PRINCIPLES OF STAINING

(Staining) (contrast and visualization) (live and dead cells) (internal and extern structures)

(___________) is the process whereby tissue components are made visible]in microscopic sections by direct interaction with a dye or staining solution.

• to enhance (________&______) of the cell or certain cellular components under a microscope (has different parts of the cell such as your nubilus, Cytoplasm, extracellular structure)

• to differentiate between (_______&______) in a specimen (which is useful in research and diagnostics)

• to demonstrate the relationship between (_________&_________) of the cells (staining allows us to observe interactions between organelles and how cellular are arranged in. Tissues)

• to identify different types of cells

(acidic) (affinity for basic dye)

PRINCIPLES OF STAINING

Certain parts of cells and tissues that are (_____) in character (e.g. nucleus) have greater (___________), while basic constituents (e.g. cytoplasm) take more of the acid stains.

TYPES OF STAINING

Histological staining is the process whereby the tissue constituents and general relationship between cell and tissue are demonstrated in sections by direct interaction with a dye or staining solution, [producing coloration| of the active tissue component. Example: Micro-anatomic stains, bacterial stains and specific tissue stains

TYPES OF STAINING

Histochemical staining is the process whereby various constituents of tissues are studied thru chemical reactions that will permit microscopic localization of a specific tissue substance.

xample:

Perl's Prussian blue - hemoglo Periodic Acid Schiff-carbohydr

(Immunohistochemical (IHC) staining) (immunologic and histochemical)

TYPES OF STAINING

(_____________________) is a combination of (________&________) techniques using a wide range of polyclonal or monoclonal, fluorescent labeled or enzyme-labeled antibodies to detect and demonstrate tissue antigens| (e.8-, proteins) and phenotypic markers |under the microscope.

(XYLENE (1) (XYLENE (2) (ABS ALCOHOL (1) (ABS ALCOHOL (2) (90% ALCOHOL) (70% ALCOHOL) (WATER) (70% ALCOHOL) (90% ALCOHOL) (ABS ALCOHOL (1) (ABS ALCOHOL (2) (XYLENE (1) (XYLENE (2) (MORDANT)

STAINING OF PARAFFIN

(DEPARAFFINIZATION):

• (______________) (_______________)^

Down

• (_______________) (_______________)(CLEARNING)^

Down

(SECTION TO ALCOHOL):

• (______________) (_______________)(CLEARNING)^

Down

• (______________) (_______________)(DEHYDRATION)^

Down

• (_____________) (______________)(DEHYDRATION)^

Down

• (______________) (______________)(DEHYDRATION)^

Down

• (_______________) → (STAIN) → (___________)(DEHYDRATION)

(Proper Drying:) (thoroughly dried) (bone and nervous tissue)

Precautions During Staining Procedure

(_________) After mounting the section on the slide, it must be (__________) to remove all moisture and ensure strong adhesion. If drying is incomplete, sections especially from (________________) may detach during staining, particularly after exposure to acid differentiators.

(Prolonged Alcohol Exposure:) (prolonged alcohol immersion) (Prevent Floating Sections:) (dirty slides, greasy surfaces, or inadequate drying) (30 minutes)

Precautions During Staining Procedure

(____________________) Stained sections should not remain in alcohol for long periods, as many stains fade or are removed by (_______________).

(_____________) If sections float off the slide during staining, it could be due to (________, ___________, ___________) in the paraffin oven. Sections must be dried in an oven for at least (__________) before staining to ensure proper adhesion.

(Direct staining) (aqueous or alcoholic) (30-60)

METHODS OF STAINING

(__________) is the process of giving color to the sections by using (__________) dye solutions. In simple (or direct) staining, only one dye is used, which is washed away after (______) seconds, prior to drying and examination. Example: Methylene blue

(Indirect staining) (Potassium alum with hematoxylin) (Iron) (accentuator) (Potassium hydroxide) (Phenol)

METHODS OF STAINING

(____________) is the process whereby the action of the dye is intensified by adding another agent or a mordant which serves as a link or bridge between the tissue and the dye, to make the staining reaction possible. Examples of mordants: (________________________) in Ehrlich's hematoxylin, (___________) in Weiaert's hematoxylin.

An (________), on the other hand, is not essential to the chemical union of the tissue and the dye. It does not participate in the staining reaction, but merely accelerates the reaction. Examples: (_____________) in Loeffler's methylene blue, (______) in carbol thionine and carbol fuchsin

(Progressive staining) (Regressive staining)

METHODS OF STAINING

(___________) is the process whereby tissue elements are stained in a definite sequence, and the staining solution is applied for specific periods of time or until the desired intensity of coloring of the different tissue elements is attained.

(_____________), the tissue is first overstained |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. Example: Routine Hematoxylin and Eosin (H&E) staining

(desired intensity) (overstained and decolorized ) (staining process) (differentiating agent) (nuclear staining) (H&E Staining) (Methyl green pyronin) (Cresyl violet stain) (Toluidine blue stain) (Feulgen stain) (Hematoxylin in H&E staining) (Ziehl-Neelsen stain) (Perl's Prussian Blue)

METHODS OF STAININ

	Progressive Staining	Regressive Staining
Definition	Staining technique where the specimen is stained until the (____________ is reached	Staining technique where the specimen is deliberately (________&_______) to the correct intensity
Staining Control	Controlled by monitoring the (____________)	Controlled by decolorization using a (______________)

Commonly Used	Stain where precision is needed (___________)	Stains where contrast enhancement is required (_________)
Examples	(__________) stain • (__________) for neurons (___________) for mast cells • (__________) for DNA	• (_____________) (Mayer's, Harris Hematoxylin • (_____________) for acid fast Gram stain • (______________) for iron

(Differentiation (decolorization)

METHODS OF STAINING

(__________________) is the selective removal of excess stain from the tissue during regressive staining in order that a specific substance may be stained distinctly from the surrounding tissues.

