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HISTOPATH Staining.pptx

This document provides information on tissue processing and histopathologic techniques, focusing on staining. It defines staining as applying dyes to tissue sections to facilitate microscopic study. It then classifies stains based on pH, function, source, dye application technique, sequence, and color contrast. Specific staining techniques and commonly used stains are described for carbohydrates, lipids, proteins, and nucleic acids. Hematoxylin and eosin staining is explained as the most widely used staining procedure.

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1 TISSUE PROCESSING 1. NUMBERING 2. FIXATION 3. DECALCIFICATION 4. DEHYDRATION 5. CLEARING 6. IMPREGNATION 7. EMBEDDING 8. TRIMMING 9. SECTIONING 10.STAINING 11.MOUNTING 12.LABELING
HISTOPATHOLOGIC TECHNIQUES STAINING Presented by : Ronel Wynlor D. Abarca, RMT
Creator of all things, true source of Light and Wisdom, lofty source of all being, graciously let a ray of light of Your brilliance penetrate into the darkness of our understanding and take from us the double darkness in which we have been born, the darkness of sin and ignorance. Give us a sharp sense of understanding, a retentive memory, and the ability to grasp things correctly, and fundamentally. Grant us the talent of being exact in our explanations, and the ability to express ourselves with thoroughness and charm. Point out the beginning, direct the progress, help in the completion. We ask these through Christ, your Son, our Lord, Amen.
4 LEARNING OUTCOMES 1. Define and Differentiate an Accentuator from a Mordant. 2. Enumerate the different types of stains. 3. List the stains used for carbohydrates, proteins, nucleic acid. 4. List the stains used for connective, muscle, skeletal, and nervous tissues. 5. List the stains used to visualize microorganisms in tissues. 6. Know which stain is best used for a certain tissue specimen. 7. Explain the principles and process of H&E staining.
5 STAINING • Process of dye application on tissue sections to facilitate the study of tissue architectural patterns and cells • Visualization and Enhancement of contrast of cells for microscopic studies • Exploits affinity of tissues and cells to acid and basic solutions
6 Classifications • According to pH  simplest yet most important classification  Acidic Dyes  Affinity towards the Cytoplasm which is a basic structure (Acidophilic)  Ex. Eosin, Picric Acid  Basic Dyes  Affinity towards the Nucleus which is an acidic structure (Basophilic)  Ex. Methylene Blue  Neutral Dyes  Combined Acidic and Basic Dyes  Stains Cytoplasm and Nucleus simultaneously but with different colors  Ex. Romanowsky Stain (Eosin and Azure B or Methylene
7 Classifications • According to Function  Histologic Stains  Stains that directly interact with the tissue or cell  Histochemical Stains  Stains that chemically react with tissue constituents  Ex. Perl’s Prussian Blue reacts with Iron constituents  Ex. Periodic Acid Schiff reacts with Glycogen components  Immunohistochemical Stains  Stains that are labelled with antibodies to target specific tissue antigens
8 Classifications • According to Source  Natural Dyes  Stains that are derived from plant and animal products  Synthetic Dyes  Stains that are chemically generated, usually from hydrocarbons like benzene
9 Natural Dyes • Hematoxylin  Derived from Hematoxylum campechianum (logwood)  Most commonly used for histologic studies  Not in itself a DYE!  Hematin  active coloring agent obtained from ripening  Natural Ripening  Expose the substance to sunlight and air  Slow, 3-4 months  Ex. Ehrlich’s Hematoxylin, Delafield’s Hematoxylin  Artificial Ripening  Induced by oxidation via Hydrogen peroxide mercuric oxide, or potassium permanganate, sodium perborate and sodium iodate  Ex. Mayer’s Hematoxylin, Harris Hematoxylin
10 Natural Dyes • Hematoxylin  BLUING  process of converting soluble red component of hematoxylin into insoluble blue complexes  To ensure that hematoxylin will stain the nucleus blue.  Bluing Agents  Ammonia Water  Scott’s Tap Water  Lithium Carbonate
11 Natural Dyes • Cochineal Dyes  Extracted from Coccus cacti bug  With alum  Carmine dye  Chromatin and nuclear stain for fresh tissue and smear preparation  With picric acid  Picrocarmine  Neuropathological stain  With aluminum chloride  Best’s carmine  Demonstration of glycogen
12 Natural Dyes • Orcein Dyes  Extracted from lichens  Used to stain elastic fibers especially on the skin (brown color)  Colorless, treated with ammonia, exposed to air to produce blue/violet color  Ex. Taenzer-Unna’s  Tenzer’s orcein + Unna’s Polychrome methylene blue
13 Techniques • According to Dye Application  Direct Staining  Stain is applied directly to tissue as is  Use of aqueous or alcoholic dye solutions like Methylene Blue to directly impart a color  Indirect Staining  Stain is applied with a mordant to bind the dye to the tissue or cell  Mordant  serves as the bridge between tissue and dye Accentuator  hastens staining process Ex. Potassium Hydroxide in Loeffler’s Methylene Blue Ex. Phenol in Carbol Fuchsin
14 Techniques • According to Sequence  Progressive Staining  Tissues are stained in a definite and specific sequence  Staining proceeds with specific time intervals until desired color is achieved.  Differentiator is not REQUIRED (may be present)  Differentiator is used to remove background staining only  Retrogressive Staining  Tissue is overstained and a differentiator is applied until the desired color is achieved  Alters the nuclear stain  Differentiation process observed under microscope
15 Techniques • According to Color Contrast  Simple Staining  Tissues are stained with a single dye and therefore will all appear similar in color  Differential Staining  Use of two dyes, the primary stain which imparts color to the tissue element of concern, and the counterstain which imparts a contrasting color to the other structures.
