Staining Methods in Histology
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Questions and Answers

What is the process of applying dyes on tissue sections called?

Staining

What is the primary objective of staining?

To see and study the architectural pattern of the tissue and physical characteristics of cells

Which of the following is a method of staining where the action of the dye is intensified by adding another agent?

  • Direct Staining
  • Accentuator Staining
  • Mordant Staining
  • Indirect Staining (correct)

    • A mordant serves as a bridge between the tissue and the dye to facilitate the staining reaction.

    <p>True (A)</p> Signup and view all the answers

    An accentuator accelerates the speed of the staining reaction by reducing the staining power and selectivity.

    <p>False (B)</p> Signup and view all the answers

    What happens when alum hematoxylin stains nuclei with red color and is washed in a weak alkali solution?

    <p>The red color is converted to blue-black</p> Signup and view all the answers

    Describe metallic impregnation.

    <p>Metallic impregnation is a technique where colorless solutions of metallic salts are reduced by tissues to produce an opaque deposit, often black, on the tissue's surface.</p> Signup and view all the answers

    What is negative staining?

    <p>Negative staining involves stains that are not taken up by the tissue targets.</p> Signup and view all the answers

    How is vital staining defined?

    <p>Vital staining involves the selective staining of living cell constituents.</p> Signup and view all the answers

    What is the purpose of counterstaining?

    <p>Counterstaining involves using a different color for contrast and background.</p> Signup and view all the answers

    Where is hematoxylin extracted from?

    <p>The core wood of a Mexican tree called <em>Haematoxylon campechianum</em></p> Signup and view all the answers

    What is the source of cochineal dyes?

    <p>An extract from the female cochineal bug <em>Dactylopius coccus costal Coccus cacti</em></p> Signup and view all the answers

    Orcein is a vegetable dye extracted from what source?

    <p>Lichens</p> Signup and view all the answers

    From what is saffron derived?

    <p>The dried stigmata of <em>Crocus sativus</em></p> Signup and view all the answers

    What are chromophores?

    <p>Substances capable of producing visible color</p> Signup and view all the answers

    Define auxochromes.

    <p>Substances that alter the properties of chromogens when added</p> Signup and view all the answers

    Describe the basic structure of a dye.

    <p>A dye consists of an auxochrome and a chromophore group attached to a hydrocarbon benzene ring</p> Signup and view all the answers

    Which of the following is NOT a type of hematoxylin?

    <p>Calcium Hematoxylin (C)</p> Signup and view all the answers

    What is the most widely used cytoplasmic stain?

    <p>Eosin</p> Signup and view all the answers

    What is the simplest method for the differential staining of collagen?

    <p>Acid Fuchsin-Picric Acid</p> Signup and view all the answers

    Which fluorochrome is commonly used to demonstrate DNA and RNA?

    <p>Acridine Orange</p> Signup and view all the answers

    How does Alcian Blue stain acidic mucopolysaccharides?

    <p>By forming salt linkages</p> Signup and view all the answers

    What is the primary use of Aniline Blue in staining?

    <p>Counterstaining of epithelial sections</p> Signup and view all the answers

    Basic Fuchsin stains acid-fast organisms, mitochondria, and smooth muscle differentiation, but only when used with picric acid.

    <p>True (A)</p> Signup and view all the answers

    What does Benzidine stain?

    <p>Hemoglobin</p> Signup and view all the answers

    What is the primary target of Bismarck Brown?

    <p>Diphtheria organisms</p> Signup and view all the answers

    Malachite Green can be used as both a decolorizer and a counterstain.

    <p>True (A)</p> Signup and view all the answers

    Which stain is used to visualize elastic fibers?

    <p>Orcein</p> Signup and view all the answers

    What is Celestine Blue recommended for?

    <p>Routine staining of fixed sections</p> Signup and view all the answers

    Congo Red is used for staining what in embryos?

    <p>Axis cylinders</p> Signup and view all the answers

    Crystal Violet is a stain for what?

    <p>Amyloid in frozen sections</p> Signup and view all the answers

    List the substances that Iodine stains.

    <p>Amyloid, cellulose, starch, carotenes, and glycogen</p> Signup and view all the answers

    What is Janus Green B used for?

    <p>Mitochondria are organelles responsible for energy production in cells, and Janus Green B helps visualize them.</p> Signup and view all the answers

    The first step of the routine H&E staining for paraffin embedded sections is clearing the section in a xylene bath for 3 minutes.

