolorization is not performed

Not favored by laboratories

Can lead to diffused colors, Can lead to obscured details

Disadvantages of Progressive Staining

Regressive Staining

The tissue is first overstained and the excess dye is removed selectively

There is excess dye

Decolorization is performed

Decolorization

Also called differentiation

Decolorization

Selective removal of excess dye

Commonly used is acid alcohol as it can both remove acid and basic dyes

Basic solution

What solution is used to remove acid dye?

Acid solution

What solution is used to remove basic dye?

Metachromatic staining

Not a routine

For demonstration of cartilage, epithelial mucins, mast cell granules, connective tissues

Staining tissue with a color that is different from the the stain itself

Orthochromatic

the stain or dye imparts a color with the same shade or hue as that of the dye but not exactly the color of the dye

Methyl violet or crystal violet, Cresyl blue, Safranin, Bismarck brown, Basic fuchsin, Methylene blue, Thionine, Toluidine blue, Azure A, B, C

Examples of Metachromatic staining

For reticulocytes

What use is cresyl blue in metachromatic staining?

Counterstaining

Involves application of a different color to produce contrast and background

Most commonly used is eosin which is a red acid dye and a cytoplasmic stain; hematoxylin is a basic dye

Basic Dye

What dye to use if Nucleus?

Acid dye

What dye to use if Cytoplasm:

What color does the cytoplasmic stain Eosin Y produce?

Red

What color does the cytoplasmic stain Eosin B produce?

Red

What color does the cytoplasmic stain Phloxine B produce?

Red

What color does the cytoplasmic stain Picric acid produce?

Yellow

What color does the cytoplasmic stain Orange G produce?

Yellow

What color does the cytoplasmic stain Rose Bengal produce?

Yellow

What color does the cytoplasmic stain Light Green SF produce?

Green

What color does the cytoplasmic stain Lissamine Green produce?

Green

What color does the nuclear stain Neutral red produce?

Red

What color does the nuclear stain Safranin O produce?

Red

What color does the nuclear stain Carmine produce?

Red

What color does the nuclear stain Hematoxylin produce?

Red

What color does the nuclear stain Methylene blue produce?

Blue

What color does the nuclear stain Toluidine blue produce?

Blue

What color does the nuclear stain Celestine blue produce?

Blue

Vital Staining

Involves demonstrating living, viable, fresh cells

Freshly removed from the body

Intravital

Injecting the dye to any part of the living body

Lithium, Carmine, India Ink

Examples of Intravital

Supravital

Stain is applied immediately to tissue after its removal from living body

Neutral red, Janus green, Trypan blue, Nile blue, Thionine, Toluidine blue

Examples of Supravital Stains

Neutral red

best vital dye

Janus green

recommended for mitochondria demonstration

Thionine

for metachromatic and supravital staining

Differential Staining

Uses more than 1 chemical stain to differentiate microorganisms or structures / cellular components of single organisms

Gram stain, Acid-fast

Example of Differential Staining

Metallic Impregnation

For tissues that cannot absorb dye

Tissue elements are demonstrated NOT by stains but by colorless solutions of metallic salts

Black deposit on the surface of the tissue

Usually done on spiral organisms

Gold chloride, silver nitrate, ammonical silver

Examples of MetallicImpregnation

H&E

Routine tissue stain

Papanicolaou

Routine cytological stain

Slotted staining dishes

can accommodate 5-19 slides

Coplin jars

can accommodate 5-9 slides

Slide carrier

up to 30 slides, used in labs

ROUTINE HEMATOXYLIN & EOSIN STAINING

Most common method utilized for microanatomical studies of tissues

Histological, regressive staining method

The slide with the tissue will be subjected to different reagents in less than 1 hour

Initial xylene bath

3 minutes (2x)

To further remove the wax

Descending grades of alcohol

Ethanol

To hydrate or add water to the tissue to allow the it to absorb the stain

Hematoxylin

Basic dye, nuclear stain, primary dye

Acid alcohol

Decolorizer

Ammonia water / alcohol

1-2 minutes

Bluing agent

To restore the faded color

Eosin

Counterstain

To color the cytoplasm

Ascending grades of alcohol

To dehydrate tissue

Removal of the absorbed chemicals

Last xylene bath

For clearing prior to mounting

To increase refractive index

blue to blue black

H&E result for nuclei

dark blue

H&E result for Karyosome

pale pink

H&E result for Cytoplasm

bright orange red

H&E result for RBCs, eosinophilic granules, keratin

purplish blue

H&E result for calcium and decalcified bone

pink

H&E result for Decalcified bone matrix, collagen and osteoid

deep pink

H&E result for muscle fibers

Staining methods for Frozen Section

Used when dealing with fresh tissues

Requires immediate cutting

H&E, Thionine method, Polychrome methylene blue, Alcoholic pinacyanol method

Methods of Staining for Frozen Section