Histopathologic Techniques - 05 Staining and Mounting

STAINING & MOUNTING OUTLINE • Introduction • Special and Additional Stains o Staining of Carbohydrates • Staining o Proteins o Staining Categories o Fats of Lipids o Staining Techniques o Enzyme o Routine Hematoxylin & Eosin o Bone Marrow & Blood Staining Elements o Natural Dyes o Connective Tissue o Synthetic Dyes o Nucleic Acid • Mounting o Muscle and Bone o Types of Mountant o Central Nervous System o Ringing o Tissue Pigments & Deposits o Labelling o Microorganisms • Additional Info o Other Special Stains • STAINING TECHNIQUES • INTRODUCTION • • Staining is the process of applying dyes on the sections to see and study the architectural pattern of the tissue and physical characteristics of the cells. This is made possible because different tissues and cells display varying affinities for most dyes and stains during the process, so that they become more visible, morphologic changes are more easily identified, and the presence or absence of disease process can be established. Physical characteristics and structural relationships of tissues and their cells are thereby studied and evaluated. In order to create permanent slides for histoparhology, mounting of stained tissue section must be done. Mounting media holds the specimen in place, in between the slide and coverslip, and allow the specimen to be stored for years. Mounting media may also provide the maximum degree of transparency to stained tissue section. • STAINING • • Process of applying dyes on the section to see and study the architectural pattern of the tissue and physical characteristics of cells. Involves application of dyes or stains on tissue sections to allow their study under the microscope • STAINING CATEGORIES • • Histological o Tissue components are demonstrated by direct interaction with a dye or staining solution o Active tissue component is colored o Micro-anatomical stains, bacterial stains, Specific tissue stains o If there is a need to demonstrate nucleus or cytoplasm of cells, this is used o Gram stain, acid fast stain, Wright stain, Giemsa stain, Methylene Blue, carbol fuchsin, crystal violet o For bacteria: methylene blue carbol fuchsin, crystal violet Histochemical/Histochemistry o Tissue components are studied through chemical reaction o Demonstrate chemical components o Demonstration of fats and carbohydrates Perl’s Prussian blue reaction - used for hemoglobin Periodic acid Schiff - used for carbohydrates Sudan Dyes - used for fats ▪ Sudan Black, Sudan III Immunohistochemical staining o Instead of stains/dyes, we use antibodies o Combination of histochemical and immunologic technique o Detection of phenotypic markers that are detected by antibodies o Used to demonstrate tissue antigens o i.e. monoclonal, polyclonal o o o HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES • • Direct Staining o Uses aqueous or alcoholic dye solutions to produce a color o Color of the dye is also the resulting color o Uses only 1 dye o Tissue + dye = color o i.e., Methylene Blue (resulting color is blue) Indirect staining o Uses a dye with help of either mordant or accentuator o Mordant ▪ Substance that serves as link or bridge between the dye and the tissue ▪ i.e. Potassium alum – Meyer’s Hematoxylin stain ▪ i.e. Iron (ferric ammonium chloride) – Weigert’s Hematoxylin stain ▪ other examples: copper, phosphotungstic acid ▪ general: alum, iron, copper, PTAH ▪ specific: potassium alum, ferric ammonium sulfate, ferric ammonium chloride o Accentuator ▪ Substance that heightens the color intensity and selectivity of the dye ▪ Increases the staining power of the dye ▪ i.e. KOH in Loeffler’s Methylene Blue ▪ i.e. Phenol in Carbol Fuchsin and Carbol Thionine Progressive Staining o