    <p>True (A)</p> Signup and view all the answers

    What is the duration for the blueing step in the routine H&E staining procedure?

    <p>The blueing step involves adjusting the pH of the solution to achieve the desired blue color for the stained nuclei.</p> Signup and view all the answers

    How long is the counterstaining step with 5% Aq. Eosin in the routine H&E staining procedure?

    <p>The counterstaining step provides a contrasting color, often pink or red, to highlight different cellular structures.</p> Signup and view all the answers

    What is the final step in the routine H&E staining procedure?

    <p>This step involves removing water from the tissue, clearing it for final visualization, and mounting it onto a slide for observation.</p> Signup and view all the answers

    What is the purpose of immunohistochemistry?

    <p>Identifying patterns of antigen distribution within an organism or tissue (C)</p> Signup and view all the answers

    Immunohistochemical techniques are currently used primarily for analysis of frozen tissues whereas paraffin-embedded tissues require other methods.

    <p>False (B)</p> Signup and view all the answers

    How are polyclonal antibodies produced?

    <p>By immunizing an animal with an immunogen containing the antigen of interest</p> Signup and view all the answers

    How are monoclonal antibodies made?

    <p>Products of an individual clone of plasma cells</p> Signup and view all the answers

    Which immunohistochemistry technique involves conjugating the primary antibody directly to a label?

    <p>Direct technique (C)</p> Signup and view all the answers

    What is the key difference between the direct and indirect immunohistochemistry techniques?

    <p>The direct technique uses a labeled primary antibody, while the indirect technique employs unlabeled primary antibody followed by a labeled secondary antibody directed against the first one.</p> Signup and view all the answers

    What is the principle behind the peroxidase-antiperoxidase (PAP) technique?

    <p>It involves the use of a soluble peroxidase-antiperoxidase complex bound to the primary antibody by a bridging antibody, resulting in amplified signal.</p> Signup and view all the answers

    Describe the basic sequence of staining in the avidin-biotin complex technique.

    <p>It involves the sequence of primary antibody, biotinylated secondary antibody, and preformed avidin-biotin-enzyme complex or labeled streptavidin.</p> Signup and view all the answers

    What is the staining sequence in the labeled streptavidin avidin biotin technique?

    <p>It consists of primary rabbit antibody, biotinylated anti-rabbit immunoglobulin, and streptavidin-enzyme conjugate.</p> Signup and view all the answers

    Which staining method utilizes PAS reagent?

    <p>Periodic Acid Schiff staining (A)</p> Signup and view all the answers

    What are the major components stained by PAS reagent?

    <p>Glycogen, mucin, and fibrin</p> Signup and view all the answers

    What is the color of the tissue constituents when stained with Langhan's Iodine method?

    <p>Yellow</p> Signup and view all the answers

    What is the color of acid mucin when stained with Alcian Blue?

    <p>Blue</p> Signup and view all the answers

    What is the color of glycosaminoglycans when stained with metachromatic toluidine blue?

    <p>Red-purple</p> Signup and view all the answers

    What is the color of acid mucopolysaccharides when stained with the fluorescent acridine orange?

    <p>Black</p> Signup and view all the answers

    What is the color of acid mucin in Hale's Dialyzed Irone stain?

    <p>Dark blue</p> Signup and view all the answers

    Which staining method is specifically used for the detection of lipids?

    <p>All of the above (D)</p> Signup and view all the answers

    The Feulgen technique stains DNA in a red-purple color while the cytoplasm appears green.

    <p>True (A)</p> Signup and view all the answers

    What is the color difference between stained DNA and RNA when using Methyl Green?

    <p>DNA appears green/blue-green, while RNA appears rose-red</p> Signup and view all the answers

    What is the color of DNA when stained with Acridine Orange?

    <p>Yellow-green fluorescent</p> Signup and view all the answers

    Describe the range of colors seen in RNA when stained with Acridine Orange.

    <p>Brick to orange red</p> Signup and view all the answers

    What is the significance of the Alkaline Fast staining method?

    <p>It is a method for staining basic proteins.</p> Signup and view all the answers

    Which amino acids are stained blue-green by Peracetic Acid?

    <p>Cystine and cysteine</p> Signup and view all the answers

    What is the color reaction of arginine when stained with Sakaguchi's Test?

    <p>Orange red</p> Signup and view all the answers

    Gomori Calcium stain renders Alkaline Phosphatase (ALP) brown-black, with nuclei appearing green.

    <p>True (A)</p> Signup and view all the answers

    Describe the color appearance of Acid Phosphatase (ACP) when stained with Gomori Lead.