16 Counterstains Cytoplasmic Nuclear Red Yellow Green Red Blue Eosin Y Picric Acid Light Green SF Neutral Red Methylene Blue Eosin B Orange G Lissamine Green Safranin O Toluidine Blue Phloxine B Rose Bengal Carmine Celestine Blue Hematoxylin
17 Techniques • Metachromatic Staining  Impartment of a color to the tissue that is different from the original color of the dye • Vital Staining  staining of cells or tissues without killing them  Intravital Staining  Dye is injected to a body part  Ex. India Ink, Lithium, and Carmine  Supravital Staining  Dye is applied to the removed tissue specimen  Ex. Neutral Red, Janus Green, Trypan Blue, Thionine, and Toluidine Blue
18 Solvents • Distilled Water • Alcohol • Aniline Water • Phenol
19 Hematoxylin Stains • Aluminum Hematoxylin  Routinely used for Hematoxylin-Eosin staining  Mordant: Aluminum  Examples  Harris Hematoxylin  Routinely used in nuclear staining  Ripened with Mercuric Oxide (HgO)  Nuclear stain in Papanicolaou  Stains sex chromosomes  Addition of glacial acetic acid gives a precise nuclear stain  Employs progressive staining
20 Hematoxylin Stains Aluminum Hematoxylin  Ehrlich’s Hematoxylin  Recommended for bone and cartilage  Recommended nuclear stain for immunohistochemistry and cytochemical staining  Not applicable for frozen sections  Ripened with Sodium Iodate  Glycerin  slows oxidation; prolongs shelf-life  Mayer’s Hematoxylin  Ripened with Sodium Iodate  Primarily a regressive stain
21 Hematoxylin Stains Aluminum Hematoxylin  Delafield’s Hematoxylin  Recommended for blood films  Recommended stain for Microfilariae following Knott’s Concentration  Naturally Ripened (Sunlight)  Corazzi’s Hematoxylin  Used for Frozen Sections  Artificially Ripened with Potassium Iodide  Cole’s Hematoxylin  Ripened with Alcoholic Iodine
22 Hematoxylin Stains • Iron Hematoxylin  Iron  Mordant and Ripening Agent  All are employed in regressive staining  Examples  Weigert’s Hematoxylin  Mordant/Oxidizer: Ferric Ammonium Chloride  Standard Iron Hematoxylin solution  Used in demonstrating muscle fibers and connective tissues  Heidenhain’s Hematoxylin  Mordant/Oxidizer: Iron Alum  For Nuclear and cytoplasmic inclusions  Cytological stain  Result: Gray-Black  Loyez Hematoxylin  for frozen sections
23 Hematoxylin Stains • Tungsten Hematoxylin  Mordant: 1% aqueous phosphotungstic acid  Oxidizer: Potassium Permanganate  Examples  Phosphotungstic Acid Hematoxylin  Naturally Ripened  Progressive stain  For CNS and general staining of tissues • Lead Hematoxylin  For demonstration of granules of endocrine cells of alimentary tract • Copper Hematoxylin  For spermatogenesis studies
24 Eosin Stain • Red acidic dye (xanthene) • Routinely used as a counterstain after hematoxylin and before methylene blue • Stains connective tissues and cytoplasm differentially • Three forms:  Yellow (Eosin Y)  Most common  Water-soluble  Green yellow fluorescence  Eosin B, Erythrosin B  Red color  Eosin S, Ethyl Eosin  Soluble in alcohol
25 H&E Staining Procedure 1. Xylene – 2 mins 2. Xylene – 2 mins 3. Absolute Alcohol – 2 mins 4. Absolute Alcohol – 2 mins 5. 95% Ethanol – 2 mins 6. Wash with water for 2 mins 7. Hematoxylin – 3 mins 8. Wash for 1 min 9. Differentiator (mild acid) – 1 min 10.Wash for 1 min 11.Bluing Agent – 1min 12.Wash for 1 min 13.95% Ethanol – 1 min 14.Eosin – 45 sec 15.95% Ethanol - 1 min 16.100% Ethanol – 1min 17.100% Ethanol – 1min 18.Xylene – 2 mins
26 Lipid Stains • Lysochromes (Oil Soluble Dyes)  Not a real dye  Lack auxochrome  Gives color to lipids because they are more soluble in lipid medium of tissues than in 70% Alcohol  Sudan Black B  most sensitive; Black, stains Triglycerides and Phospholipid  Sudan III  First sudan dye, stains fat in CNS, light orange  Sudan IV  Scharlach R; Most commonly used  Stains neutral fats (TG)  Addition of benzoic acid enhances staining  Color: Red • Oil Red O  neutral fats, lipofuscin • Osmic Acid  unsaturated fats in frozen sections • Nile Blue Sulfate  Red Oxazone  dissolve neutral fats
27 Carbohydrate Stains • Carbohydrates  Forms: Monosaccharides, Polysaccharides  Glycogen  stored form  Mucins  polysaccharides bound to other substances  Acid Mucopolysaccharides  Alcian Blue +  Periodic Acid Schiff Neg  Neutral Mucopolysaccharides (Mucoproteins)  Alcian Blue Neg  Periodic Acid Schiff +
28 Carbohydrate Stains Stain Use Color Unique Feature Periodic Acid Schiff Stains glycogen, mucoproteins, glomerular basement membrane Magenta Intensity of PAS is proportional to content PAS with Diastase Control Method of Choice for Glycogen Red Control – only nuclei is stained Addition of Diastase serves as control Best Carmine Glycogen Bright Red Selective and Highly Specific Langhans Iodine Glycogen Mahogany Brown Oldest Glycogen Stain
29 Carbohydrate Stains Stain Use Color Unique Feature Alcian Blue Acid MPS Blue Most popular method for Acid MPS Azure A Acid MPS Crimson or Red Violet Most useful metachromatic dye; Fixative: Mercuric Chloride Uranyl Acetate Azure Acid MPS Crimson or Red Violet Metachromatic Stain Toluidine Blue Acid MPS Red Purple Metachromatic Stain
