HCT - Routine Staining and Mounting PDF

HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES A QUINOID GROUP/STRUCTURE QUALITIES OF GOOD STAINING TOPIC 05: ROUTINE STAINING ▪ where two hydrogen...

HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES A QUINOID GROUP/STRUCTURE QUALITIES OF GOOD STAINING TOPIC 05: ROUTINE STAINING ▪ where two hydrogen atoms on the benzene ring have been replaced specificity/selectivity sensitivity Learning Objectives: by oxygen atoms PURPOSE/IMPORTANCE OF STAINING (DYEING) o Define the process known as staining ▪ QUINOID chromophore may occur alone To enable one to study and see the physical characteristics and o Discuss the principle of Histological Staining Technique triphenyl methane dyes Basic Fuschin 1 relationship of the tissue components, which do not retain enough o Name the two main types of stains as to source AZO group Congo Red color after processing o State the importance of staining Xanthene EOSIN To allow the tissue and cellular components to be outline and be o Discuss the theoretical aspects of staining B QUINONE-IMENE GROUP 2 visible by bringing out differences in refractive index to that trained o Differentiate the methods of staining pathologist or histologist may be able to identify tissues ▪ Interbenzene bonds o Discuss the basic procedure for staining paraffin infiltrated & embedded To establish the presence or absence of disease processes, be they ▪ OXAZINS where O2 atom is incorporated 3 sections congenital, inflammatory, degenerative or neoplastic ▪ e.g. as in cresyl fast violet o Define the process known as mounting thiazines where sulphur atom is incorporated → toluidine blue MAJOR CLASSIFICATION OF STAINING TISSUES o Name the two methods of mounting/cover slipping 1 HISTOLOGIC STAINING 2 AUXOCHROMES o State the purpose/s of mounting ▪ tissue components are demonstrated in sections by direct interaction ▪ are group responsible for tissue to bind firmly to a given dye thus the o Discuss the two types of mounting media dye possesses auxochromic groups with a dye/staining solution ▪ are groups on the benzene ring which impart a net charge to the e.x. Bacterial stains, specific tissue stains HEMATOXYLIN & EOSIN (H & E) STAINING METHOD molecules by conferring the property of electrolytic dissociation (muscles, connective tissue & neurologic stains) the process that renders the different tissue components ▪ electrical charge is an important mechanism in the attachment of dyes ▪ micro-anatomical/histologic staining: demonstrate the general more visible through variation in color, thereby promoting on tissues relationship of tissue & cells with differentiation of nucleus & easier optical differentiation & identification of the cell & ▪ for a substance to be a dye, it should possess a chromophore, which is cystoplasm staining tissue components the one responsible for the color and auxochrome for tissue binding e.x H&E accomplished by the use of dyes or stains that imparts a 2 HISTOCHEMICAL STAINING (HISTOCHEMISTRY) amino group (-NH2) important cationic auxochrome characteristic color to a usually transparent tissue ▪ whereby various constituents of tissues are studies through chemical hydroxyl (OH-) and components important anionic auxochrome reactions that will permit microscopic localization of specific tissue carboxyl (COOH-) groups PRINCIPLES OF HISTOLOGICAL TECHNIQUE FOR DISTINCTION OF TISSUE substance COMPONENTS ARE BASED ON: CHROMOGEN e.g. Hemoglobin – Perl’s Prussian Blue a substance possessing a chromophore + auxochrome = DYE CHO’s – PAS (Periodic Acid Schiff) ▪ alteration of contrast ▪ alteration of color TWO MAIN TYPES OF STAINS ▪ enzyme histochemistry: active reagent serves as a substrate upon DYES/STAINS which the enzyme act → final opacity/coloration produced from the ▪ essentially aromatic benzene ring compounds 1 NATURAL STAINS/DYES obtained from plants and animals substrate rather than the tissues (or derivatives) which possess the twin 3 IMMUNOHISTOCHEMICAL STAINING ▪ Hematoxylin ▪ Saffron properties of: ▪ combination of immunologic & histochemical techniques that allow ▪ Cochineal Dyes ▪ Carmine o color band o able to