    <p>ACP appears black, with nuclei appearing green.</p> Signup and view all the answers

    What color are 5' nucleotidase deposits when stained with the Lead method?

    <p>Blackish brown</p> Signup and view all the answers

    What are the colors of Esterase and nuclei when stained with A-Naphthyl Acetate?

    <p>Esterase appears reddish brown, while nuclei appear green</p> Signup and view all the answers

    What are the colors of Esterase activity and nuclei, respectively, when stained with Indoxyl Acetate?

    <p>Indoxyl Acetate is another method used to visualize Esterase in tissues, producing a distinct blue color reaction specific for the enzyme.</p> Signup and view all the answers

    Which enzyme is visualized when using the Tetrazolium staining method?

    <p>Tetrazolium staining is a technique for visualizing Monoamine oxidase, an enzyme involved in neurotransmitter metabolism, which produces a bluish black staining pattern in the enzyme's location.</p> Signup and view all the answers

    What is the color of the reticulin fibers when stained with Gomori's Silver Impregnation?

    <p>Black</p> Signup and view all the answers

    What are the colors of collagen and nuclei when stained using Van Gieson's method?

    <p>Collagen appears pink/deep red, with nuclei appearing brownish black.</p> Signup and view all the answers

    Describe the colors of muscle, red blood cells (RBC), keratin, and nuclei in Masson's Trichrome stain.

    <p>Muscle, RBC, and keratin appear red, with nuclei appearing blue/black.</p> Signup and view all the answers

    What is the characteristic staining pattern for elastic fibers in Weigert's stain?

    <p>Elastic fibers are stained dark blue or black, with a clear background.</p> Signup and view all the answers

    What colors do Verhoeff's stain show for elastic fibers and nuclei?

    <p>Elastic fibers appear black, while nuclei appear gray to black.</p> Signup and view all the answers

    How do elastic fibers and nuclei appear when stained with Orcein?

    <p>Elastic fibers are stained dark brown, while nuclei appear blue.</p> Signup and view all the answers

    What colors do elastic fibers, fibrin, connective tissue (CT), red blood cells (RBC), and neuroglia, muscle striation, amoeba appear when stained with Kajian's Technique?

    <p>Elastic fibers appear bright red, fibrin and CT appear dark blue, RBC appear orange yellow, neuroglia, muscle striation, and amoeba appear dark blue.</p> Signup and view all the answers

    What is the color reaction of fibrin, neuroglia, muscle striation, and amoeba when stained with PTAH stain?

    <p>They appear dark blue.</p> Signup and view all the answers

    What colors do amyloid, nuclei, cytoplasm, and connective tissue (CT) appear when stained with Methyl Violet-Crystal Violet?

    <p>Amyloid appears purplish red, nuclei and cytoplasm appear shades of violet, and CT appears shades of violet.</p> Signup and view all the answers

    What are the colors of amyloid and nuclei when stained with Krajian Amyloid stain?

    <p>Amyloid appears red on a clear background, while nuclei appear blue.</p> Signup and view all the answers

    What are the colors of neurofibril, axon, and dendrites in Bielschowsky's Technique?

    <p>Neurofibril, axon, and dendrites appear black on a grayish background.</p> Signup and view all the answers

    What are the primary targets of Sevier-Munger stain?

    <p>Neural tissues</p> Signup and view all the answers

    What type of tissues are typically stained with Cresyl-Fast Violet?

    <p>Paraffin sections</p> Signup and view all the answers

    What is the color of myelin when stained with Luxol Fast Blue-H&E stain?

    <p>Blue green</p> Signup and view all the answers

    Describe the colors of the myelin sheath and background in Weil's Method.

    <p>The myelin sheath appears black, while the background appears yellow.</p> Signup and view all the answers

    What colors do astrocytes and the background appear in Cajal's Gold Sublimate stain?

    <p>Astrocytes appear black on a light brownish background.</p> Signup and view all the answers

    Describe the colors of the normal myelin sheath and cells in Weigert-Pal stain.

    <p>The normal myelin sheath appears blue black, with cells appearing brown.</p> Signup and view all the answers

    What is the primary purpose of the Kluver and Barrer's Luxol Fast Blue staining method?

    <p>It stains myelin with a Nissl counterstain.</p> Signup and view all the answers

    What is the color of hemosiderin and ferric salts when stained with Perl's Prussian Blue?

    <p>Blue</p> Signup and view all the answers

    Which stain helps visualize reducing substances?