30 Carbohydrate Stains Stain Use Color Unique Feature Combine Alcian Blue PAS Technique Acid and Neutral MPS Acid Mucin  Blue Neutral Mucin  Magenta Separate Acid and Neutral Mucin Combined Gomori’s Aldehyde Fuchsin – Alcian Blue Mucins Sulfated Mucins  Purple Carboxylated Mucin  Blue Greater Affinity for Sulfated Mucins Mucicarmine Mucins Red Addition of Aluminum Hydroxide - Southgate’s Mucicarmine Technique stains encapsulated fungi such as Cryptococcus neoformans Hale’s Colloidal (Dialyzed) Iron Technique Mucin Acid Mucin – Dark blue Greater Sensitivity and Intensity compared to Alcian Blue Fluorescent Acridine Orange Acid Mucins Brilliant Orange Fluorescence Uses Fluorochrome Stains Temporarily
31 Protein Stains • Proteins  Histochemically identified using amino acid content  Neutral Buffered Formol Saline – most commonly used fixative  Enzyme Histochemistry  Metal Precipitation – most common technique
32 Protein Stains Stain Use Color Unique Feature Alkaline Fast Green Histones and Protamines Green Proteins Peracetic Acid – Alcian Blue Cystine and Cysteine Blue Green Amino Acids Sakaguchi’s Arginine Orange Red Amino Acids
33 Protein Stains Stain Use Color Gomori Calcium ALP Brownish Black Gomori Lead ACP Black Lead Method 5’-nucleotidase Orange Red Metal Precipitation ATPase Light Brown / Dark Brown
34 Protein Stains Stain Use Color Alpha naphthyl acetate Non-specific esterase Reddish brown Indoxyl Acetate Non Specific esterase Blue Acetylthiocholine Acetylcholinesterase Dark Brown to Blue Tetrazolium Monoamine Oxidase Bluish-Black
35 Nucleic Acid Stains Stain Use Color Unique Feature Fuelgen’s Technique Nuclear DNA Red Purple Most Reliable and Specific Biochemical Technique for DNA Methyl Green – Pyronin DNA and RNA DNA – Green RNA - Red Differential Stain Fluorescein DNA and RNA Apple Green Fluorescent Dye Rhodamine DNA and RNA Orange Red Fluorescent Dye
36 Nucleic Acid Stains Stain Use Color Unique Feature Acridine Orange DNA and RNA DNA – yellow Green RNA – Brick to Orange Red Most common fluorochrome Acriflavine DNA Yellow Alternative to Basic Fuchsin
37 Connective Tissue Stains • Connective Tissue  Produced by Fibroblasts  3 Types of CT Fibers  Reticular  Collagen  Elastic  Deposits  Firbrin  Fibrinoid – eosinophilic, seen in hypersentivity  Hyaline  Amyloid  Semi-translucent, ground-glass, or hyaline eosinophilic substance  Composed of chondroitin sulfate protein complex
38 Connective Tissue Stains Stain Use Color Unique Feature Gomori’s Silver Reticulin Black Van Gieson’s Collagen Pink or Deep Red Simplest Method Masson’s Trichrome Collagen Blue Mallory’s Aniline Blue Collagen, Elastic, Reticular, Hyaline, Amyloid Collagen – Red Elastic – Pale Pink or Yellow Not specific for collagen; excellent and colorful method
39 Connective Tissue Stains Stain Use Color Unique Feature Azocarmine Amyloid and Mucus Colloid Deep Blue Renal Glomerular Membrane Weigert’s Elastic Dark Blue Verhoeff’s Elastic Black Orcein (Tanzer-Unna) Elastic Dark Brown Elastic Fibers of skin Krajian;s Elastic, Fibrin, Amyloid Elastic – Bright Red Fibrin and CT – Dark Blue Rapid Method for Elastic Fibers
40 Connective Tissue Stains Stain Use Color Unique Feature Martius Scarlet Blue Fibrin Red Mallory’s Phosphotungstic Acid Hematoxylin Firbin Dark Blue Gram’s Iodine Amyloid Delicate Purple or Blue Congo Red Amyloid Red Alkaline Congo Red – method of choice Methyl Violet – Crystal Violet Amyloid Purplish Metachromatic Staining Thioflavine Amyloid Silver Blue / Yellow Fluorescence
41 Muscle and Bone Stains Stain Use Color Modified Gomori’s Trichrome Muscle Fibers Red Mallory’s PTAH Muscle Fibers Blue Heidenhain’s Iron Hematoxylin Muscle Fibers Grey Black Lissamine Fast Red Tartrazine Muscle Fibers Red
42 CNS Stains Stain Use Color Unique Feature Bielschowsky’s Neurons, Axons, Neurofibrils Black on Grayish Background Bodian’s Nerve Fibers and Nerve Endings Black Demonstrates neuritic plaques and neurofibrillary tangles for Alzheimer’s Sevier Munger Neuritic Plaques and Neurofibriliary Tangles Black Cresyl Fast Violet Nissl Granules Purple – Dark Blue Weigert Pal Myelin Sheath Blue Black Brilliant stain but time consuming
43 CNS Stains Stain Use Color Unique Feature Kluver and Barrera Luxol Fast Blue Myelin Blue or Green Has counterstain for Nissl Granules Luxol Fast Blue Myelin Blue Green Weil’s Myelin Black Cajal’s Gold Sublimate Astrocytes Black on a light background Schleifsteins Negri Bodies Deep Magenta Victoria Blue Neuroglia For frozen section
44 Microorganism Stains Stain Use Color Unique Feature Wade Fite Mycobacterium leprae Nocardia spp Red Acid-Fast Stain Fite Faraco M. leprae Auramine-Rhodamine M. tuberculosis Golden Yellow Fluorescent dye Toluidine Blue Helicobacter pylori Blue
45 Microorganism Stains Stain Use Color Unique Feature Dieterle Spirochetes Legionella pnemophila Dark Brown to Black Levaditi’s Spirochetes Black Modified Steiner- Steiner Spirochetes Black Also stains Donovan Bodies and Fungi Warthin Starry Spirochetes Black
46 Microorganism Stains Stain Use Color Unique Feature Grocott Methenamine Silver Fungi Black PAS Hyphae, Yeast Magenta Lenundrum’s Phloxine Tartrazine Viral Inclusions Bright Red Orcein HBsAg Brown-Black Giemsa Blood and Marrow Parasites Recommended for Toxoplasma