Tissue elements re stained in a definite sequence. o Involves gradual application of dye (little by little) to tissues until the desired color is achieved o Tissue will not be overstained o There will be no excess dye o Decolorization is not performed because there is no excess dye Regressive Staining o Tissue is first overstained therefore, there will be excess dye o The excess dye is removed selectively o Excess dye must be removed by decolorization o Decolorization/Differentiation ▪ Commonly used is acid alcohol because it can remove both acid and basic dye ▪ Selective removal of dye ▪ RULE: Use basic solution in order to remove the excess acid dye or vice versa Counterstaining o Involves application of a different dye/color to produce contrast in background o Acid dye, cytoplasmic stain Eosin – a red acid dye and also classified as the cytoplasmic stain (most commonly used) o RULE: acid structure = basic dye (or vice versa) o Forms: ▪ Eosin Y - yellowish ▪ Eosin B - Bluish ▪ Eosin S - Ethyl Eosin Metachromatic Staining o Uses a dye that produce a different color o Staining tissue with a color that is different from the stain itself o Examples : Methyl violet or crystal violet; Cresyl blue for reticulocytes; Safranin; Bismarck brown; Basic fuchsin; Methylene blue; Thionine; Toluidine blue ; Azure A, B, C o For epithelial mucins, amyloid, cartilage, and mast cell granules Orthochromatic Staining o Tissues is stained with the same shade/hue as that of the dye, not exactly the color of the dye Vital Staining o Used to demonstrate living cells—cells that are viable and fresh o 2 vital staining methods o Intravital ▪ Dye/ stain is injected to any part of the living body (animal) ▪ i.e lithium, carmine, india ink o Supravital – stain is applied immediately to tissue after its removal from living body ▪ Neutral red - considered as the best vital stain ▪ Janus green - is used for mitochondria ▪ Trypan blue; Nile blue ; Thionine ; Toluidine blue Metallic Impregnation o Tissue elements are demonstrated NOT by stains but by colorless solutions of metallic salts o When applied to tissues, these are not absorbed by the tissues o Metallic salts can leave black deposits on tissue surface o i.e., ammoniacal silver, silver nitrate, and gold chloride o Can be used for spirochetes in microbiology o • • • • o o o o o • • • • • • • • • • • • Routine fixative: 10% formalin Routine dehydrating: ascending concentrations of ethyl alcohol Routine clearing: xylene Routine decalcification: 5-10% nitric acid Routine infiltrating and embedding medium: paraffin wax (MP: 56C) Routine sectioning microtome: rotary microtome Routine stains: H&E H & E staining consists of different reagents Most common method utilized for microanatomical studies of tissues Regressive staining method Reagents in H & E Staining: o Initial Xylene bath - For further deparaffinization or to completely remove the wax prior to staining o Descending grades of alcohol - High to lower concentration; to hydrate tissues o Hematoxylin - Primary dye; nuclear stain; classified as the basic dye Set up of H & E staining Tissue sections will pass through different reagents H & E Result: o Nuclei – blue to blue black o Karyosome – dark blue o Cytoplasm – pale pink o RBCs, eosinophilic granules, keratin-bright orange red o Calcium and decalcified bone-purplish blue o Decalcified bone matrix, collagen and osteoidpink o Muscle fibers- deep pink NATURAL DYES • • derived from plants and animals i.e. Hematoxylin, Cochineal dyes, orcein, saffron HEMATOXYLIN • • • ROUTINE HEMATOXYLIN & EOSIN STAINING • • Acid alcohol - Acts as decolorizer Ammonia water - Bluing agent Eosin - Counterstain, as to pH it is the acid dye; used for cytoplasmic staining Ascending grades of