    <p>Schmorl's Ferric-Ferricyanide stain</p> Signup and view all the answers

    What color does hemofuscin pigment appear when stained with Mallory's Fuchsin stain?

    <p>Red</p> Signup and view all the answers

    Which stain is used to detect melanin and argentaffin cell's granule?

    <p>Masson-Fontana stain</p> Signup and view all the answers

    What is the color of calcium when stained with Von Kossa's Silver Nitrate?

    <p>Black</p> Signup and view all the answers

    What is the color of copper when stained with Linquist's Modified Rhodanine?

    <p>Reddish brown</p> Signup and view all the answers

    What is the color of calcium salts when stained with Calcium Dye Lake?

    <p>Intense reddish orange</p> Signup and view all the answers

    What is the color reaction of liver bile pigments when stained with Modified Fouchet's Technique?

    <p>Emerald to blue green</p> Signup and view all the answers

    What is the color of lipofuscin when stained with Gomori's Aldehyde Fuchsin?

    <p>Purple</p> Signup and view all the answers

    What are the targets of the Wade-Fite stain?

    <p>Leprosy bacilli and nocardia</p> Signup and view all the answers

    What is the color of Helicobacter when stained with Toluidine Blue?

    <p>Dark blue</p> Signup and view all the answers

    Which stain is used to detect Legionella pneumophila?

    <p>Dieterle stain</p> Signup and view all the answers

    What are the targets of the Levaditi's Method?

    <p>Spirochetes</p> Signup and view all the answers

    Which stain is designed for visualising spirochetes?

    <p>Warthin-Starry stain</p> Signup and view all the answers

    What is the colour of fungi when stained with Grocott Methenamine Silver?

    <p>Black</p> Signup and view all the answers

    What colour does bacteria appear when stained with Rapid Giemsa Stain?

    <p>Blue</p> Signup and view all the answers

    What is the Ziehl-Neelsen method used for?

    <p>Staining acid-fast bacteria (AFB)</p> Signup and view all the answers

    What are the colors of acid-fast bacteria (AFB), cells, nuclei, and red blood cells (RBC) when stained with Ziehl-Neelsen?

    <p>AFB appears red, cells and nuclei appear blue, and RBC appear pink.</p> Signup and view all the answers

    Study Notes

    Staining Methods

    • Staining is a process of applying dyes to tissue sections to study their architectural pattern and cellular characteristics.

    Methods of Staining

    • Direct Staining: A method of coloring tissue sections using aqueous dye solutions.
    • Indirect Staining: Enhancing dye action by adding a mordant.
      • Mordant: A substance that acts as a bridge between the tissue and the dye, enabling better staining.
      • Accentuator: Increases staining power and selectivity, accelerating the staining process.

    Specific Staining Techniques

    • Blueing: Alum hematoxylin stains nuclei red, turning blue-black after a weak alkali wash. Use saturated lithium carbonate, ammonia in distilled water or Scott's tap water substitute as needed.
    • Metallic Impregnation: Using metallic salts to highlight specific tissue elements through reduction creating an opaque deposit on the tissue surface (usually black).
    • Negative Staining: Stains do not bind to the tissue targets.
    • Vital Staining: Stains living cell constituents.
    • Counterstaining: Using a different color to highlight features and contrast the background.

    Stains and Staining Solutions

    • Natural Dyes: Derived from natural sources.

      • Hematoxylin: A valuable stain extracted from the corewood of the Haematoxylon campechianum tree.
      • Cochineal Dyes: Derived from the female cochineal bug Dactylopius coccus costal Coccus cacti.
      • Orcein: A vegetable dye extracted from lichens.
      • Saffron: Derived from the dried stigma of Crocus sativus.

    • Synthetic Dyes: Man-made dyes.

      • Chromophore: Substances producing visible color.
      • Chromogens: Simple benzene compounds containing chromophores.
      • Auxochrome: Substances added to chromogens to alter their properties.
      • A dye combines an auxochrome and chromophore group attached to a hydrocarbon benzene ring.

    • Common Staining Solutions:

      • Hematoxylin: Several types exist (Ehrlich's, Harris', Delafield's, Cole's, Mayer's, Gill's, Weigert's, Heidenhain's, Loyez, Verhoeff's, Phosphotungstic acid hematoxylin, Copper hematoxylin).

