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H&E staining
Nuclear and cytoplasmic staining in the histology lab.
| Question | Answer |
|---|---|
| Two majors parts of a cell | Nucleus and Cytoplasm |
| Resting nucleus is typically in what phase? | Interphase |
| The nucleus appears to be what color when being stained with the standard H&E stain? | Dark blue to purple |
| Electron microscopes are able to view subcellular particles which include | Nuclear membrane, nuclear pores, nucleolous, chromatin, plasmalemma, mitochondria, ribosomes, endoplasmic reticulum, Golgi apparatus, centriole, and lysosomes |
| Most likely the avenues of communication between the cytoplasm and the nucleus | Nuclear pores |
| Usually intensely basophilic but with a good H&E stain it is acidophilic | Nucleolous |
| Produces most of the ribosomal RNA | Nucleolous |
| Heterochromatin | Stainable |
| Euchromatin | Non stainable |
| Consists of chromosomes or DNA and attached protein | Chromatin |
| Intensely basophilic | Heterochromatin |
| This type of tissue will exhibit the most intense nuclear staining of all cells because heterochromatin dominates. | Lymphocytes |
| Euchromatin is predominantly present in this type of tissue and will not stain well with heme. | Neuronal nuclei |
| Energy producing powerhouses of the cell | Mitochondria |
| Site of protein synthesis | Ribosomes |
| Some are on the endoplasmic reticulum while others are located in the cytoplasm | Ribosomes |
| Responsible for the blue color in the cytoplasm | Ribosomes |
| Packaging apparatus of the cell | Golgi apparatus |
| Responsible for spindle formation in cell division | Centriole |
| Occur in pairs | Centrioles |
| Part of the bodies defense mechanism | Lysosomes |
| Aid in the digestion of food taken into the cell | Lysosomes |
| Long lived or permanent cells such as neurons, cardiac muscle, and hepatocyte accumulate a large amount of residual bodies. it is referred to as | Lipofuscin (wear and tear pigment) |
| Exogenous materials | Carotene, dusts, and minerals |
| Melanin and hemoglobin breakdown | endogenous pigments |
| Attraction for minute particles from the surrounding solution, by the surface of certain tissue components; the dye is then bound to the tissue primarily by ionic, covalent, or hydrogen bonds | Adsorbtion |
| Ionic bonding | Different charges that become attracted to one another |
| hydrogen bonding | Covalently bonded hydrogen is attracted to atoms that have a strong electronegative charge. |
| Hydrogen frequently bonds to what elements | Oxygen and Nitrogen |
| Covalent bonding | When atoms share electrons |
| Elements that typically form covalent bonds | Carbon, hydrogen, and oxygen |
| Van der Waals forces | Caused by electrostatic attraction of a molecule for the electrons of its neighboring molecules. These are weak physical forces that are effective over only very short distances. |
| Nuclear staining is performed in what two ways? | 1. Done with basic (cationic or positively charged) dyes 2. Done with dyes combined with or followed by metal mordants |
| Staining performed with a basic dye depends on what? | Presence of the nucleic acids (DNA and RNA) to form dye salt type unions |
| Staining performed by combining the dye with or followed by a mordant depends on what? | Nucleic acids have been removed (decalcified tissue) and also may occur in tissue that is not negatively charged. |
| Non nuclear staining is primarily cause by | proteins or charged groups on the side chains of amino acids constituting the proteins |
| Proteins that can be positive or negative | Amphoteric |
| Eosin must be kept below pH of 6 or else | the -COO group of eosin recombines with hydrogen and the result is the free acid, uncharged form of eosin. Non specific staining will occur. |
| A group that confers the property of color | Chromophore |
| if reduction occurs to a chromopohore what happen? | Color is lost |
| A benzene derivative containing chromophoreic groups | Chromogen |
| An ionizing group that links firmly to the tissue | Auxochrome |
| Amino (NH2) | Auxochrome |
| Chromophore | C=O, C=S, C=N, N=N, N=O,NO2 |
| Picric acid is | anionic or an acid dye because of the 1 auxochrome group |
| Anionic auxochromes | Sulfonic, carboxyl, and hydroxyl groups |
| Cationic dyes | Chloride salts |
| Basic dyes | Crystal violet and safranin |
| Acid dyes | Orange G and picric acid |
| Amphoteric dyes | Hematein and lithium carminate |
| Factors that affect dye binding | 1.pH 2. Temp 3. Concentration 4. Salts 5. Fixative used |
