1. MAJOR GROUPS AND METHODS OF STAINING

Histological Staining

Process whereby the tissue constituents are demonstrated in sections by direct interaction with a dye or staining solution producing coloration of the active tissue component. We allow the tissue to come in contact with a dye in order to produce a color.

Acid fast stain, Gram stain, Stain used in hematology to stain blood cells

What are examples of Histological Staining?

Histochemical Staining

Process whereby various constituents of tissues are studied through the chemical reaction that will permit microscopic localization of a specific tissue substance. Used to demonstrate the fat or carbohydrates.

Perls prussian blue (for hemoglobin), Periodic acid shift (for carbohydrates)

What are examples of Histochemical Staining?

Immunohistochemical Staining

Combination of immunologic and histological techniques that allow phenotypic markers to be detected and demonstrated under the microscope using a wide range of polyclonal or monoclonal, fluorescent-labeled or enzyme-labeled antibodies. We used antibodies instead of antibodies.

Direct Staining

Process of giving color to the sections by using aqueous or alcoholic dye solution. Uses only one dye and usually the color of the dye is the resulting color.

Methylene blue (color blue)

What are examples of Direct Staining?

Indirect Staining

Process whereby the action of the dye is intensified by adding another agent called mordant. In here, the tissue components is demonstrated using a dye but with the help of either mordant or accentuator. Mordant is like sya yung bridge para magkaroon ng kulay and para mas matingkad pa yung kulay is lagyan mo ng accentuator.

Mordant

Serves as a link or bridge between the tissue and the dye, to make the staining reaction possible.

Phosphotungstic Acid, Iron, Lead, Aluminum, Molybdenum, Copper, Potassium alum

What are examples of Mordants?

Meyers Hematoxylin

Example of stain with potassium alum as a mordant.

Weigert's Hematoxylin

Example of stain with Iron (ferric ammonium chloride) as a mordant.

Accentuator

Accelerates or hastens the speed of the staining reaction by increasing the staining power and selectivity of the dye.

Hydrogen peroxide, Phenol

What are examples of Accentuators?

Phenol

Used as an accentuator whenever we used carbolfuscin and carbolthionine.

Lake

The combination of a dye and a mordant.

Progressive Staining

Process whereby tissue elements are stained in a definite sequence and the staining solution is applied for a specific periods of time or until the desired intensity of coloring of the different tissue elements is attained. Staining technique that involves gradual application of the dye. We apply the dye little by little to the tissues until the desired color is achieved. Since it is applied gradually, it follows a definite sequence and there will be no excess dye since it is applied little by little too. DECOLORIZATION is not applied here since we apply the stain gradually and little by little and so there will be no excess dye.

Regressive Staining

The tissue is to obliterate the cellular details, and the excess stain is removed or decolorized from unwanted parts of the tissue, until the desired intensity of color is obtained. Routine hematoxylin and eosin staining is the most common method using the regressive staining which consists of overstaining the nuclei, followed by removal of excessive color of the tissue constituent by the acid differentiation. When using the regressive staining method, the nucleus is deliberately overstained with non-acified hematoxylin. The tissue is first overstain therefore there will be excess dye and the excess dye must be removed by doing decolorization or differentiation.

Differentiation or Decolorization

Selective removal of excess stain from the tissue during regressive staining.

Acid Alcohol

A commonly used decolorizer because it can remove both acid and basic dyes.

Rule for Decolorization

If you remove excess acid stains or dye, you need to use a basic solution. If you remove excess basic stains or dye, you need to use an acid solution.

Washing out

Removal of unwanted artifacts during staining.

Metachromatic Staining

Technique using a specific dyes which differentiates particular substances by staining them with a color that is different from the stain itself. This is particularly employed for staining cartilage, connective tissues, epithelial mucins, mast cell granules and amyloids. We use a dye that will produce different color from the color of the dye itself.

Methyl violet, Crystal violet, Cresyl blue

What are examples of dyes used in Metachromatic Staining?

Metallic Impregnation

Specific tissue elements are demonstrated not by stains, but by colorless solutions of metallic salts which are thereby reduced by the tissue, producing an opaque, usually a black deposit on the surface of the tissue or bacteria.

Gold (gold chloride), Silver (silver nitrate)

What are the most valuable metals used in Metallic Impregnation?

Negative Staining

An acidic anionic dye and is used to change the color of the background, not the cells, causing the cells to standout. This can be achieved by smearing the sample onto the slide and then applying nigrosin—a black synthetic dye or india ink, an aqueous suspension of carbon particles.

Nigrosin, India ink

What are examples used in Negative Staining?

Counterstaining

Application of a different color or stain to provide contrast and background to the staining of the structural components to be demonstrated. Staining technique that involves application of a different color using an another dye to produce a contrast background.

Eosin

Commonly used counterstain and is available in 3 forms.

Yellow

What is the color of EOSIN Y

Ethyl Eosin

What is the color of EOSIN S

Blue

What is the color of eosin B

Nuclear Counterstain

Counterstains used for nuclei.

safranin O

neutral red

carmine

hematoxylin

Nuclear Chounterstain with Red Color

methylene blue

toluidine blue

celestine blue

Nuclear Chounterstain with Blue Color

Cytoplasmic Counterstain

Counterstains used for cytoplasm.

floxine B

Eosin Y

Eosin B

Cytoplasmic Stain with Color Red

picric acid

orange G

Rose bengal

Cytoplasmic Stain with Color Yellow

Light green SF

lissamine green

Cytoplasmic Stain with Color Green

Vital Staining

The selective staining of a living cell (cells that are viable) constituents.

Intravital Staining

Staining done by injecting the dye to any part of the the living body (inside).

Lithium, India Ink, Carmine

What are examples of Intravital Staining dyes?

Supravital Staining

Staining done after removal from the body. Applied immediately to the tissue after its removal to the living body.

Toluidine blue

Trypan blue

Thionine

Nile blue,

Janus Green—used for mitochondria

Neutral Red—regarded as the best vital stain

What are examples of Supravital Staining dyes?