alcohol - Low to high concentration; used for dehydration Last xylene bath - Used in clearing prior to mounting • • • Most important natural dye Not a stain, active coloring agent is hematein Formed from the oxidation of hematoxylin either by ripening (expose material to sunlight) or by adding oxidizing agents like: Hydrogen peroxide; Mercuric peroxide ; Sodium iodate ; Sodium perborate; Potassium permanganate Source of hematoxylin is from a tree o Remove hemoxylin from tree, from hematoxylin, the active agent hematein is extracted o To extract hematein, ripening is done where material is exposed to sunlight ▪ To accelerate process, oxidizing agents are used o After ripening and oxidizing agents, the active coloring agent hematein or ripened hematoxylin is produced Ripened hematoxylin is weak and therefore cannot be used on its own o Ripened hematoxylin requires incorporation of mordants (potassium alum and iron) Used in combination with alum, iron, chromium & copper salts • • • • • • Alum Hematoxylin o Uses potassium alum as mordant Ripening agent/Notes Ehrlich’s hematoxylin Sodium iodate as ripening agent Harris hematoxylin Mercuric oxide as ripening agent; Used for exfoliative cytology and demonstrating sex chromosomes Cole’s hematoxylin Alcoholic iodine; Mayer’s hematoxylin Sodium iodate as ripening agent; With citric acid & chloral hydrate as preservative; Preferred for immunohistochemistry Delafield’s Glycerol to stabilize oxidation process; smells like wine; gives a purple-red shade/hue Gill’s Sodium iodate as ripening agent; to stain mucin in goblet cells Carazzi’s Potassium iodate as ripening agent Iron Hematoxylin o In general for photomicrography o Uses iron as mordant Mordant Weigert’s Hematoxylin Ferric ammonium chloride (For Muscle fibers and CT) (also the ripening agent) Heidenhain’s Hematoxylin Ferric ammonium sulfate (For nuclei & cytoplasmic (also the ripening agent) Inclusion i.e. mitochondria) Copper Hematoxylin - uses copper as mordant o Used to demonstrate spermatogenesis Tungsten Hematoxylin - mordant is phosphotungstic acid Lead Hematoxylin – demo of granules of endocrine cells of alimentary tract Molybdenum Hematoxylin - demo of collagen, reticulin and argentaffin cells LYSOCHROME DYES • • • • • • MOUNTING • • • • • • • OTHER NATURAL DYES • • • Cochineal dyes – extracted from bug treat ed w/ alum to produce carmine o With picric acid will produce picrocarmine (for neuropathological studies) o With Aluminum chloride will produce dye Best’s carmine (for Glycogen) Orcein – vegetable dye extracted from lichens, used for staining elastic fibers Litmus - vegetable dye extracted from lichens, not used as cytological stain but used as pH indicator due to its poor staining properties a.k.a Coal Tar dyes or Aniline dyes Artificially made Two Components o Chromophore - responsible for coloring property (imparts color) o Auxochrome - responsible for dyeing property (retains imparted color) Regarded as the last step in tissue processing o In practice, labelling is the last step Process of applying a mounting medium / mountant between the stained tissue section and the coverslip To protect tissues from damage/injury For permanent safekeeping and to facilitate easy handling and storage To prevent bleaching of sections Mounting medium/mountant should have a refractive index close to that of the slide which is 1.518 o Mountant should be as clear as coverslip and slides o Mountant is place between the tissue and coverslop Processed slides have retention period (5-10 years) TYPES OF MOUNTANT • SYNTHETIC DYES • • • Aka oil-soluble dyes Dyes without auxochrome component Used to demonstrate fats Sudan Black B - classified as histochemical stain o Most sensitive among oil-soluble dyes Scharlach R - classified as histochemical stain o a.k.a Sudan IV o For neutral lipids, staining them red Sudan III - classified as histochemical stain o First Sudan dye to be introduced in histochemistry o Good fat stain for CNS tissues • Resinous mountant o Used for preparations that have been dehydrated and cleared in xylene o Permanent mounting media o Can be synthetic or natural o Canada balsam (1.524) ▪ Natural mountant extracted from Abus Balsamea ▪ Commonly used ▪ Disadvantage: darkens with age and it causes gradual fading of many stains o DPX (1.532) o XAM (1.52) o CLARITE (.544) Aqueous mountant o Designed to mount water-miscible preparations o Considered semi-permanent or temporary o Glycerin jelly (1.47) - aka Kaiser’s o Farrant’s (1.43) - aka Gum Arabic o Apathy’s (1.52) - for methylene blue stained nerve preparations o Bruns Fluid - for mounting frozen sections directly from water o Water - not used in practice ▪ Has very low refractive index, Evaporates easily ▪ Not allow examination of tissues under OIO • • • • RINGING • Not a mandatory step in tissue processing Process of sealing the margins of the coverslip To prevent escape of fluid; Prevents sticking of slides upon storage; and to immobilize coverslip (to keep coverslip from moving) Uses ringing media o Kronig cement and Durofix • • LABELLING • Indicating year and specimen number for proper identification • ADDITIONAL INFO • • • • • • • • • Zinc sulfate - Substitute for mercuric chloride (metallic fixative) o Zinc sulfate is toxic Types of Fixative as to composition (Simple & Compound) o Simple - with only one component substance ▪ Eg. glacial acetic acid o Compound - combine two or more simple fixative Factors to be considered when choosing the right decalcifier/decalcifying agent o Urgency of the case o Staining technique to be used o Degree of mineralization Four ways to decalcify tissues o Electrolytic method o Ion-exchange resin o Use of EDTA o Use of acid Carnoy’s - Fixative to be avoided prior to decalcification o It contains chloroform that can be toxic Factors affecting decalcification rate o Tissue structure o Size and consistency of specimen ▪ Large specimen = longer decalcification o Temperature (18-30C) ▪ Increase temp (37C) = shorten process o Volume and concentration of decalcifying ▪ Increased concentration and volume = shorten process Types of Autotechnicon (automatic tissue processor) o Can only do fixation to infiltration o 1 - tissue transfer type / dip and dunk type ▪ Tissue is transferred from one reagent to another ▪ Most common type o 2 - fluid transfer type or enclosed type ▪ Tissue remains within a chamber; the reagents required in each process is pumped in and out of the chamber Type of dyes according to pH (acid dyes, basic dyes, neutral dyes or amphoteric dyes) o Stain acid structure with basic dye (and vice versa) o Neutral dye - Wright stain o Basic dye - Hematoxylin o Acid dye - Eosin Stains for frozen sections o H & E stain o Thionine o Polychrome methylene Blue o Alcoholic Pinacyanol • • Classification of Dyes as to pH o Acid dye o Basic o Neutral / amphoteric dye - Giemsa, Leishman, Irishman Solvents Used for Stains o Water, Alcohol (ethyl or methyl), Aniline water, Phenol Collodionization - Process of coating slide with thin celloidin solutions. Done if sections will be subjected to strong alkali or acid solutions and for tissues containing glycogen for demonstration Re-Staining of Old Sections 1. Immersion in xylene for 24 hrs 2. Removal of coverslip 3. Immersion in xylene 30 minutes 4. Immersion in 0.5% potassium permanganate 5-10 minutes, rinse with tap water 5. Immersion in 5% oxalic acid 6. Re-stain Retention Period o Guidelines For Record And Specimen Retention (Source: Henry’s) o Records: ▪ Clinical pathology laboratory reports - 10 yrs ▪ Autopsy forensic reports - indefinitely ▪ Surgical pathology and bone marrow Reports - 10 years ▪ Cytogenetics report - 20 years o Specimens: ▪ Pathology /Bone marrow slides - 10 years ▪ Pathology blocks - 10 years ▪ Cytogenetic slides - 3 years ▪ Cytogenetic diagnostic images - 20 years Tissue Processing SPECIAL AND ADDITIONAL STAINS STAINING OF CARBOHYDRATES • • Periodic Acid Schiff - Glycogen o used for the demonstration of glycogen specially when it uses the Diastase Method; may also stain Mucoproteins, Mucin, Carbohydrates, Glycoproteins, Glycolipids, Unsaturated Lipids and Phospholipids. (+Red) Best Carmine Method - Glycogen, Mast Cell Granules, Fibrin And Mucin o has a good affinity to glycogen (bright red) due to alkaline carminic acid ; weakly stains mast cell granules, fibrin and mucin • • • • • • • • • • Langhan’s Iodine Method - Glycogen, and Amyloid o obsolete stain, not specific for glycogen, rapid but tends to fade after a few months Metachromatic Staining- Azure A - Glycosaminoglycans o gives a red-purple stain Metachromatic Toluidine Blue - Glycosaminoglycans o gives a red-purple stain Colloidal (Dialyzed) Iron Technique - Acid Mucin o is absorbed onto tissue containing acid mucin at low ph, and visualized by it’s conversion to ferric cyanide. Alcian Blue Technique - Acid Mucopolysaccharide o most popular method for acid mucin (+Blue) Combined Alcian Blue-pas-hematoxylin Technique Differentiate Acid Mucin from Neutral Mucin o Neutral mucins are stained by PAS while acid mucin are stained by Alcian blue. Southgate’s Mucicarmine Technique - Encapsulated fungi: C. Neoformans Gomori’s Aldehyde Fuchsin - Sulfated Mucins o demonstrates sulfated mucins, staining it purple Mucicarmine Stain - Mucin o demonstrates mucin by adding aluminum hydroxide to carmine. (+) red Fluorescent Acridine Orange Technique - Acid Mucin And Mucopolysaccharide o Gives selective brilliant orange fluorescence. It is temporary and lasts for 2 hours once it is mounted. • • • STAINING OF BONE MARROW AND BLOOD ELEMENTS • • • • • Alkaline Fast Green - Basic proteins : Protamines & Histones Peracetic Acid-Alcian Blue - Cystine and Cysteine Sakaguchi’s Test - Arginine • • • • • • • STAINING OF FATS OR LIPIDS • • • • • • • • Sudan Black - Lipids Sudan IV (Scharlach R) - Triglycerides o Intensified and preserved w/ addition of Benzoic Acid Oil Red O - Fats Nile Blue Stain - Neutral Fats, Cholestrin Esters, Cholestrin Fatty Acids, Cerebrosides, Fatty Acids, Soap o Differentiates lipid by its oxazone; Red(Neutral Lipids), Blue (Basic Lipids) Osmic Acid Stain - Unsaturated Fats o Unstable oxide used as fixative for EM and Histochemistry Toluidine Blue Acetone Method - Sulfatide deposits o aka Bodian and Lake Borohydride Periodic Schiff - Gangliosides Perchloric acid-napthoquinone - Cholesterol • • • • • • • STAINING OF ENZYME • • • Gomori – Calcium Method - Alkaline Phosphatase Activity o (+) brownish- black Gomori – Lead Method - Acid Phosphatase Activity o (+) black precipitate Wachstein & Meisel - Lead Method for 5-nucleotidase o (+) blackish-brown deposit. For unfixed free floating cryostat sections. Prefixed sections are not preferred due to loss of enzyme activity Rapid Toluidine-Eosin Stain - Glycol Methacrylate Section Wright’s-Giemsa-Jenner stain - Improves Staining Of Cytoplasmic Granules o combination of Romanowsky stain w/ other stain Peroxidase Reaction - Myeloid Cells Except Basophils o (+) green to dark blue granules o (-) Basophil, Lymphocytes, Erythroblasts. STAINING OF CONNECTIVE TISSUE STAINING OF PROTEINS • Alpha-naphthyl Acetate Method - Nonspecific Esterase o (+) reddish brown color due to alpha naphthol with diazonium salt which produces insoluble dye