bind tissues phenotypic markers to be detected and demonstrated under the ▪ Orcein ▪ Brazilin ▪ all dyes are organic compounds, mostly microscope 2 SYNTHETIC/ARTIFICIAL DYES derivatives of coal tar or benzene ▪ utilizes a wide range of: ▪ known as “coal tar dyes” ▪ derived from the hydrocarbon benzene (C6H6) o polyclonal/monoclonal antibodies ▪ collectively known as aniline dyes o fluorescent labels Abs INTEGRAL COMPONENTS OF DYES ▪ classification next table o enzyme – labeled Abs 1 CHROMOPHORES CLASSIFICATION OF SYNTHETIC DYES GENERAL METHODS OF STAINING ▪ the group on the Benzene Ring which confers color: ▪ azocarmine ▪ vital staining ▪ direct staining C=C, C=O, C=S, C=N, N=N, N=O, and NO2 ▪ janus green ▪ specific/special/selective ▪ indirect staining ▪ it alters the light resonance properties of the compound so that azo dyes ▪ congo red ▪ routine staining ▪ metachromatic staining unequal absorption occurs when white light is passed through ▪ sudans ▪ progressive staining ▪ counterstaining ex. Red Color Seen → Blue-Green Components is absorbed out ▪ regressive staining ▪ aniline blue (490-500mm) 1 VITAL STAINING triphenylmethanes ▪ crustal violet ▪ a benzene containing chromophoric groups is called a chromogen ▪ selective staining of living cells constituents ▪ acid & basic fuchsin thiazine dyes ▪ methylene blue ▪ demonstrate cytoplasmic structures by phagocytosis of dye particle ▪ eosins (cytoplasmic phagocytosis) ▪ demonstration of nuclear structure during vital staining suggests ▪ pyronine xanthene dyes permeability of nuclear membrane, signifying the death of the cell as ▪ phloxine the nucleus of a living cell is resistant to vital stains ▪ fluoresceins ▪ e.g. ▪ brilliant cresyl blue o Janus Green (true vital stain): stains mitochondria oxazine dyes ▪ cresyl violet o Tryphan Blue: for RES ▪ celestine blue tE __E thank u luv ANJELA SOLIS for constantly being there for me, stay strong, we can do this! ily ♡ AIRAH M.  TWO WAYS OF DOING VITAL STAINING selective removal of excess stain from the not a mordant & does not participate in the 1 INTRAVITAL STAINING tissue during regressive staining staining reaction but hasten the speed of ▪ process of injecting the dye into any part of the animal body either purpose: is to stain specific substances (intensity) and selectivity of the dye intravenously, intraperitoneally, or subcutaneously, producing differentiation or differentially from the rest of the surrounding examples include: specific coloration of certain cells particularly the RES decolorization tissue ▪ KOH in Leoffler’s Methylene Blue ▪ commonly used dyes: differentiation is controlled by following exact ▪ Phenol in Carbol Thionine & Carbol Fuchsin o India Ink times specified or by microscopic examination ▪ Glacial Acetic Acid o Carmine of the section ▪ Aniline used with Gentian Violet o Lithium are not mordants, too, so do not from lakes DIFFERENTIATION is carried out by: 2 SUPRAVITAL STAINING with the stain ① washing section in simple solution (e.g water or alcohol) accelerators ▪ process of staining living cells immediately after removal from the ② the use of acids, bases, oxidizing agents & mordants (classes of when used with a stain, they increase the rate living body differentiators for mordant dyes) of the staining action ▪ commonly used dyes: e.g. acid alcohol in Harris Hematoxylin staining method used in 8 METACHROMATIC STAINING o Neutral Red (Best vital Stain) o Nile Blue regressive staining staining process in which certain dyes stain certain tissues in a color or o Janus Green: especially. o Thionine hue which is quite different from that of the stain itself (metachromasia) o Toluidine Blue acts by combining with the metal thus breaking recommended for mitochondria ▪ Crystal Violet ▪ Toluidine Blue (commonly o Tryphan blue the latter’s union with the tissue or cell ▪ Methyl violet employed) acid components 2 SPECIFIC/SPECIAL/SELECTIVE STAINING ▪ Safranine ▪ New Methylene blue differentiators example: during H&E staining, the decolorizer ▪ Thionine (commonly employed) ▪ Cresyl Blue ▪ the basis of histochemistry in which the identification of certain used is the acid alcohol ▪ Azure, A, B, C structures and out a final color at the site or location of the structures/substances in the cells/tissues e.g. HCl & acetic acid 9 COUNTERSTAINING ▪ it has little/no affinity for other tissue elements act by oxidizing the dye to a colorless substance application of a different stain to provide contrast and background to the ▪ e.g. (leukoform) staining of the structural components to be demonstrated (e.g. Eosin) o Oil Red O: For neutral fats component holding least dye will be bleach first HEMATOXYLIN o Perl’s Prussian Blue: for hemosiderin examples include: oxidizing a natural stain from the tree Haematoxylon campechianum o Periodic Acid Schiff (PAS): For polysaccharides ▪ potassium ferricyanide differentiators o Weigert’s Elastic Stain: elastic fibers ▪ potassium permanganate THREE METHODS OF RIPENING OR OXIDATION OF HEMATOXYLIN STAINS 3 ROUTINE STAINING ▪ chromic acid ripening is where your hematoxylin is oxidized as hematin ▪ is one that stains the various tissue elements with little differentiation ▪ picric acid 1 AIR OXIDATION except between nucleus and cytoplasm ▪ potassium dichromate ▪ Ehrlich’s hematoxylin ▪ commonly used routine stain is: mass action phenomenon: binding a dye to the ▪ Delafield’s hematoxylin o Hematoxylin & Eosin Stain (H&E) tissue, and on the other hand removing this 2 ADDITION OF OXIDIZING AGENTS ✓ nuclei: Bright Clear Blue same dye from its combination with the tissue ▪ sodium iodate ✓ cytoplasm & other tissue component: Pink to red mordant the tissue components which contain the least ▪ mercuric oxide 4 PROGRESSIVE STAINING differentiators dye will be decolorized first ▪ potassium permanganate ▪ staining is continued until the desired intensity of the color of the ▪ hydrogen peroxide the structures containing most dye (nuclear different tissue element is attained chromatin) will be deeply stained ▪ calcium hypochlorite ▪ the intensity of color of the tissue components becomes greater with WITHOUT AN OXIDIZING AGENTS, the rate of conversion of 6 DIRECT STAINING 3 time hematoxylin to hematein is determined: ▪ during progressive staining the mordant precedes the stain or with the ▪ staining of tissue/sections with a simple aqueous or alcoholic solution of the dye ▪ pH of the solvent stain as in H & E staining method ▪ type of the solvent ▪ e.g. ▪ e.g. Methylene Blue o Cole’s Hematoxylin (useful) 7 INDIRECT STAINING pH & TYPE OF THE SOLVENT o Phosphotungstic acid Hematoxylin staining tissues with the addition of mordant/accentuator so that the rate of the oxidation of hematoxylin to hematein in aqueous neutral solution 5 REGRESSIVE STAINING action of the dye is intensified as compared to other solvents ▪ tissues are overstained and the excess dye is then removed selectively a substance which enhances the combination 1 AQUEOUS SOLVENT until the desired intensity is obtained of the dye with the tissues forming a colored mordant ▪ Neutral pH: forms in a few hours ▪ once the tissue is overstained, it is then partially decolorized or “lake” → tissue mordant – dye complex → ▪ Acid pH: hematein forms more slowly differentiated insoluble in aqueous and alcoholic solvents ▪ Alkaline pH: hematein forms more rapidly ▪ e.g. a substance that can cause no chemical union 2 ALCOHOLIC SOLVENT o Alum Hematoxylin (recommended, although can also be used for between the tissue and dye ▪ Hematein forms more slowly progressive) accentuator hastens the speed of intensity of the dye and 3 ALCOHOLOC SOLVENT with GLYCERIN o Harris Hematoxylin has no chemical union between the tissue and ▪ Hematein forms even more slowly with glycerin o Mayer’s Hematoxylin the dye o Iron Hematoxylin EFFICIENCY OF A HEMATOXYLIN SOLUTION o Heldenhain’s Hematoxylin 1 MORDANT Doft thank u luv ANJELA SOLIS for constantly being there for me, stay strong, we can do this! ily ♡ AIRAH M.  