47 Tissue Pigments & Deposits • Endogenous Pigments – physiologic  Minerals (Iron, Calcium, Copper)  Hematogenous  Hemosiderin  Iron-containing Hb pigment (yellow to brown)  Hematoidin  Iron-free Hb (bright yellow)  Hematin  Heme pigment  Hemozoin Malarial inclusion  Hemofuscin (Lipofuscin + Hemoglobin)  wear and tear pigment; iron-free, brownish  Nonhematogenous  Lipofuscin  Lipid  Melanin  Chromaffin
48 Tissue Pigments & Deposits • Exogenous – foreign  Carbon  most common  Het black on lungs and adjacent lymph nodes (Anthracotic)  Anthracosis  carbon deposition in lungs  Can be confused with melanin  Melanin dissolves in bleaching agents  Silica  Coal Worker’s Pneumoconiosis
49 Tissue Pigments & Deposits Stain Use Color Unique Feature Perl’s Prussian Blue Hemosiderin (Ferric Iron) Deep Blue Turnbull’s Blue Hemosiderin (Ferrous Iron) Bright Blue Benzidine Nitroprusside Hemoglobin and Oxidase Dark Blue Carcinogenic Modified Fouchet’s Bile Pigments Emerald to Blue Green Most common method Gmelin Bile Pigments Blue, Green, Violet Diagnostic for Bile Pigments Stein’s Iodine Bile Pigments Green
50 Tissue Pigments & Deposits Stain Use Color Unique Feature Schmorl’s Ferric Ferricyanide Reducing Substances (bile, lipofuscin, melanin) Dark Blue Gomori’s Aldehyde Fuchsin Lipofuscin Purple Mallory’s Fuchsin Hemofuscin Red Masson Fontana Melanin and Argentaffin Black Calcium Dye Lake Calcium With H&E  Deep Purplish Blue With Sodium Alizarin Red S  Orange Red Skeletal system of Embryos
51 Tissue Pigments & Deposits Stain Use Color Unique Feature Gypsum and Oxalate Soluble Calcium Von Kossa’s Silver Nitrate Calcium Black Indirect Lindquist’s Modified Rhodamine Copper Red to Orange-Red Apathy’s Mountant – recommended mounting medium to prevent copper fading Birefringence Monosodium Urate Calcium Pyrophosphate MSU (-) Pyrophosphate (+) Not a stain but a phenomenon observed under Polarizing Microscope
52 Electron Microscopy Stains • Phosphotungstic Acid  General • Uranyl Acetate  Best stain • Lead  Primary or Secondary Stain
HISTOPATHOLOGIC TECHNIQUES Mounting and Labelling Presented by : Ronel Wynlor D. Abarca, RMT
54 LEARNING OUTCOMES 1. Understand the process of mounting. 2. Know the refractive index of the glass slide and the different mounting media discussed. 3. Differentiate an aqueous mounting media from a resinous mounting media. 4. Explain the process and importance of ringing. 5. Explain the process and importance of labelling. 6. Perform mounting properly.
55 Mounting & Labelling • Mounting  use of a medium and a coverslip to facilitate handling, storage, protection of the tissue section  Media must have a RI almost near to RI of Glass (1.510) • Ringing  Sealing of margins of coverslips to prevent escape of fluid and sticking of slidrd  Kronig Cement (2 parts paraffim mixed with 4-9 parts powdered colophonium resin  Durofix or Dunoyer’s (Cellulose Adhesives) • Labelling  Year  Specimen Number
56 Mounting Media • Aqueous  Composed of Gelatin, Glycerin Jelly, or gum Arabic (Solidifiers)  Glycerol prevents drying and cracking  Sugar  increases refractive index  Merthiolate  Preservative • Resinous  More Viscous  Mostcommon type
57 Aqueous Mounting Media • Water • Glycerol (semi-permanent)  Sections are kept preserved for years if sealed with paraffin  Standard mounting Medium  1.46 RI  Requires ringing • Farrant’s Medium (Gum Arabic Medium)  1.44 RI • Apathy’s Medium  For methylene blue stained nerve preparations • Brun’s Fluid  frozen sections • Levulose (Fructose)
58 Resinous Mounting Media • Canada Balsam - natural  Transparent, colorless, for whole mounts and thick sections  1.524 RI  Diluted in Xylene/Toluene • DPX  Synthetic, For small tissue sections; dries rapidly  1.52 RI • XAM  Synthetic resin mixture dissolved in xylene (75:25) • Clarite  Synthetic, Preferred over DPX • Permount, HSR, Clearmount
59 Resinous Mounting Media • Eukitt® • Poly(butyl methacrylate-co- methyl methacrylate) • 45% resin, 55% xylene • 1.50 RI • Routinely used in both manual and automated coverslipping
60 Coverslip • Histopath Coverslip is rectangular and longer.  1.0 mm thick  24 mm x 50 mm to 24 mm x 60 mm
61 Mounting Process • Soak the stained slide in Xylene. • Pick a slide to be mounted, slightly remove excess xylene, just enough in which the slide is not dripping with xylene. • Add a drop of mounting medium at the a bit lower from the center of the section. • Place cover slip.  Either drop the coverlip and let the medium spread, or  Start at the edge of the slide (away from the frosted end) and gently guide the cover slip by slightly bending it and gently adding pressure as you press the cover slip against the glass slide. • Wipe the edges and the bottom. • Remove excess mounting media and bubbles using applicator stick.
62 Mounting Process
63 Remounting • Soak the mounted slide in Xylene. • Check from time to time if the cover slip can be easily detached from the slide. • Remove the cover slip. • Soak again the slide in Xylene. • Begin the remounting process.
HISTOPATH Staining.pptx

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HISTOPATH Staining.pptx

  • 1.
    1 TISSUE PROCESSING 1. NUMBERING 2.FIXATION 3. DECALCIFICATION 4. DEHYDRATION 5. CLEARING 6. IMPREGNATION 7. EMBEDDING 8. TRIMMING 9. SECTIONING 10.STAINING 11.MOUNTING 12.LABELING
  • 2.
  • 3.