    • Additional Solutions (by function):

      • Eosin: Common cytoplasmic stain—best at pH 4.6 to 5.
      • Acid Fuchsin-Picric Acid: A method for differential staining of collagen (simplest method).
      • Acridine Orange: Fluorochrome used to detect DNA and RNA.
      • Alcian Blue: Stains acid mucin.
      • Aniline Blue: A cytoplasmic stain for epithelial tissue.
      • Basic Fuchsin: Stains acid-fast organisms, mitochondria, and smooth muscles.
      • Benzidine: Stains hemoglobin.
      • Bismarck Brown: Stains diphtheria.
      • Malachite Green: Decolorizer and counterstain.
      • Orcein: Stains elastic fibers.
      • Celesitine Blue: Routine stain for fixed sections.
      • Congo Red: Stains amyloid, particularly useful for frozen sections.
      • Crystal Violet: Stains amyloid.
      • Iodine: Stains amyloid, cellulose, starch, carotenes, glycogen.
      • Janus Green B: Shows mitochondria.

    Routine H&E Staining Procedure

    • Clear paraffin-embedded sections in xylene.
    • Transfer to second xylene bath.
    • Immerse in absolute ethanol, then 95% ethanol.
    • Rinse with running water.
    • Stain with Harris hematoxylin for 5 minutes.
    • Wash with running water.
    • Decolorize using acid alcohol (10 - 30 seconds)
    • Rinse in tap water.
    • Blue with ammonia water (5 minutes) or 1% aqueous lithium carbonate until blue.
    • Wash with running water.
    • Counterstain with 5% aqueous eosin (5 minutes), followed by a quick counterstaining in alcoholic eosin.
    • Dehydrate, clear, and mount.

    Immunohistochemistry

    • Uses antibodies to identify antigen distribution in tissues (frozen or paraffin embedded).
    • Polyclonal antibodies: Produced by immunizing an animal with an immunogen containing the target antigen.
    • Monoclonal antibodies: Products of an individual plasma cell clone.

    Immunohistochemistry Techniques

    • Direct: Conjugating primary antibody with a label (e.g., fluorochrome or peroxidase).
    • Indirect: A multistep procedure where unlabeled primary antibody is followed by a labeled secondary antibody that targets the primary antibody.
      • Peroxidase-Antiperoxidase (PAP): Indirect technique using an enzyme complex.
      • Avidin-Biotin: An indirect method using a bridging antibody to a preformed complex for staining.
      • Labeled Streptavidin: Uses biotin for conjugate step with avidin and streptavidin reagents.

    Carbohydrates

    • Periodic Acid Schiff (PAS): Stains carbohydrates, often showing glycogen as bright red granules.
    • Best Carmine: Stains glycogen a deep mauve.
    • Langan's Iodine Method: Shows glycogen as a mahogany brown.
    • Alcian Blue: Stains acid mucin.
    • Metachromatic Toluidine Blue: Stains glycosaminoglycans.
    • Fluorescent Acridine Orange: Shows acid mucopolysaccharides

    Fats/Lipids

    • Sudan IV: Stains lipids.
    • ** Oil Red O:** Stains lipids.
    • Osmic Acid: Stains fats.

    Nucleic Acids

    • Feulgen Technique: Stains DNA purple, cytoplasm often appears green.
    • Methyl Green: Stains DNA.
    • Acridine Orange: Stains DNA yellow/green, and RNA brick to orange.
    • Fluorescent Staining: Visualizes nucleic acids utilizing fluorescent dyes.

    Proteins

    • Alkaline Fast: A method used to stain proteins.
    • Peracetric Acid: Used to stain proteins, useful with stains requiring denaturation of proteins and peptides.
    • Sakaguchi's Test: Used to detect arginine-containing amino acid residues within proteins.

    Enzymes

    • Gomori Calcium: Stains calcium.
    • Gomori Lead: Stains calcium
    • Lead Method: Stains specific enzymes.
    • A-Naphthyl Acetate: Stains esterase activity.
    • Indoxyl Acetate: Shows esterase activity
    • Tetrazonium: Shows monoamine oxidase.

    Connective Tissue

    • Various techniques exist to stain different connective tissue components.

    Central Nervous System

    • Various techniques stain specific components.

    Tissue Pigments and Deposits

    • Various techniques are used to view different tissue pigments and deposits.

    Microorganisms

    • Specific stains are use for visualization of diverse organisms.

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    Description

    This quiz covers various staining techniques used in histology, including direct and indirect staining methods. Learn about specific techniques like blueing, metallic impregnation, and negative staining. Test your knowledge on the principles and applications of these methods.

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