at the site of enzyme. Indoxyl Acetate Method - Nonspecific Esterase o This uses bromo-indoxyl acetate producing bromoindoxyl and oxidized to an insoluble azo dye giving the enzyme activity blue. Tetrazolium Method - Monoamine Oxidase o uses unfixed cryostat sections. The enzyme activity is demonstrated by oxidized tetra nitro-blue tetrazolium giving the activity (+) bluish black color. • Gomori’s Silver Impregnation - Reticulin Fibers Gordon’s and Sweets - Reticulin Fibers Van Gieson - Stains Collagen o simplest method of differential staining of collagen; demonstrates CT Masson’s Trichrome Stain - Stains Collagen Fibers Weigert’s Elastic Tissue Stain - For Elastic Fibers Weigert’s Resorcin-fuchsin - For Elastic Fibers Orcein (Taenzer-unna-orcein Method) - For Elastic Fibers o natural vegetable dye from lichens, used mainly in dermatology due to its ability to demonstrate fine and delicate fibers Kraijan’s Technique - Rapid Method For Staining Elastic Fibers, Fibrin And Amyloid Mallory’s Phosphotungstic Acid Hematoxin(PTAH) Method - Amyloid o used as a stain for both CNS and general tissue structures Highman’s Congo Red - Amyloid Alkaline Congo Red Technique - Amyloid o Method of choice for Amyloid Kraijan’s Amyloid Stain (Modified Bennhold Method) Amyloid Methyl Violet-crystal Violet Method - Amyloid Induced Fluorescent Staining With Thioflavine T Amyloid o uses ultraviolet or Quart iodine lamps for demonstration; used as a screening method; does not require microscopic differentiation Sirius Red - Amyloid STAINING OF NUCLEIC ACID • • Feulgen's Technique - Nuclear DNA o most reliable and specific histochemical staining technique Methyl Green-Pyronin Method - DNA and RNA • Acridine Orange Fluorescent Staining - DNA and RNA o most commonly used fluorochrome technique for DNA (orange) and RNA (red) demonstration STAINING OF MUSCLE AND BONE • • • • • • Modified Gomori’s Trichrome - Muscle Fiber and Collagen Mallory’s Phosphotungstic Acid Hematoxylin (PTAH) Muscle, Neuroglia, Myelin and Collagen Heidenhain’s Iron Hematoxylin - Muscle striations, Mitochondria, Myelin & Chromatin o resistant to fading if the section is washed well in running tap water after differentiation to remove all traces of iron alum Lissamine Fast Red – Tartrazine Method - Muscle and Bones o Routine staining method of choice in bone histology Schmorl’s Picro Thionine Method - Bones: Lacunae, Canaliculi,& Bone Matrix o staining may be intensified by adding one to two drops of conc. ammonia before use Goldner’s Method - Bone Cells: Osteoblast and Osteoclast STAINING OF CENTRAL NERVOUS SYSTEM • • • • • • • • • • • • • Neurons, Axons, and Neurofibrils - Bielchowsky Technique Myelin sheath - Weigert Pal Technique, Weil’s Method Myelin - Kluver & Barrera Luxol Fast stain w/ Nissl Counterstain, Luxol Fast Blue – H&E stain, Luxol Fast Blue – PAS Hematoxylin Stain, Page’s Solochrome Cyanine Technique Nissl substance, Neurons - Cresyl Fast Violet Astrocytes, Nerve Cells & Nerve Fibers - Cajal’s Gold Sublimate Astrocytic Processes - Modified Holzer’s Method Astrocytes - Modified PTAH stain Neural Tissues - Sevier Munger Technique Nerve Fibers & Nerve Endings - Bodian’s Stain Bielchowsky – pioneered the current staining methods for axons and neuronal processes. Bodian’s Stain and Sevier-Munger are used for demonstration of Neuritic plaques and Neurofibrillary tangles for patients with Alzheimer’s Disease Modified Holzer’s should only be performed under a fume hood due to the hazardous nature of aniline Cajal’s Gold Sublimate stain for metallic impregnation made up of HgCl2 and AuCl3 STAINING OF TISSUE PIGMENTS & DEPOSITS • • • • • • Hemosiderin - Perl’s Prussian Blue, Turnbull’s Blue Reaction Lipofuscin - Schmorl’s Ferric Ferricyanide, Gomori’s