    Creator of allthings, true source of Light and Wisdom, lofty source of all being, graciously let a ray of light of Your brilliance penetrate into the darkness of our understanding and take from us the double darkness in which we have been born, the darkness of sin and ignorance. Give us a sharp sense of understanding, a retentive memory, and the ability to grasp things correctly, and fundamentally. Grant us the talent of being exact in our explanations, and the ability to express ourselves with thoroughness and charm. Point out the beginning, direct the progress, help in the completion. We ask these through Christ, your Son, our Lord, Amen.
  • 4.
    4 LEARNING OUTCOMES 1. Defineand Differentiate an Accentuator from a Mordant. 2. Enumerate the different types of stains. 3. List the stains used for carbohydrates, proteins, nucleic acid. 4. List the stains used for connective, muscle, skeletal, and nervous tissues. 5. List the stains used to visualize microorganisms in tissues. 6. Know which stain is best used for a certain tissue specimen. 7. Explain the principles and process of H&E staining.
  • 5.
    5 STAINING • Process ofdye application on tissue sections to facilitate the study of tissue architectural patterns and cells • Visualization and Enhancement of contrast of cells for microscopic studies • Exploits affinity of tissues and cells to acid and basic solutions
  • 6.
    6 Classifications • According topH  simplest yet most important classification  Acidic Dyes  Affinity towards the Cytoplasm which is a basic structure (Acidophilic)  Ex. Eosin, Picric Acid  Basic Dyes  Affinity towards the Nucleus which is an acidic structure (Basophilic)  Ex. Methylene Blue  Neutral Dyes  Combined Acidic and Basic Dyes  Stains Cytoplasm and Nucleus simultaneously but with different colors  Ex. Romanowsky Stain (Eosin and Azure B or Methylene
  • 7.
    7 Classifications • According toFunction  Histologic Stains  Stains that directly interact with the tissue or cell  Histochemical Stains  Stains that chemically react with tissue constituents  Ex. Perl’s Prussian Blue reacts with Iron constituents  Ex. Periodic Acid Schiff reacts with Glycogen components  Immunohistochemical Stains  Stains that are labelled with antibodies to target specific tissue antigens
  • 8.
    8 Classifications • According toSource  Natural Dyes  Stains that are derived from plant and animal products  Synthetic Dyes  Stains that are chemically generated, usually from hydrocarbons like benzene
  • 9.
    9 Natural Dyes • Hematoxylin Derived from Hematoxylum campechianum (logwood)  Most commonly used for histologic studies  Not in itself a DYE!  Hematin  active coloring agent obtained from ripening  Natural Ripening  Expose the substance to sunlight and air  Slow, 3-4 months  Ex. Ehrlich’s Hematoxylin, Delafield’s Hematoxylin  Artificial Ripening  Induced by oxidation via Hydrogen peroxide mercuric oxide, or potassium permanganate, sodium perborate and sodium iodate  Ex. Mayer’s Hematoxylin, Harris Hematoxylin
  • 10.
    10 Natural Dyes • Hematoxylin BLUING  process of converting soluble red component of hematoxylin into insoluble blue complexes  To ensure that hematoxylin will stain the nucleus blue.  Bluing Agents  Ammonia Water  Scott’s Tap Water  Lithium Carbonate
  • 11.
    11 Natural Dyes • CochinealDyes  Extracted from Coccus cacti bug  With alum  Carmine dye  Chromatin and nuclear stain for fresh tissue and smear preparation  With picric acid  Picrocarmine  Neuropathological stain  With aluminum chloride  Best’s carmine  Demonstration of glycogen
  • 12.
    12 Natural Dyes • OrceinDyes  Extracted from lichens  Used to stain elastic fibers especially on the skin (brown color)  Colorless, treated with ammonia, exposed to air to produce blue/violet color  Ex. Taenzer-Unna’s  Tenzer’s orcein + Unna’s Polychrome methylene blue
  • 13.
    13 Techniques • According toDye Application  Direct Staining  Stain is applied directly to tissue as is  Use of aqueous or alcoholic dye solutions like Methylene Blue to directly impart a color  Indirect Staining  Stain is applied with a mordant to bind the dye to the tissue or cell  Mordant  serves as the bridge between tissue and dye Accentuator  hastens staining process Ex. Potassium Hydroxide in Loeffler’s Methylene Blue Ex. Phenol in Carbol Fuchsin
  • 14.
    14 Techniques • According toSequence  Progressive Staining  Tissues are stained in a definite and specific sequence  Staining proceeds with specific time intervals until desired color is achieved.  Differentiator is not REQUIRED (may be present)  Differentiator is used to remove background staining only  Retrogressive Staining  Tissue is overstained and a differentiator is applied until the desired color is achieved  Alters the nuclear stain  Differentiation process observed under microscope
  • 15.
    15 Techniques • According toColor Contrast  Simple Staining  Tissues are stained with a single dye and therefore will all appear similar in color  Differential Staining  Use of two dyes, the primary stain which imparts color to the tissue element of concern, and the counterstain which imparts a contrasting color to the other structures.
  • 16.
    16 Counterstains Cytoplasmic Nuclear Red YellowGreen Red Blue Eosin Y Picric Acid Light Green SF Neutral Red Methylene Blue Eosin B Orange G Lissamine Green Safranin O Toluidine Blue Phloxine B Rose Bengal Carmine Celestine Blue Hematoxylin
  • 17.
    17 Techniques • Metachromatic Staining Impartment of a color to the tissue that is different from the original color of the dye • Vital Staining  staining of cells or tissues without killing them  Intravital Staining  Dye is injected to a body part  Ex. India Ink, Lithium, and Carmine  Supravital Staining  Dye is applied to the removed tissue specimen  Ex. Neutral Red, Janus Green, Trypan Blue, Thionine, and Toluidine Blue
  • 18.
    18 Solvents • Distilled Water •Alcohol • Aniline Water • Phenol
  • 19.