CLASSIFICATION OF MORDANT EOSIN OTHER COUNTERSTAINS ▪ mordant w/ iron ▪ mainly a synthetic stain and is used as a counterstain to the nuclear stain acid fuchsin magenta color ▪ mordant containing other metallic ions such as: ▪ a plasma stain, staining cytoplasm of the cells and other tissue components aniline blue blue color o aluminum red or pink congo red red o potassium ▪ most common & widely used either as an aqueous/alcohol solution at orange G orange o ammonium concentrations 0.5 – 1% phloxine B red purple ▪ mordant w/ sulfate ions ▪ the speed and intensity of eosin depends on the fixative at pH use * alum is a mordant that contains aluminum salts KINDS OF STAINS ▪ it stains rapidly & brilliantly depending on: 2 BLUEING acidic/anionic Stains basic/cationic Stains o fixative used BLUEING SOLUTIONS ✓ Mercuric salts of Xenker’s fixative attach to the (-) charged groups TYPES OF TISSUE COMPONENTS ▪ ammonia (ammonia H2O) used in routine H&E staining method of the tissue component. This increases the attraction of the 1 Basophilic Tissue Components ▪ warm H2O (40-50°C) or distilled H2O negatively charged eosin stain to the positively charged groups of 2 Acidophilic Tissue Components ▪ lithium carbonate ▪ bicarbonate the tissue components. 3 Neutrophilic Tissue Components ▪ potassium or sodium acetate ✓ Formalin fixed tissue: place tissue sections in a saturated mercuric CHEMICAL BASIS OF HISTOLOGICAL STAINING/FACTORS THAT ▪ Scott’s tap h2o substitute (Na or K bicarbonate plus Mg sulfate) chloride 2-3 minutes prior to counterstain to eosin INFLUENCE CHEMICAL STAINING o pH of the eosin solution CLASSIFICATION OF HEMATOXYLIN SOLUTION ✓ stains best if solution is slightly acidic at optimum pH of 5.4 -5.6 1 CONCENTRATION OF STAIN based on whether the mordant is mixed or ▪ increasing concentration of stain increases ionization of stain STABILITY OF THE ACIDIFIED COUNTERSTAIN ▪ greater amount of stains become bound to tissue components that not mixed with hematoxylin stain o gives good results for about 2 weeks, then pH will rise & the stain is no accept stain 1 “DIRECT” HEMATOXYLIN STAIN longer useful ▪ increasing the amount of dissolved salts in the stain → decreases the mordant plus hematoxylin are mixed together o eosin is rapidly removed during dehydration in 95% & absolute alcohol, staining action of the stain as salt ions compete w/ ionized stain for A ALUM MORDANT HEMATOXYLIN thus, sections should be slightly overstained & rapidly dehydrated through reactive sites on the tissue chemical components A.1. HARRIS HEMATOXYLIN COMPOSITIONS the alcohols to the clearing agent 2 AMOUNT OF IONIZATION OF STAIN hematoxylin stain pH OF THE ENVIRONMENT in which the tissue chemical components KINDS OF EOSIN 3 ethyl alcohol (95%) stain solvent and the stain are found EOSIN Y (yellowish) potassium alum mordant EOSIN B (bluish) ▪ each chemical components of the tissue, especially CHON & Nucleic most commonly used distilled water mordant solvent Acids, there is a pH of the environment at which each molecule has mercuric oxide oxidizing solvent TYPES OF EOSIN equal number of (+) or (-) charges glacial acetic acid accentuator ALCOHOLIC EOSIN AQUEOUS EOSIN EOSIN-ORANGE G ▪ pH is termed isoelectric point (IEP) A.2. EHRLICH’S HEMATOXYLIN EOSIN Y or EOSIN B ▪ At the IEP → net algebraic charge is zero. The molecule is at zwitterion. sodium iodate oxidizing agent 95% ethyl alcohol - - Since the zwitterion has zero or neutral charge on itself, it has little glacial acetic acid affinity or attraction for charged stain molecules. glycerin A.3. DELAFIELD’S HEMATOXYLIN is the most common counterstain to alum o e.g. H&E stain at pH 7.0, nucleus has a net (-) charge attracts hematoxylin in H&E method hematoxylin stain (+) or basic/cationic stain B IRON MORDANT HEMATOXYLIN it stains satisfactorily from both aqueous and ▪ cytoplasm has a net (+) charge attracts eosin stain (-) or acidic/anionic B.1. WEIGERT’S IRON HEMATOXYLIN stain ethanolic sol’n SOLUTION A (MORDANT) SOLUTION B (STAIN) 4 FIXATION TREATMENT OF TISSUES it is one of the dyes in Papanicolaou’s EA solution ferric chloride ▪ fixation may cause reorganization of tissue chemical components to EOSIN Y for staining exfoliative ferrous sulfate hematoxylin render certain chemical more accessible to the stain molecules thus used as a counterstain in the Gram-Weigert’s hydrochloric acid ethyl alcohol, 95% increases staining affinity method for gram (+) bacteria & fibrin, & can also distilled water ▪ fixation might change permeability of tissue components allowing be used to make Romanowsky stain mix equal volume of solution A and B before use for best results certain stains to have increase/decrease rates of penetration sometimes called EOSIN Y, spirit soluble because 2 “MORDANT” HEMATOXYLIN: IRON MORDANTS the free coloured acid radical of this dye is Eosinol ▪ fixation might bind to certain chemical component of the tissue and where your mordant is not mixed together with your hematoxylin Y, can be made from Eosin Y, the sodium salt, by leave other components free to accept the stain treatment with HCl o e.g. formalin fixatives increase affinity of the chemical component A. HEDENHAIN’S IRON HEMATOXYLIN of tissue for basic stain, therefore combines to amino groups (NH2) soluble in ethanol, slightly soluble in xylene but not SOLUTION A (MORDANT) SOLUTION B (STAIN) of tissues leaving the carboxyl (COO4) group free in H2O hematoxylin o e.g. mercuric chloride fixative heavy metal fixatives increase ferric ammonium alum sometimes useful for staining difficult tissues ethyl alcohol, 95% affinity of the tissue for acidic stains. As fixative combines with the distilled water sometimes used instead of Eosin Y in staining distilled water EOSIN B COOH leaving the NH2 group free techniques Solutions A & B are not mixed. If they are, stain deteriorates. 5 TEMPERATURE commonly used to compound Romanowsky stains During staining procedure, solution A, the mordant, precedes ▪ increases temperature increases the rate of penetration of the stain the free colored radical of this dye, Eosin B, can be solution B, the stain. and staining action made from Eosin B, the sodium salt, by treatment OTHER NUCLEAR STAINS: o action: this swells the tissue & facilitates diffusion of dye molecules with HCl for effective dye action & rapid staining time o▪ Toluidine Blue ▪ Methylene Blue ▪ Crystal Violet sometimes called Eosin B, spirit soluble NMNNENehTe__ thank u luv ANJELA SOLIS for constantly being there for me, stay strong, we can do this! ily ♡ AIRAH M.  ▪ 70,95% 2 or 3 changes of 100% alcohol before passing to xylene REASON: Dehydrating in ascending grades of alcohol as stained sections are mounted in a mounting medium soluble in an organic solvent, such as xylene ▪ time in each alcohol is very short (10 dips) as many stains are soluble in low alcohol concentration & therefore can be extracted from tissue A B C sections during the process ▪ other remedy: the use of an alcoholic counterstain and differentiating fluids E 7 CLEARING ▪ assures the removal of the alcohol, thereby making tissue transparent CHARACTERISTICS OF A GOOD MOUNTING MEDIUM ▪ XYLENE: the most commonly used clearing agent and it serves as the 1 the refractive index of the mountant should be as near as possible to solvent for the mounting medium that of the glass slide which is 1.518 D ▪ 2 or 3 changes of xylene for 1 minute (10 dips) which are placed in different staining dishes to prevent carry-over 2 3 it should be freely miscible with xylene and toluene it should not quickly dry (A&D) slide carrier; (B) staining jar; (C&E) staining jar with stain 8 MOUNTING it should not crack or produce artefactual granularity on the slide 4 upon drying BASIC PROCEDURES FOR STAINING PARAFFIN PRECAUTIONS IN STAINING 5 it should not dissolve out of fade tissue sections INFILTRATED & IMBEDDED TISSUES 1 STAINS ON THE SKIN SHOULD BE AVOIDED 6 is should not cause shrinkage and distortion of tissues 1 DEPARAFFINIZATION (DECERATE) not just a sign of poor technique but are health hazards per se, being 7 it should not leach out any stain or affect staining slowly absorbed by the skin eventually producing side effects 8 it should not change in color or pH ▪ we remove the paraffin so that the stain can FAILURE OF SECTIONS TO REMAIN ON THE SLIDE DURING STAINING penetrate 2 it should set hard, thereby producing permanent mounting of DUE TO: 9 ▪ removal of paraffin so the stain can sections ▪ dirty/oily slide permeate the tissue section w/ highly ▪ faulty solution, hematoxylin may not have been properly and METHODS OF COVER SLIPPING (HAND COVER SLIPPING) organic solvents sufficiently