(Differential Staining)

METHODS OF STAINING

(_____________) uses more than one chemical stain to better differentiate between various microorganisms or structures/cellular components of a single organism.

(Metachromatic staining)

METHODs OF STAININC

(_______________) technique entails the use of specific dyes which differentiate particular substances by staining them with a color that is different from that of the stain itself (metachromasia).

Example: Methyl violet (crystal violet), Cresv| blue (for reticulocytes, Safranin, Bismark brown, Basic fuchsin, Methylene blue, Thionine, Toluidine blue, Azure A, B, C

(Metalic Impregnation) (gold (gold chloride) and silver (silver nitrate)

METHOD OF STAINING

(______________) is a process where 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 black deposit on the surface of the tissue or bacteria. Example: (______________________)

(Vital staining) (true vital staining) (Intravital staining) (intravenous, intraperitoneal or subcutaneous) (Supravital staining)

METHOD OF STAINING

(___________) iS the selective staining of living cell constituents, demonstrating cytoplasmic structures by phagocytosis of the dye particle (cytoplasmic phagocytosis), or by staining of pre-existing cellular components (_______________).

(______________) of living cells is done by injecting the dye into any part of the animal body (either (_____, ______, ______).

Example: Lithium, Carmine and India ink

(____________) is a method of staining used in microscopy to examine living cells that have been removed from an organism

Example: Neutral red, Janus green, Trypan blue, Nile blue, Thionine, Toluidine blu

(living organism) (vivo) (freshly removed living cells) (in vitro) (physiological processes and cell functions) (structure and function) (Injected or ingested) (Directly applied)

METHODS OF STAINING

	Intravital Staining	Supravital Staining
Definition	Staining technique where dyes are introduced into a (________) to stain tissues or cells in (____)	Staining technique where (______________) or tissues are stained outside the body (______) before they die
Application	Used to study (_______&_______) in living organisms	Used to examine the (_______&______) of living cells outside the body

Mode of Administration	(____/_____) into the organism	(________) to isolated cells or tissues

(macrophage activity) (blood circulation) (organ function and disease pathology) (blood smears) (Sperm viability) (nerve and muscle tissues) (Lithium.) (Carmine) (India ink) (Neutral red) (Janus green) (Trypan blue) (Nile blue) (Thionine) (Toluidine blue)

METHODS OF STAINING

	Intravital Staining	Supravital Staining
Commonly Used in	Studying (_______), observing (__________) Research on (______&_____)	Reticulocyte count in (_______) (_______) testing Vital staining of (____&____)
Example of Dyes	(________) (______) (______) (Used in macrophage studies)	• (______) (probably best vital dye) • (_______) (recommended for mitochondria) (________) . (______) (______). (_________)

(tissue-based diagnosis) (tissue morphology) (Regressive Staining)

(osmic acid solutions) (Rapid Diagnosis (Progressive Staining,)

HEMATOXYLIN AND EOSIN (H + E) STAINING

the corner stone of (______________)

• stains thin tissue sections to visualize (__________)

• Routine H&E staining in Paraffin-Embedded Section (____________).

Note: Most fixatives can be used except (____________) which inhibit hematoxylin

• H & E staining of Frozen Sections for (_________________(____________)

(clearly visible) (very reliable) (Hematoxylin-Eosin Method) (Thionine Method) (Polychrome Methylene Blue Method) (Alcoholic Pinacyanol Method)

HEMATOXYLIN AND EOSIN ( + E) STAINING

• plays a significant role in tissue-based diagnosis by coloring otherwise transparent tissue sections allowing cell structures to be (_______).

• (__________) with some variation based on thickness of the sections.

• thicker sections take up Staining methods for Frozen sections:

○ (_____________)

○ (_____________)

○ (______________)

○ (_________________) (used also for supravital staining of mitochondria)

(avoided) (0.5% acid alcohol) (water) (paraffin, fixative or decalcifying solution) (discarded) (dirty or oily slide) () () () () () () () () () () ()

HEMATOXYLIN AND EOSIN (H + ) STAINING Precautions in staining

• stains on the skin should be (____)

○ may be removed using (_____________) followed by (_______)

• failure of staining may be due to (_______, _______, _______) not thoroughly washed out stains may be saved and used again

• formation of precipitates and poor staining results should be (_________).

• failure of sections to remain on slide during staining could have been due to a (______/_____).

(xylene for 24 hour) (0.5% potassium permanganate solutions for 5-10 minutes) (5% oxalic acid for 5 minutes) (Old, bleached, faded sections)

RE-STAINING OF OLD SECTIONS

• The slide is usually immersed in (__________) or gently heated (until mounting medium bubbles).

• The coverslip is lifted with a dissecting needle, then the section is placed in xylene for 3 minutes.

• Then brought down to water. Then placed in, (_______________) rinse again in tap.

water.

• Then immerse in (______________) or until completely decolorized.

• Wash in running tap water for another 5 minutes, then stain.

Re-stained:

○ (______, _______, ____).