HCT Routine Staining Part 1 PDF

HCT LESSON #5: ROUTINE STAINING PART 1 MYT | A.Y 2023 - 2024 | MR. NESTOR M. POMPA JR....

HCT LESSON #5: ROUTINE STAINING PART 1 MYT | A.Y 2023 - 2024 | MR. NESTOR M. POMPA JR. ROUTINE STAINING P1,P2,&P3 GENERAL PROCEDURE Begins with complete removal of paraffin in fixed sections of slides PART 1: INTRODUCTION TO STAINING Dewaxing starts with: ○ heating the slides in 60C for at least 30 mins to soften the wax STEPS PRIOR TO STAINING Slides then immersed in a couple of changes of xylene then 1 Slide Preparation descending concentrations of alcohol to distilled water prior to Initial steps in tissue processing: primary stain Fixation 2 Dehydration STAINING MECHANISM Clearing Impregnation/Infiltration PRINCIPLES OF HISTOLOGICAL TECHNIQUE FOR DISTINCTION OF TISSUE MANUAL EMBEDDING TECHNIQUE: COMPONENTS ARE BASED ON: Using the pop-out mold Alteration of CONTRAST Alteration of COLOR ○ Make a tissue block from the processed tissue using Alter the contrast between the After tissue mounting, you will have a paraffin wax parts of the tissue colorless transparent tissue 3 ○ Important factor is the centering or orientation, in order NOTE: especially on the diagnostic purposes, because of markers to have a good position in mounting ○ During this process, you are expected to produce a 1 NUCLEAR STAINING block, a cube-like block Very important to exhibit the nucleus CUTTING ○ To differentiate it to cytoplasm Familiarize the parts of the microtome, its uses and how to Nuclei are acidic, anionic or negatively charged 4 use the microtome properly during the cutting Stain with basic dye (blue) under the microscope, cationic or positively Expected to produce tissue ribbons as the final product charged dyes 5 Manual Method of Getting Individual Tissue Sections from the ○ Remember that opposite poles attract, since the nucleic acid Ribbon present in your nucleus is anionic it will be attracted to cationic MOUNTING USING THE FLOATATION BATH: and they will produce a: NOTE: the way of the fish-out method Basophilic reaction by forming dye-salt unions that is why it is Do not forget to place the albumin as an adhesive to the Bluish to purple in color tissue section 6 2 CYTOPLASMIC STAINING Without albumin, tissue section may wash-out during the staining process Cytoplasm are amphoteric due to the NOTE:Make sure to make enough slides or an extra slides in ○ terminal amino group to one end and order to have complete slides, in case of washout ○ carboxyl group to other end 7 HEAT FIX That is why they may appear basophilic or acidophilic; they are metachromatic depends on the substance that is present STAINING in your cytoplasm that is why: Process that renders the different tissue components more visible There may have a net positive or negative charge spending on ○ Through variation in color thereby; the pH of the solution Promoting easier optical differentiation and Identification of the cell and tissue components. Characteristics Below pH 6 Above pH 6 Treating tissue or cells with a series of reagents Net charge of Positive (+) that has high Negative (-) that has high ○ So that it acquires a color; cytoplasmic affinity to negative (-) affinity to positive charged ○ No particles are seen and the stained element is transparent proteins charged dye such as eosin (+) or cationic dyes To have a very good slides, rendering a reddish to orange rendering purplish to It must not possess those stain precipitates and in color bluish in color The stained element must transparent. NOTE: It must be able to differentiate your cytoplasm from nucleus or other To distinguish cytoplasm if they are in the positive or attracted to cationic cytoplasmic parts dye they have lighter color compared to your nucleus (darker bluish color) Accomplished by the use of Dyes or Stains DYES/STAINS ○ That imparts a characteristic color to a usually transparent tissue components (which is unstained) but once it is stained, it is now Chemical or reagents with aromatic benzene ring compounds(or visible under the microscope derivatives) which posses the twin properties of: STAINS - Chemical substances used to achieve visible color contrast in ○ Color band the microscopic picture of a prepared tissue ○ Able to bind the tissues All dyes are organic compounds, mostly derivatives of coal tar or PURPOSE OF STAINING benzene Outlines tissue and Cellular components ○ Now there are synthetic dyes Identification of tissues: ○ But in H&E, we’ll be using organic dye ○ In Histology, we were presented with normal (non-pathologic) slides INTEGRAL COMPONENTS OF DYES ○ In HCT, we are presented with pathologic slide 1 CHROMOPHORES Establishes the presence or absence of disease processes The group on the Benzene ring which confers color: C=C, C=O, C=S, COMMON STAINING METHODS N=N, N=O, and NO2 ○ It alters the light resonance properties of the compound so that H&E Staining Histopathology unequal absorption occurs when white light is passed through. Gram’s Stain & Ziel-Neelsen Microbiology Under the microscope you will have a contrast & see the Staining different colors Romanowsky Staining Hematology Example: Giemsa Staining Parasitology Red color seen Papanicolaou Staining Cytology Blue-green component is absorbed out (490-500 nm) GOLVIOGO, GUILLAR, LATONIO, MACOPIA, MAHUSAY, MANTOS  HCT LESSON #5: ROUTINE STAINING PART 1 MYT | A.Y 2023 - 2024 | MR. NESTOR M. POMPA JR. Chromagen A benzene derivative containing chromophoric groups CLASSIFICATION BASED ON THE PH OF THE DYE Where 2 hydrogen atoms on the benzene ring have been replaced by oxygen atoms CATEGORIES ACTIONS IN TISSUES EXAMPLES Quinoid May occur alone Group/ E.g. Stains basic components such as Eosin, Structure ○ Triphenyl methane dyes- Basic Fuchsin Acidic dyes cytoplasm, acidophil granules, Acid Fuchsin ○ AZO groups- Congo Red etc. ○ Xanthene- Eosin Stains acidic components such as Hematoxylin, Basic Interbenzene bonds Basic dyes nucleus, basophil granules, etc. Fuchsin, Methylene blue Oxazins where O2 atoms is incorporated Consist of mixtures of basic and Quinone- Romanowsky dyes E.g. acidic dyes. Imine Group formed by interaction of ○ Cresyl Fast Violet, thiazines where sulphur is Neutral dyes Stains both cations and anions, incorporated toluidine blue polychrome methylene contain chromophoric groups blue and eosin and both have colored radicals. 2 AUTOCHROMES Are group responsible for tissue to bind firmly to a given dye thus the CLASSIFICATION OF SYNTHETIC DYES dye possess auxochromic groups Are groups on the benzene ring which impart a net charge to the Categories ACTIONS IN TISSUES EXAMPLES molecules by conferring the property of electrolytic dissociation Azocarmine Used in textiles, cosmetics, and Electrical charge is an important mechanism in the attachment of dyes Janus green Azo dyes food coloring. on tissues Congo red Stains muscle fibers Sudan dyes Autochrome Groups: Used in polyamide, wool, and silk Aniline blue, Amino Group (-NH2): important cationic auxochrome Triphenyl - modified acrylic fibers. crystal violet, Hydroxyl (OH-) & Carboxyl (COOH-) Group- important anionic methane Used as metachromatic stains in Acid & Basic auxochrome tissues. Fuchsin Chromogen: Used as different stains in Thiazine Methylene blue A substance possessing a chromophore + auxochrome= DYE histology, cytology, and dyes Toluidine blue hematology. DYES AND ITS CLASSIFICATION Eosins, Xanthene Used as fluorescent stains in Pyronine, dyes tissues and tumor markers. Phloxine, CATEGORIES CLASSIFICATION EXAMPLES Fluorescence Organic fluorescent dyes used in Brilliant cresyl blue, Oxazine chemotherapies and radioactive Cresyl violet, dyes 1.Natural Dyes Hematoxylin, dyes. Celestine blue Cochineal, Orcein, Came from the nature, found in QUALITIES OF A SUCCESSFUL STAINING Saffron, Carmine, Origin plants In order for us to produce a successful staining, the choice of stain is very Brazilin important. 2. Synthetic/Artificial Dyes Coal Tar Dyes, 1. SPECIFICITY/SELECTIVITY Came from the lab (man-made) Aniline dyes Can stain specific parts or target parts for the pathologists to 1.Fluorescent dyes observe. If you are using a phase contrast Acridine orange 2. SENSITIVITY microscope Must catch even the smallest amount of your target component, 2. Leuco dyes even the specific portion. Physico- Leuco Methylene To differentiate your (nucleotides, CLASSIFICATIONS OF TISSUE STAINING chemical Blue WBC, or nucleated cells) Properties 3. Metachromatic dyes BASED ON FUNCTIONS/ACTIONS IN TISSUES Basic or acidic dye depends on the Toluidine Blue pH of solution CATEGORIES ACTIONS IN TISSUES EXAMPLES 4. Neutral dyes Azure-eosinate Histologic / Direct interaction between 1.Azo dyes orange Micro- dye & staining solution. H&E, Paps anatomical Differentiate tissue Al or Fe complexes Structure 2. Metal Complex dyes Staining components. of Hematein Demonstrate chemical 3. Xanthene dyes Pyronine Y reactions in specific tissue Histochemical Perl’s Prussian Blue, 1. Fat Stains Oil Red O substance such as Staining Periodic Acid Schiff (PAS) Use in hemoglobin and 2. Fluorescent Probe YOYO-1 (used as Biological carbohydrates Used in immunochemistry synthetic dye) Staining Exhibits immunologic & 3. Mucin stain Alcacian Blue histochemical processes in Polyclonal/monoclonal 1. Acid stain Eosin tissues. antibodies, Immuno - Detection of phenotypic Fluorescent labeled Use in Textile 2. Basic stain Safranine histochemical markers. antibodies, Dyeing Staining If you want to exhibit a Enzyme-labeled 3. Direct stain Congo Red tumor marker, you may antibodies Supposed Gallacyanine chrome use this stain. 