| Tissue binds less with Eosin when this type of fixative is used | Formalin |
| Tissue binds less with Heme when this type of fixative is used | Potassium dichromate |
| Tissue will lose its nuclear staining properties when stained with these types of fixatives | Zenker, Bouin, unbuffered formalin, (all acidic fixatives) |
| Types of fixatives that increase tissue basophilia or the uptake of cationic or positively charged dyes | Formaldehyde, mercuric chloride, and osmium tetroxide |
| increases the binding of anionic or negatively charged dyes | Picric acid |
| Metals that act as a link between the tissue and the dye | Mordants |
| Ways to differentiate | 1. When basic or cationic dyes are used differentiate by weak acid 2. Excess mordant 3. oxidizers |
| Potassium permanganate and chromium trioxide | Oxidizers |
| An example of what stain uses excess mordant to differentiate | Iron heme stains |
| Aluminum hemes can be differentiated with | Dilute hydrochloric acid |
| Eosin can be differentiated with | Dilute solution of Ammonium hydroxide |
| Hematein | Oxidized heme |
| Hematoxylin can be oxidized by | air, light, sodium iodate, mercuric oxide, and potassium permanganate |
| Harris Heme | Hematoxylin Absolute Alcohol Ammounium aluminum sulfate Distilled water Mercuric Oxide |
| Original formula of Harris Heme used to use mercuric oxide. What do they use now? | Sodium Iodate |
| Harris Hematoxylin mordant and oxidizer | Mordant: Aluminum Oxidizer: Sodium Iodate |
| Harris hematoxylin is used | Progressively and acidified |
| Delafield hematoxylin | Solution A Ammonium aluminum sulfate Distilled water Solution B Hematoxylin 95% alcohol Glycerol |
| Delafield hematoxylin mordant and oxidizer | Mordant: Aluminum Oxidizer: light and air |
| Glycerol does what in the Delafield hematoxylin | Stabilizes the solution against over oxidation and prevents rapid evaporation |
| To check for over oxidation of Delafield heme | Drop a few drops into a container of water. If it is blue-black it can still be used. If the solution turns into a red or red brown then it will be over oxidized. |
| How to check for under oxidation of Delafield heme | Drop some heme onto filter paper if the drop has a purple edge it is ready to use. If the purple edge is absent then it is under oxidized. |
| Delafield heme is used | Regressively |
| Mayer heme | hematoxylin Distilled water Sodium Iodate Ammonium or potassium aluminum sulfate Citric Acid Chloral hydrate |
| Mayer heme mordant and oxidizer | Mordant: Aluminum Oxidizer: Sodium Iodate |
| Citric Acid and Chloral hydrate do what in the Mayer heme solution? | Adjust the pH and helps prevent the scum and precipitates |
| Recommended heme for immunoperoxidase techniques when 3-amino-9-ethylcarbazole is used | Mayer heme is used because it does not contain alcohol and will not dissolve the reaction product. |
| Very difficult to over stain with this heme and produces very crisp nuclear staining. (Progressive) | Mayer heme |
| Ehrlich heme | Hematoxylin Alcohol 95% Distilled water Glycerol Ammonium or potassium aluminum sulfate Glacial acetic acid |
| Ehrlich heme mordant and oxidizer | Mordant: Aluminum Oxidizer: natural or chemical can be used |
| Ehrlich heme | More commonly used regressively but can be used progressively |
| Gill heme | Distilled water Ethylene glycol hematoxylin, anhydrous Sodium Iodate Aluminum sulfate Glacial acetic acid |
| Ethylene glycol does what in the Gill heme? | Prevents the formation of surface precipitate |
| Mordant and oxidizer of Gill heme | Mordant: Aluminum sulfate Oxidizer: Sodium iodate |
| Gill 2 and 3 are used for | staining tissue |
| Gill 3 is | most concentrated |
| Can be used for staining glycol methacrylate sections | Gill 3 |
| Gill 1 and 2 are used | Progressively |
| What structure is stained in tissue when using Gill heme | Goblet cells in Mucin |
| Less prone to surface precipitate | Mayer and Gill |
| Scott Solution | Magnesium sulfate Sodium bicarbonate Tap water |
| Scott solution | Changes the pH and allows the slides to blue (change color) Also stabilizes the stain. |
| Weakly alkaline solutions | Lithium carbonate, ammonium hydroxide, or Scott solution |
| Weigert heme | Ferric chloride Distilled water hydrochloric acid Hematoxylin 95% alcohol |
| Resists decolonization in acidic staining solutions | Weigert heme |
| Weigert mordant and oxidizer | Mordant and oxidizer: Ferric Chloride |
| Substitute for Weigert heme | Gallein iron heme |
| how many shade of pink should be obstained when staining with Eosin | Three (Erythrocytes, collagen, and muscle) |