    19 Hematoxylin Stains • AluminumHematoxylin  Routinely used for Hematoxylin-Eosin staining  Mordant: Aluminum  Examples  Harris Hematoxylin  Routinely used in nuclear staining  Ripened with Mercuric Oxide (HgO)  Nuclear stain in Papanicolaou  Stains sex chromosomes  Addition of glacial acetic acid gives a precise nuclear stain  Employs progressive staining
  • 20.
    20 Hematoxylin Stains Aluminum Hematoxylin Ehrlich’s Hematoxylin  Recommended for bone and cartilage  Recommended nuclear stain for immunohistochemistry and cytochemical staining  Not applicable for frozen sections  Ripened with Sodium Iodate  Glycerin  slows oxidation; prolongs shelf-life  Mayer’s Hematoxylin  Ripened with Sodium Iodate  Primarily a regressive stain
  • 21.
    21 Hematoxylin Stains Aluminum Hematoxylin Delafield’s Hematoxylin  Recommended for blood films  Recommended stain for Microfilariae following Knott’s Concentration  Naturally Ripened (Sunlight)  Corazzi’s Hematoxylin  Used for Frozen Sections  Artificially Ripened with Potassium Iodide  Cole’s Hematoxylin  Ripened with Alcoholic Iodine
  • 22.
    22 Hematoxylin Stains • IronHematoxylin  Iron  Mordant and Ripening Agent  All are employed in regressive staining  Examples  Weigert’s Hematoxylin  Mordant/Oxidizer: Ferric Ammonium Chloride  Standard Iron Hematoxylin solution  Used in demonstrating muscle fibers and connective tissues  Heidenhain’s Hematoxylin  Mordant/Oxidizer: Iron Alum  For Nuclear and cytoplasmic inclusions  Cytological stain  Result: Gray-Black  Loyez Hematoxylin  for frozen sections
  • 23.
    23 Hematoxylin Stains • TungstenHematoxylin  Mordant: 1% aqueous phosphotungstic acid  Oxidizer: Potassium Permanganate  Examples  Phosphotungstic Acid Hematoxylin  Naturally Ripened  Progressive stain  For CNS and general staining of tissues • Lead Hematoxylin  For demonstration of granules of endocrine cells of alimentary tract • Copper Hematoxylin  For spermatogenesis studies
  • 24.
    24 Eosin Stain • Redacidic dye (xanthene) • Routinely used as a counterstain after hematoxylin and before methylene blue • Stains connective tissues and cytoplasm differentially • Three forms:  Yellow (Eosin Y)  Most common  Water-soluble  Green yellow fluorescence  Eosin B, Erythrosin B  Red color  Eosin S, Ethyl Eosin  Soluble in alcohol
  • 25.
    25 H&E Staining Procedure 1.Xylene – 2 mins 2. Xylene – 2 mins 3. Absolute Alcohol – 2 mins 4. Absolute Alcohol – 2 mins 5. 95% Ethanol – 2 mins 6. Wash with water for 2 mins 7. Hematoxylin – 3 mins 8. Wash for 1 min 9. Differentiator (mild acid) – 1 min 10.Wash for 1 min 11.Bluing Agent – 1min 12.Wash for 1 min 13.95% Ethanol – 1 min 14.Eosin – 45 sec 15.95% Ethanol - 1 min 16.100% Ethanol – 1min 17.100% Ethanol – 1min 18.Xylene – 2 mins
  • 26.
    26 Lipid Stains • Lysochromes(Oil Soluble Dyes)  Not a real dye  Lack auxochrome  Gives color to lipids because they are more soluble in lipid medium of tissues than in 70% Alcohol  Sudan Black B  most sensitive; Black, stains Triglycerides and Phospholipid  Sudan III  First sudan dye, stains fat in CNS, light orange  Sudan IV  Scharlach R; Most commonly used  Stains neutral fats (TG)  Addition of benzoic acid enhances staining  Color: Red • Oil Red O  neutral fats, lipofuscin • Osmic Acid  unsaturated fats in frozen sections • Nile Blue Sulfate  Red Oxazone  dissolve neutral fats
  • 27.
    27 Carbohydrate Stains • Carbohydrates Forms: Monosaccharides, Polysaccharides  Glycogen  stored form  Mucins  polysaccharides bound to other substances  Acid Mucopolysaccharides  Alcian Blue +  Periodic Acid Schiff Neg  Neutral Mucopolysaccharides (Mucoproteins)  Alcian Blue Neg  Periodic Acid Schiff +
  • 28.
    28 Carbohydrate Stains Stain UseColor Unique Feature Periodic Acid Schiff Stains glycogen, mucoproteins, glomerular basement membrane Magenta Intensity of PAS is proportional to content PAS with Diastase Control Method of Choice for Glycogen Red Control – only nuclei is stained Addition of Diastase serves as control Best Carmine Glycogen Bright Red Selective and Highly Specific Langhans Iodine Glycogen Mahogany Brown Oldest Glycogen Stain
  • 29.
    29 Carbohydrate Stains Stain UseColor Unique Feature Alcian Blue Acid MPS Blue Most popular method for Acid MPS Azure A Acid MPS Crimson or Red Violet Most useful metachromatic dye; Fixative: Mercuric Chloride Uranyl Acetate Azure Acid MPS Crimson or Red Violet Metachromatic Stain Toluidine Blue Acid MPS Red Purple Metachromatic Stain
  • 30.
    30 Carbohydrate Stains Stain UseColor Unique Feature Combine Alcian Blue PAS Technique Acid and Neutral MPS Acid Mucin  Blue Neutral Mucin  Magenta Separate Acid and Neutral Mucin Combined Gomori’s Aldehyde Fuchsin – Alcian Blue Mucins Sulfated Mucins  Purple Carboxylated Mucin  Blue Greater Affinity for Sulfated Mucins Mucicarmine Mucins Red Addition of Aluminum Hydroxide - Southgate’s Mucicarmine Technique stains encapsulated fungi such as Cryptococcus neoformans Hale’s Colloidal (Dialyzed) Iron Technique Mucin Acid Mucin – Dark blue Greater Sensitivity and Intensity compared to Alcian Blue Fluorescent Acridine Orange Acid Mucins Brilliant Orange Fluorescence Uses Fluorochrome Stains Temporarily
  • 31.