1. Mordant Mode of alum Action of Dye 2. Reactive Dyes Mercury Orange GOLVIOGO, GUILLAR, LATONIO, MACOPIA, MAHUSAY, MANTOS  HCT LESSON #5: ROUTINE STAINING PART 1 MYT | A.Y 2023 - 2024 | MR. NESTOR M. POMPA JR. GENERAL METHODS OF STAINING 2 WAYS OF DOING VITAL STAINING BASED ON HOW THEY ARE BEING USED IN LAB INTRAVITAL STAINING SUPRAVITAL STAINING CATEGORIES ACTIONS IN TISSUES EXAMPLES Process of injecting the dye into any part of the animal Reticulocyte stain Just like your reticulocyte staining body either intravenously, Selective staining of living (identifying reticulocytes where you take out the tissue intraperitoneally, or cell constituents. in its live form), Janus from the source still in a living Vital Staining subcutaneously (muscle) Demonstrates cytoplasmic green, state, you are introducing a dye producing specific & nuclear structures. Tryphan blue, to combine with it coloration of certain cells Brilliant cresyl blue Process of staining living cells particularly the RES Basis of histochemistry, immediately after removal from You are to inject the dye for identification Oil Red O, Perl’s Prussian the living body Specific / Special / into a live person or of specific structures. Blue, Periodic Acid Schiff, Selective Staining animal’s tissue Has little or no affinity for Weigert’s Elastic Stain Commonly used dye: tissue elements. ○ Neutral red- best vital stain Used in a day to day basis. H&E Commonly used dyes: ○ Janus green- recommended Stains various tissue Nuclei: Bright clear blue ○ India ink for mitochondria Routine Staining elements with little Cytoplasm & other tissue ○ Carmine ○ Trypan blue differentiation except components: Pink to red ○ Lithium ○ Nile blue nucleus and cytoplasm. ○ Thionine BASED ON HOW YOU WILL DO IT IN THE LAB ○ Toluidine blue DIFFERENTIATION/ DECOLORIZATION Categories ACTIONS IN TISSUES EXAMPLES Selective removal of excess stain from the tissue during regressive Staining is continued until the staining desired intensity of the color of ○ This is usually done by a weak acid or alcohol the tissue element is attained. Purpose: ○ TO stain specific substances differentially from the rest of the Intensity of color in tissue Cole’s Hematoxylin, surrounding tissue Progressive components become greater in Differentiation is controlled by following exact times specified or by time. Phosphotungstic acid Staining microscopic examination of the section Usually direct method hematoxylin Differentiation is carried out by: wherein you let the dye penetrate in the tissue Washing a section in a simple solution 1 Sensitive and selectivity in a ○ E.g. water or alcohol progressive way The use of weak acids, bases, oxidizing agents & mordants (Classes of Tissues are overstained & excess Differentiators for Mordant Dyes) 2 dye is removed selectively until ○ Ex. acid alcohol in Harris Hematoxylin Staining Method used in the desired intensity is obtained. regressive staining Tissue is overstained, partially 3 Exposure to air may oxidize and improve the process colorized, and differentiated. Alum hematoxylin, Harris hematoxylin, 1 ACID DIFFERENTIATORS Regressive This is where we use a Mayer’s hematoxylin, From the word itself- you are using acid Staining mordant or accentuator. Iron hematoxylin, Acts by combining with the metal thus breaking the latter’s union with Heidenhain’s hematoxylin the tissue or cell components Overstaining then application of Examples: a secondary stain in order to ○ HCl & Acetic Acid differentiate the structures with a different color 2 OXIDIZING DIFFERENTIATORS Act by oxidizing the dye to a colorless substance (leukoform) CATEGORIES ACTIONS IN TISSUES EXAMPLES Component holding least dye will be bleach first Staining of tissue with simple Examples: Direct ○ Potassium ferricyanide aqueous or alcoholic solutions Methylene blue Staining ○ Potassium permanganate of dye. It will render one color ○ Chromic Acid Staining with addition of ○ Picric Acid mordant/accentuator so that ○ Potassium dichromate Indirect the action of the dye Gram’s stain Staining intensifies. 