| The more dilute alcohol the more _ will be removed | Eosin |
| Longer the tissue is fixed | the A The longer it may need to stain |
| Time may need to be increased in Heme and decreased in Eosin when fixed in | Helly, Zenker, or B-5 |
| Restoring tissue basophilia: If over exposed to Bouin | Place in 5% aqueous lithium carbonate for 1 hour |
| Restoring tissue basophilia: If over exposed to Zenker | Place in 5% aqueous sodium bicarbonate for 3 hours |
| Restoring tissue basophilia: Method III | 5% periodic acid for 30 minutes |
| Tissue basophilia loss may result from | -wet tissue too long in Bouin, Zenker, or unbuffered formalin -overdecalification of bone |
| Best heme to use to stain nuclear chromatin in specimens that have been over exposed to unbuffered formalin and/ or an acid-decalcifying agent | Weigert iron heme |
| White spots may be seen in tissue sections | incomplete deparaffinization |
| Ways to correct incomplete deparaffinization | -Ensuring the sections are dried properly -Allow enough time in xylene -Ensure xylene is not contaminated -If slides are stained then decolorize and restain |
| When distinct chromatin pattern cant be seen (Smudgy or muddy nuclear detail) | Nuclear staining is not crisp |
| Most frequent cause of nuclear staining not being crisp | Overfixation |
| Other causes of nuclear staining not being crisp | Too much heat during processing or drying of the microscopic sections |
| Smudgy nuclei can be prevented by | -Complete fixation of specimens -Tissues should be dehydrated and cleared completely -No heat on processor -Tissue should not remain in melted paraffin for long -Dryer is correct temp |
| Pale nuclear staining | 1. Not leaving the slides in heme long enough 2. Staining with overoxidized or depleted heme 3. Overdifferentiating the heme |
| Over decalcified specimen | Pale nuclear staining |
| Pale nuclear staining can be corrected by | -Ensuring slides are left in heme long enough -Ensure heme is not overoxidized -Ensuring differentiation step is properly times -restaining section -use of fixative |
| Dark nuclear staining most likely causes | 1. Sections left too long in heme 2. Sections too thick 3. Differentiation step too short |
| Dark nuclear staining can be corrected or prevented by | -Ensuring sections are thing -Decolorize the sections and restain -Decrease the time sections remain in heme -Increasing the time of differentiation |
| Red or brown nuclei is caused by | Either the heme is breaking down or blueing step was not properly done |
| Ways to prevent or correct red/red-brown nuclei | -Ensure sections are blued properly -Check the oxidation of the heme |
| Pale cytoplasmic staining may result from | pH of the Eosin being >5. It may also occur from sections being too thin or being left too long in dehydrating solution |
| Pale cytoplasmic staing can be prevented or corrected from | -Checking the pH of Eosin -Ensure the blueing reagent is removed before transferring the slides to eosin -Ensure that stained slides are not allowed to stand in low concentrations of alcohols after eosin -Ensure sections are not too thin |
| Dark cytoplasmic staining can be corrected/prevented by: | -Ensure the eosin solution is not too concentrated -Ensure sections are not left too long in Eosin -Ensure time in dehydration solutions allow good differentiation -Check section for proper thickness |
| Eosin not properly differentiated may be prevented or corrected by | -Ensuring timely and complete fixation -Ensure good dehydration and clearing during processing -Ensure eosin stained sections get proper differentiation -Ensure eosin is correct pH |
| Blue-black precipitate on top of sections | Caused from the metallic sheen that is formed on top of heme solutions (Filter to correct) |
| Water and slides turn milky when slides are placed in water following alcohol during deparaffination | if after rehydrating alcohols then it indicates the presence of xylene. Back the slides up and change the alcohols. The take slides from alcohol to water. Water should be clear |
| Slides are hazy or milky in last xylene before applying cover glass | Water is still present. Back up the slides and change the alcohol and xylene. Then rehydrate and clear sections. |
| Uneven H&E staining | Caused by water or fixative infiltrating paraffin caused by contamination of reagents in closed tissue processors because of equipment malfunction or absorbtion of atmospheric water by dehydrating alcohols on the open processors |