    31 Protein Stains • Proteins Histochemically identified using amino acid content  Neutral Buffered Formol Saline – most commonly used fixative  Enzyme Histochemistry  Metal Precipitation – most common technique
  • 32.
    32 Protein Stains Stain UseColor Unique Feature Alkaline Fast Green Histones and Protamines Green Proteins Peracetic Acid – Alcian Blue Cystine and Cysteine Blue Green Amino Acids Sakaguchi’s Arginine Orange Red Amino Acids
  • 33.
    33 Protein Stains Stain UseColor Gomori Calcium ALP Brownish Black Gomori Lead ACP Black Lead Method 5’-nucleotidase Orange Red Metal Precipitation ATPase Light Brown / Dark Brown
  • 34.
    34 Protein Stains Stain UseColor Alpha naphthyl acetate Non-specific esterase Reddish brown Indoxyl Acetate Non Specific esterase Blue Acetylthiocholine Acetylcholinesterase Dark Brown to Blue Tetrazolium Monoamine Oxidase Bluish-Black
  • 35.
    35 Nucleic Acid Stains StainUse Color Unique Feature Fuelgen’s Technique Nuclear DNA Red Purple Most Reliable and Specific Biochemical Technique for DNA Methyl Green – Pyronin DNA and RNA DNA – Green RNA - Red Differential Stain Fluorescein DNA and RNA Apple Green Fluorescent Dye Rhodamine DNA and RNA Orange Red Fluorescent Dye
  • 36.
    36 Nucleic Acid Stains StainUse Color Unique Feature Acridine Orange DNA and RNA DNA – yellow Green RNA – Brick to Orange Red Most common fluorochrome Acriflavine DNA Yellow Alternative to Basic Fuchsin
  • 37.
    37 Connective Tissue Stains •Connective Tissue  Produced by Fibroblasts  3 Types of CT Fibers  Reticular  Collagen  Elastic  Deposits  Firbrin  Fibrinoid – eosinophilic, seen in hypersentivity  Hyaline  Amyloid  Semi-translucent, ground-glass, or hyaline eosinophilic substance  Composed of chondroitin sulfate protein complex
  • 38.
    38 Connective Tissue Stains StainUse Color Unique Feature Gomori’s Silver Reticulin Black Van Gieson’s Collagen Pink or Deep Red Simplest Method Masson’s Trichrome Collagen Blue Mallory’s Aniline Blue Collagen, Elastic, Reticular, Hyaline, Amyloid Collagen – Red Elastic – Pale Pink or Yellow Not specific for collagen; excellent and colorful method
  • 39.
    39 Connective Tissue Stains StainUse Color Unique Feature Azocarmine Amyloid and Mucus Colloid Deep Blue Renal Glomerular Membrane Weigert’s Elastic Dark Blue Verhoeff’s Elastic Black Orcein (Tanzer-Unna) Elastic Dark Brown Elastic Fibers of skin Krajian;s Elastic, Fibrin, Amyloid Elastic – Bright Red Fibrin and CT – Dark Blue Rapid Method for Elastic Fibers
  • 40.
    40 Connective Tissue Stains StainUse Color Unique Feature Martius Scarlet Blue Fibrin Red Mallory’s Phosphotungstic Acid Hematoxylin Firbin Dark Blue Gram’s Iodine Amyloid Delicate Purple or Blue Congo Red Amyloid Red Alkaline Congo Red – method of choice Methyl Violet – Crystal Violet Amyloid Purplish Metachromatic Staining Thioflavine Amyloid Silver Blue / Yellow Fluorescence
  • 41.
    41 Muscle and BoneStains Stain Use Color Modified Gomori’s Trichrome Muscle Fibers Red Mallory’s PTAH Muscle Fibers Blue Heidenhain’s Iron Hematoxylin Muscle Fibers Grey Black Lissamine Fast Red Tartrazine Muscle Fibers Red
  • 42.
    42 CNS Stains Stain UseColor Unique Feature Bielschowsky’s Neurons, Axons, Neurofibrils Black on Grayish Background Bodian’s Nerve Fibers and Nerve Endings Black Demonstrates neuritic plaques and neurofibrillary tangles for Alzheimer’s Sevier Munger Neuritic Plaques and Neurofibriliary Tangles Black Cresyl Fast Violet Nissl Granules Purple – Dark Blue Weigert Pal Myelin Sheath Blue Black Brilliant stain but time consuming
  • 43.
    43 CNS Stains Stain UseColor Unique Feature Kluver and Barrera Luxol Fast Blue Myelin Blue or Green Has counterstain for Nissl Granules Luxol Fast Blue Myelin Blue Green Weil’s Myelin Black Cajal’s Gold Sublimate Astrocytes Black on a light background Schleifsteins Negri Bodies Deep Magenta Victoria Blue Neuroglia For frozen section
  • 44.
    44 Microorganism Stains Stain UseColor Unique Feature Wade Fite Mycobacterium leprae Nocardia spp Red Acid-Fast Stain Fite Faraco M. leprae Auramine-Rhodamine M. tuberculosis Golden Yellow Fluorescent dye Toluidine Blue Helicobacter pylori Blue
  • 45.
    45 Microorganism Stains Stain UseColor Unique Feature Dieterle Spirochetes Legionella pnemophila Dark Brown to Black Levaditi’s Spirochetes Black Modified Steiner- Steiner Spirochetes Black Also stains Donovan Bodies and Fungi Warthin Starry Spirochetes Black
  • 46.
    46 Microorganism Stains Stain UseColor Unique Feature Grocott Methenamine Silver Fungi Black PAS Hyphae, Yeast Magenta Lenundrum’s Phloxine Tartrazine Viral Inclusions Bright Red Orcein HBsAg Brown-Black Giemsa Blood and Marrow Parasites Recommended for Toxoplasma
  • 47.