ripened 1 FORCEPS METHOD ▪ 2 changes of xylene, 2 mins each ▪ paraffin, fixative or decalcifying solution 2 INVERTED SLIDE METHOD o that has not been thoroughly washed 3 INVERTED COVERSLIP METHOD 2 ABSOLUTE ALCOHOL o out and removed ▪ removes xylene from the tissue sections (xylene is not miscible with lower concentrations of alcohol & H2O) RESTAINING OF OLD SECTIONS A B ▪ one or two changes of absolute alcohol for about 1 minute is adequate old, bleach or faded sections may be restrained 3 HYDRATION (run slides to H2O) ▪ the slide is usually immersed in xylene for 24 hours, or gently heated until the mounting begins to bubble gradual “run” through a series of decreasing concentration of alcohols ▪ the coverslip may then be removed by lifting it with a dissecting (95, 80,70%) for delicate tissues, even 50 & 30% to H 2O needle PURPOSE: prevents rapid movement of fluids into & out of the tissue ▪ the section is place in xylene for 30 minutes to remove the remaining sections & consequent loss of the sections from the glass slides balsam & then brought down to H2O DURATION: A MINUTE OR 10 DIPS ▪ place in a 0.5 Potassium Permanganate sol’n for 5-10 minutes C 95% ethyl alcohol for hydration & dehydration but isopropyl alcohol or ▪ rinse in tap H2O tertiary butyl alcohol may be employed ▪ subsequently immerse in 5% oxalic acid for 5 minutes or until the 4 WASHING section is decolorized ▪ this is the step where the tissue sections are brought to the solvent of ▪ wash again in running tap water for another 5 minutes the stain, usually H2O or the concentration of alcohol in which the stain ▪ re-stain with the appropriate staining technique is dissolved MOUNTING ▪ if H2O is the solvent, the washing is accomplished in slowly dripping tap is the placing of the coverslip over the stained tissue sections using a syrupy H2O for about 3 minutes fluid applied between the section and the coverslip, setting the section firmly, 5 STAINING AND COUNTERSTAINING preventing the movement of the cover slip ▪ follow the order of applying the stain and counterstain and the use of (A) This is a forceps method where you use forceps in holding your cover slip. PURPOSE mordants, differentiating or decolorizing agents to obtain properly (B) This picture shows your inverted slide method where the mounting media 1 to make a permanent preparation stained tissue sections is placed on the slide and the cover slip is then slowly lowered to cover the to provide a clear visualization of tissue components to be studied ▪ mordant acts to hold stain properly to the tissue to prevent the stain using a medium which has a refractive index close to that of glass tissue. (C) This is another photo of your inverted slide method. from being lost from the tissue sections when they are placed in H2O or 2 slide and near the average refractive of the tissue (Rf=1.518) during low concentrations of Alcohol, as in dehydration Image below (D) This is the inverted cover slip method in which the mounting microscopic study. Rf of glass slide and tissues (Rf=1.53-1.54) 6 DEHYDRATION (run slides up) media is placed on the cover slip and the slide is then slowly lowered. The to protect the specimen from physical injury, bleaching and ▪ accomplished w/ a series of gradually increasing concentrations of 3 difference between your inverted slide and inverted cover slip is where you deterioration due to oxidation alcohol and absolute alcohol put the mounting media. 4 to facilitate easy handling and storage ostriches thank u luv ANJELA SOLIS for constantly being there for me, stay strong, we can do this! ily ♡ AIRAH M.  MOUNTING OF BROKEN SLIDES If sections are Zenker fixed, treat first with either Lugol’s sol’n or 1% 5 1 the coverslip can be removed by soaking in xylene Alcoholic Iodine sol’n for 10-15 mins. D 2 place the broken slide in the incubator (37°C) until all the mountant 6 Wash with tap H2O (4 dips). has been removed 7 Treat with 5% sodium thiosulfate for 3 mins. using a sharp scalpel blade, the hardened film is cut around the 8 Wash well with tap H2O (4dips) – (or 10 dips). 