Aldehyde Fuchsin Iron Pigments - Gomori’s Prussian Blue Hemoglobin & Oxidase Granules - Benzidine Nitroprusside Stain Bile - Schmorl’s Ferric Ferricyanide, Modified Fouchet’s Technique Calcium - Alizarin Red S, Von Kossa’s Silver Nitrate Method • • • • • • • • Copper - Rubeanic Acid, Modified Rhodamine, Lindquist’s Modified Rhodamine Thyroid Colloid - Schmorl’s Ferric Ferricyanide Argentaffin - Schmorl’s Ferric Ferricyanide Melanin - Masson Fontana Technique Skeletal system in Embryos & Fetuses - Calcium Dye Lake Reaction Prussian Blue used for demonstration of circulatory system thru intravital staining Turnbull’s Blue reaction – rarely used since ferrous Is less common than ferric iron Benzidine Nitroprusside stain – highly carcinogenic STAINING OF MICROORGANISMS • • • • • • • • • • • • • • • • • • Bacteria - Grain Twort Stain, Brown & Brenn, Rapid, Giemsa, Modified Steiner & Steiner Technique Helicobacter - Toluidine Blue Stain, Cresyl Violet Acetate Method H. pylori - Gimenez Method, Toluidine Blue in Sorenson’s Buffer Spirochete - Modified Steiner and Steiner Technique, Levaditi’s, Warthin-Starry Method, Rapid Giemsa Nocardia - Brown and Brenn Method, Wade Fite Technique Fungi - Modified Steiner and Steiner Method, Grocott Methamine Silver Actinomyces - Brown and Brenn AFB - Ziehl-Neelsen Method Mycobacteria - Auramine-Rhodamine Fluorescent Method o More sensitive but fades after prolonged exposure to UV light M. Leprae - Wade Fite Technique L. Pneumophilia - Dieterle Method Donovan Bodies - Modified Steiner and Steiner Method Diptheria - Bismark Brown Viral Inclusions - Lendrum’s Phloxine - Tartrazine Method Leshmania, Malaria & Trypanosomes - Rapid Giemsa o Differentiates WBCs Inclusion Conjunctivitis &Toxoplasma - Rapid Giemsa o Differentiates WBCs Filamentous and Non-Filamentous Bacteria - Modified Steiner Rickettsia and Viral Inclusion - Macchiavello’s Stain OTHER SPECIAL STAINS • • • • • • Acridine red 3B - Demonstration deposits of calcium salts & possible sites of phosphates activities Aniline Blue - Cytoplasmic stain for counterstaining of epithelial sections Basic Fuchsin - For deep staining of acid fast organisms o Main constituent of Feulgen's & Schiff's for aldehyde detection Mayer’s Carmalum solution - Act as a basic dye & staining acidic substances Celestine Blue - Recommended for routine staining of fixed sections Congo Red - Best known as indicator stain for axis cylinders in embryos • • • • • • • • • • • • • • Crystal violet - Nuclear stain for staining amyloid in frozen sections and platelets in blood Gentian violet - Staining solution formed by mixture of crystal violet, methyl violet & dextrin Iodine - For microscopic study of starch granules, amyloid, cellulose, starch & carotenes Malachite green - Decolorizer and contrast stain for ascaris eggs, rbcs, bacterial spore stain Methylene Violet - Metachromatic dye formed whenever methylene blue is heated Neutral red - For observing cell granules and vacuoles of phagocytic cells Night Blue - Used as substitute for carbol fuchsin in acid fast staining Osmium Tetrooxide - Used to stain fats o can be also used as a fixative for myelin and peripheral nerves for Electron Microscopy Picric acid - Used as contrast stain to acid fuchsin o may be used as stain, fixative, and decalcifying agent Rhodamine B - Used with osmic acid to fix & stain blood and glandular tissues Silver nitrate - For identification of spirochetes, reticulum & other fiber stain o most commonly used agent for impregnation Toluidine Blue - Used as substitute for thionine in fresh frozen tissues o For staining of nissle granules or chromophilic bodies o Acts as metachromatic and Supravital stain Victoria blue - Demonstration of neuroglia in frozen sections