    47 Tissue Pigments &Deposits • Endogenous Pigments – physiologic  Minerals (Iron, Calcium, Copper)  Hematogenous  Hemosiderin  Iron-containing Hb pigment (yellow to brown)  Hematoidin  Iron-free Hb (bright yellow)  Hematin  Heme pigment  Hemozoin Malarial inclusion  Hemofuscin (Lipofuscin + Hemoglobin)  wear and tear pigment; iron-free, brownish  Nonhematogenous  Lipofuscin  Lipid  Melanin  Chromaffin
  • 48.
    48 Tissue Pigments &Deposits • Exogenous – foreign  Carbon  most common  Het black on lungs and adjacent lymph nodes (Anthracotic)  Anthracosis  carbon deposition in lungs  Can be confused with melanin  Melanin dissolves in bleaching agents  Silica  Coal Worker’s Pneumoconiosis
  • 49.
    49 Tissue Pigments &Deposits Stain Use Color Unique Feature Perl’s Prussian Blue Hemosiderin (Ferric Iron) Deep Blue Turnbull’s Blue Hemosiderin (Ferrous Iron) Bright Blue Benzidine Nitroprusside Hemoglobin and Oxidase Dark Blue Carcinogenic Modified Fouchet’s Bile Pigments Emerald to Blue Green Most common method Gmelin Bile Pigments Blue, Green, Violet Diagnostic for Bile Pigments Stein’s Iodine Bile Pigments Green
  • 50.
    50 Tissue Pigments &Deposits Stain Use Color Unique Feature Schmorl’s Ferric Ferricyanide Reducing Substances (bile, lipofuscin, melanin) Dark Blue Gomori’s Aldehyde Fuchsin Lipofuscin Purple Mallory’s Fuchsin Hemofuscin Red Masson Fontana Melanin and Argentaffin Black Calcium Dye Lake Calcium With H&E  Deep Purplish Blue With Sodium Alizarin Red S  Orange Red Skeletal system of Embryos
  • 51.
    51 Tissue Pigments &Deposits Stain Use Color Unique Feature Gypsum and Oxalate Soluble Calcium Von Kossa’s Silver Nitrate Calcium Black Indirect Lindquist’s Modified Rhodamine Copper Red to Orange-Red Apathy’s Mountant – recommended mounting medium to prevent copper fading Birefringence Monosodium Urate Calcium Pyrophosphate MSU (-) Pyrophosphate (+) Not a stain but a phenomenon observed under Polarizing Microscope
  • 52.
    52 Electron Microscopy Stains •Phosphotungstic Acid  General • Uranyl Acetate  Best stain • Lead  Primary or Secondary Stain
  • 53.
    HISTOPATHOLOGIC TECHNIQUES Mounting andLabelling Presented by : Ronel Wynlor D. Abarca, RMT
  • 54.
    54 LEARNING OUTCOMES 1. Understandthe process of mounting. 2. Know the refractive index of the glass slide and the different mounting media discussed. 3. Differentiate an aqueous mounting media from a resinous mounting media. 4. Explain the process and importance of ringing. 5. Explain the process and importance of labelling. 6. Perform mounting properly.
  • 55.
    55 Mounting & Labelling •Mounting  use of a medium and a coverslip to facilitate handling, storage, protection of the tissue section  Media must have a RI almost near to RI of Glass (1.510) • Ringing  Sealing of margins of coverslips to prevent escape of fluid and sticking of slidrd  Kronig Cement (2 parts paraffim mixed with 4-9 parts powdered colophonium resin  Durofix or Dunoyer’s (Cellulose Adhesives) • Labelling  Year  Specimen Number
  • 56.
    56 Mounting Media • Aqueous Composed of Gelatin, Glycerin Jelly, or gum Arabic (Solidifiers)  Glycerol prevents drying and cracking  Sugar  increases refractive index  Merthiolate  Preservative • Resinous  More Viscous  Mostcommon type
  • 57.
    57 Aqueous Mounting Media •Water • Glycerol (semi-permanent)  Sections are kept preserved for years if sealed with paraffin  Standard mounting Medium  1.46 RI  Requires ringing • Farrant’s Medium (Gum Arabic Medium)  1.44 RI • Apathy’s Medium  For methylene blue stained nerve preparations • Brun’s Fluid  frozen sections • Levulose (Fructose)
  • 58.
    58 Resinous Mounting Media •Canada Balsam - natural  Transparent, colorless, for whole mounts and thick sections  1.524 RI  Diluted in Xylene/Toluene • DPX  Synthetic, For small tissue sections; dries rapidly  1.52 RI • XAM  Synthetic resin mixture dissolved in xylene (75:25) • Clarite  Synthetic, Preferred over DPX • Permount, HSR, Clearmount
  • 59.
    59 Resinous Mounting Media •Eukitt® • Poly(butyl methacrylate-co- methyl methacrylate) • 45% resin, 55% xylene • 1.50 RI • Routinely used in both manual and automated coverslipping
  • 60.
    60 Coverslip • Histopath Coverslipis rectangular and longer.  1.0 mm thick  24 mm x 50 mm to 24 mm x 60 mm
  • 61.
    61 Mounting Process • Soakthe stained slide in Xylene. • Pick a slide to be mounted, slightly remove excess xylene, just enough in which the slide is not dripping with xylene. • Add a drop of mounting medium at the a bit lower from the center of the section. • Place cover slip.  Either drop the coverlip and let the medium spread, or  Start at the edge of the slide (away from the frosted end) and gently guide the cover slip by slightly bending it and gently adding pressure as you press the cover slip against the glass slide. • Wipe the edges and the bottom. • Remove excess mounting media and bubbles using applicator stick.
  • 62.
  • 63.
    63 Remounting • Soak themounted slide in Xylene. • Check from time to time if the cover slip can be easily detached from the slide. • Remove the cover slip. • Soak again the slide in Xylene. • Begin the remounting process.

Editor's Notes

  • #2 AFTER SECTIONING  SECTION IS CLEARED WITH XYLENE  DEHYDRATED IN DESCENDING GRADE OF ALCOHOL - STAINING  dehydration, clearing  mounting
  • #13 Also the source of litmus paper (pH paper indicators)
  • #66 https://youtu.be/uazEYTbMPZ0