3 section, and the slide is placed in cold H2O until the film and section 9 Stain for 15 mins. in Harris Hematoxylin. float off 10 Rinse with tap H2O (4 dips) - (or 10 dips). the film containing the section is mounted on a clean slide, placed in 11 Differentiate in 1% acid alcohol (3-10 dips). 4 the 37°C incubator until dry 12 Rinse in tap H2O (4 dips). Dip in ammonia water or sat. lithium carbonate (6 dips) until MOUNTING PROCEDURE TYPES OF MOUNTING MEDIA 13 sections are bright blue. WATER-INSOLUBLE mounting media or resinous mounting media A. HAND COVERSLIPPING SLIDES 1 Rinse in distilled water for 15 mins. or in running water for 10-20 (permanent mounting media) 14 the slide is taken from the last xylene station & excess xylene is wipe mins (or in 10 dips). ▪ NATURAL RESINOUS MOUNTING MEDIA 1 of the back of the slide (opposite of the specimen) and the edges of Stain with eosin for 15 seconds to 2 mins depending on the age of o Canada Balsam (Rf index 1.524) 15 the tissue sections the eosin and the depth of counterstain desired (or 10 dips). ✓ medium of choice until 1939 the slide is placed horizontally on a tabletop, with the tissue section Dehydrate by passing through 2 changes of absolute alc. for 1 min. 2 o Gum Damar 16 facing upwards in each dish (or 10 dips each). drop one or two drops of mounting medium onto the coverslip or ▪ SYNTHETIC RESINOUS MOUNTING MEDIA 17 Mount in Permount or Balsam. beside the tissue section on the glass slide o Permount or Clarite (Rf index 1.544) ✓ most widely used in N. America RESULTS o if too much mounting medium is used: it will ooze out at the sides o Histoclad NUCLEI: BLUE CYTOPLASM: PINK 3 of the cover slip, should be carefully wiped away with the fingernail covered by a fine cloth dipped in xylene o Kleermount o if too little or too dilute mounting medium: it will draw away from o Lukitt the edges of the cover slip and the section must be remounted 2 WATER-SOLUBLE or aqueous Mounting Media (Temporary Mounts) Lower a slide carefully onto the long edge of the coverslip and allow ▪ Kaiser’s Glycerol jelly (Rf index 1.47) it to cover the slide, spreading the mounting medium evenly and ▪ Von Apathy’s Gum Syrup (Rf Index 1.52) 4 ▪ Farrant’s Medium (Gum Arabic) (Rf index 1.43) without air bubbles. If air bubbles form under the leading edge of the cover slip, move the cover slip up slightly and remove the air bubbles. ▪ Liquid Paraffin – best for Romanowsky stains 5 carefully wipe the back of the slide with a gauze pad or lint-free tissue 6 the slides are kept flat overnight (ideally) REFERENCES B. AUTOMATED COVERSLIPPING 1 Make sure the machine is on and length is set to “short”. 2 Check mounting medium level in bottle to make sure it is sufficient. Remove slide carriage from clearing agent and place in loading rack. 3 check that labels/patient name face towards you. Press xylene check and start button. make sure that it processes the 4 first few slides properly before leaving. When the machine signals that it is finished, remove slide rack and 5 move to fume hood or well-ventilated area with air vent. Replace receiving rack in cover slipper. 6 Remove from the slide rack to tray up and label. ROUTINE H&E STAINING PROCEDURE NOTE: for our purpose in the laboratory, proceed with steps 1-4, omit steps 5-7, then proceed with steps 8 to the end Arrange the sections to be stained in the bottomless glass slide 1 carrier, one slide to each slot. Immerse in the first xylene bath for 2 RE-COVER SLIPPING HAND COVERSLIPPED SLIDE mins, if a slide needs to be re-covered, place the slide into the Xylene Coplin Transfer to the 2nd xylene bath for another 2 mins. Excess solutions 1 2 should always be drained off by tilting the slide carrier and scarping Jar and secure the lid slide may need from a couple of minutes to several days depending it on the edge of the glass dish. 2 Pass through 2 changes of absolute alc. for 1 min. in each dish (or on how old the slide is 3 3 gently remove the cover slip nce it has been loosened by the xylene 10 dips each). 4 if necessary, re-cover slip by hand 4 Immerse in the 2 changes of 95% alc. for 1 min in each dish. Broadest thank u luv ANJELA SOLIS for constantly being there for me, stay strong, we can do this! ily ♡ AIRAH M.