3 MORDANT DIFFERENTIATORS Target is different from its Mass action phenomenon: background ○ Binding a dye to the tissue, and on the other hand removing this Wright’s stain, Crystal same dye from its combination with tissue Meta- Staining using certain dyes to violet, Methyl violet, The tissue components which contain the least due will be decolorized chromatic exhibit a color different from Safranine, New first Staining the stain itself methylene blue, Azure A, The structures containing most dye (nuclear chromatin) will be deeply B, C stained Application of different stain to A substance which enhances the combination of Counter- provide contrast and Eosin, the dye with the tissues forming a colored “lake” staining background to the tissue Zheil-Neelsen Mordant tissue mordant dye complex insoluble in aqueous component in target & alcoholic solvents GOLVIOGO, GUILLAR, LATONIO, MACOPIA, MAHUSAY, MANTOS  HCT LESSON #5: ROUTINE STAINING PART 1 MYT | A.Y 2023 - 2024 | MR. NESTOR M. POMPA JR. Polyvalent metal ion All dyes should be filtered before use ○ which forms coordination complexes with ○ You won’t be doing the filtering but what you will have is the certain dyes prepared reagent Intermediary between dye & tissue increasing the Specially designated bench affinity between them Staining bench should be facing the window Applicable to salts & hydroxides of divalent & ○ There will be fumes that are sometimes carcinogenic trivalent metals ○ This is opposite to cutting because the windows should be closed Should NOT be used to indicate any substance that Slide washing tray made of stainless steel improves in staining like actions of accentuators Bunsen burner or slide warmer to heat up the stained slides and accelerators ○ This is for the dewaxing process A substance that can cause no chemical union Thermostatically controlled hot plate to melt the wax between the tissue and dye Microscope to control staining reaction Not a mordant and does not participate in the SLIDES ARE STAINED USING: staining reaction For individual slide staining ○ but hasten Small grooved coplin “Dip Method” Speed (intensity) and jars with lids Accentuator ○ Using the coplin jars Selectivity of the dye For barch staining Staining Carriers/ Examples: Capable of holding 10-50 slides Racks ○ KOH in Leoffler’s Methylene blue We do this in the laboratory ○ Phenol in Carbol Thionine & Carbol Fuchsin Staining machine Automated individual or batch staining 👉🥺👈 ○ Glacial Acetic Acid ○ Aniline used with Gentian Violet Pompa pompa pompa papasara kami Are not mordants, too, so do not form lakes with stain When used with a stain, they INCREASE the rate of the staining action Accelerator Used in metallic impregnation technique for nervous system E.g. ○ Chloral Hydrate Agents which holds dye combination with tissue or bacteria E.g. Trapping ○ Tannic acid Agents ○ Iodine This is just to have differentiation between your target extracellular components like bacteria, fungi, and parasite TYPES OF STAINING REACTIONS Absorption/ Tissue penetrated by dye solution Direct Staining Indirect Staining Using intermediate treatment with mordant Physical Staining Simple solubility of dye in element of tissues Formation of new substances such as in PAS (Periodic Acid Schiff) staining Chemical Staining ○ PAS combine with a specific compound in the tissue will form a salt or cloud which is very visible under microscope Accumulation on the surface of the compound Adsorption to exhibit its presence Phenomenon Unlike absorption, this will attract a certain component in a superficial manner REQUIREMENTS IN STAINING All glassware should be thoroughly cleaned ○ This is to eliminate any carry-overs Correct solvent should be used Silver and osmic acid solutions should be kept in dark bottles ○ This is because of the oxidation process and can affect with the performance of your reagent Solutions like dilute ammonia should be freshly prepared ○ Ammonia is a very volatile substance ○ Anything volatile in nature would evaporate faster so it should be freshly prepared Constituents of stain dissolved should follow the formula ○ In making a stain, you must observe the manufacturer’s recommendation Alcoholic solutions of the stain should be kept in dark stoppered bottles GOLVIOGO, GUILLAR, LATONIO, MACOPIA, MAHUSAY, MANTOS