| Uneven H&E staining cant be corrected but can be prevented by | -Change from xylene to toluene in areas of high humidity if using an open processor -Have equipment checked for malfunctions |
| Dark basophilic staining of nuclei and cytoplasm (Especially around the edges | Laser and electrocautery techniques (No remedy) |
| Poor contrast between nuclei and cytoplasm causes | 1. nucleus is too pale to contrast with the cytoplasm 2. the cytoplasm is overstained and masks nuclei 3. the nuclear stain is too dark for the cytoplasmic stain 4. the cytoplasmic stain is too pale for the cytoplasmic stain |
| Poor contrast between the nuclei and the cytoplasm can be prevented or corrected by: | -Determine which stain is the problem -Check the pH -monitor water pH |
| DNA is found in the | nucleus |
| RNA is found in the | nucleolous and ribosomes |
| Feulgen reaction purpose | Demonstration of DNA |
| Feuglen reaction principle | Based on the mild hydrolysis of DNA by hydrochloric acid. Removes purine bases but leaves sugars and phosphates of DNA intact. Hydrolysis generates an aldehyde group that can be demonstrated with Schiff. |
| Feuglen reaction fixative | Anything but Bouins |
| Feuglen reaction results | DNA: Reddish purple Cytoplasm: Light green |
| Methyl green-pyronin Y purpose | Demonstrates DNA and RNA |
| Primarily used to identify plasma cells and immunoblasts in tissue | Methyl green-pyronin Y |
| Methyl gree-pyronin Y principle | DNA stains with green, while RNA is colored red with pyronin. Differential staining caused from differing degrees of polymerization. Methyl green is bound by more highly polymerized DNA. |
| Methyl green pyronin Y fixative | 10% NBF is preferred. B-5, Helly, or Zenker is fine |
| Methyl green pyronin Y results | DNA: Green to blue-green RNA: rose Goblet cells: Mint green Background: Pale pink to colorless Immunoblast and plasma cell cytoplasm: Intense red Nuclei: Green to blue-green |
| Polychromatic stains | A compound dye or dye mixture that contains components of different colors |
| A process in which a dye forms other dyes spontaneously | Polychroming |
| Romanowsky type stain | Giemsa |
| May Grunwald Giemsa stain Purpose | To permit differentiation of cells present in hematopoietic tissue. Also used to stain certain microorganisms |
| May Grunwald Giemsa stain Principle | "neutral" dyes combine the basic dye methylene blue and the acid dye eosin give a wide color range when staining blood smears. Because of impurities present in dye solution. |
| May Grunwald Giemsa stain Fixative | Zenker or B-5 preferred, 10% NBF is fine |
| May Grunwald Giemsa control | Spleen |
| May Grunwald Giemsa Results | Nuclei: Blue Cytoplasm: Shades of pink, gray, blue Bacteria: Blue |
| Tissue fixed in aldehydes will have | increased basophilia |
| What are two types of mounting medium? | Resinous and Aqueous |
| Resinous media tends to be | natural |
| Examples of resinous media | Canada balsam and gum dammar |
| Natural resins dissolve in what? | Xylene |
| Natural resins | Take a long time to harden, turn yellow over time, and tend to cause fading in stains over time due to their acidity. |
| Most resinous media are dissolved in | Toulene |
| Tissues average refractive index | 1.53-1.54 |
| Synthetic resins refractive index | 1.51-1.55 |
| Best preservative to protect Romanowsky stain when using a natural resin | Mineral oil |
| After cover slipping you notice that the slide has numerous microscopic water droplets all over the slide. How do you fix this? | Remove coverslip and any remaining medium with xylene. Rehydrate with the appropriate reagent, clear with xylene, and remount with synthetic resin |
| Aqueous mounting media is used when | Dehydrating and clearing will adversely affect the stain |
| Aqueous mounting media refractive index | 1.41-1.43 |
| Ideal range for photomicrography | 1 1/2 (180 micrometers thick), number 1 coverslips (~150 micrometers thick) |
| As thickness increases | Section transparency is reduced |
| Thickening of the mounting medium does what to transparency? | Decreases |
| Cloudiness of the section | Results from thickened medium or excess medium between the section and cover glass |
| If drying artifact is noted what should be done to correct it? | Rehydrate the slides by placing in water for 15-20 minutes. Restain if needed, and dehydrated, cleared and mounted with synthetic resin |
| Exhibits brown stippling that resembles pigment or nucleus appears ti have a glossy black structure | Drying artifact |
Created by:
Ziek98
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