UND 362 H&E Staining
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| List 3 components of a cell | Membrane, Nucleus, Cytoplasm
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| Nucleus contains ____________ acids and the two types are? | Nucleic acids (DNA and RNA)
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| RNA can be found in what two places | Nucleolus (in nucleus), but most is in the cytoplasm
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| Nucleic acids react to what stain and become what color | Hematoxylin (dark blue to purple)
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| Nucleus contains ______________ /give the two types | chromatin, Heterochromatin and Euchromatin
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| Heterochromatin is active or inactive? and will stain what color | Inactive and will stain dark blue
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| Euchromatin is active or inactive? and will stain? | active but will only stain lightly
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| Name three nuclear stains | Hematoxylin, feulgen reaction, methyl green pyronin Y
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| cytoplasm helps to differentiate what and how | differentiates types of cell by amount and appearance of cytoplasm
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| cytoplasm contains what that cannot be seen by a light microscope | organelles
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| aBsorption staining is done how | soaks up the dye (PHYSICAL)
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| aDsorption staining is done how | via attraction to particles in dye by surface components
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| name three adsorption dye binders | covalent, ionic, or hydrogen bonds
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| Staining is typically a combination of what two types of reactions | physical (absorption) and chemical (adsorption)
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| Lipids are non ionic and therefore the stain would be? | absorption (physical) absorbed and dissolved in lipid.
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| Adsorption takes place in 2 stages. what are they | 1. dye is placed on the material 2. dye is bound via covalent, ionic, or hydrogen bonds
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| Name 4 chemical bonds for staining (1 is more uncommon) | Ionic (aka electrostatic or salt linkage), covalent, hydrogen bonds, VanderWall forces
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| Describe ionic adsorption | opposites attract, the donor becomes positively charged (cation) and the recipient becomes negative (anion)
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| NA with 1 electron and CL with 7 becomes NACL, where the NA becomes positive and the CL becomes negative. This is what type of bond? | Ionic (the donor NA loses 1 electron to become positive) and the CL gains 1 electron (to 8) and becomes negative
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| Covalent bonds is best described as? | two chemicals sharing electrons
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| Hydrogen bonds are what type of bond | weak covalent bond (ie H-O-H)
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| Describe Vanderwall Forces | Attraction of Non polar molecules due to high pressure/low temp (the bigger the molecule the stronger the attraction)
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| Describe how the basic Nuclear dyes work | Use positive (cationic) dyes to for a salt linkage with nucleic acids.
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| When are mordants used? | When nucleic acides have been removed through decal or when tissue has no negative charge (Usually caused by fixative splitting phosphate group)
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| Describe the typical cytoplasmic stain | Anionic (neg.) charged with affinity for pos. charged tissue
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| Amphoteric stains can be what, depending on PH of dye solution | positive or negative
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| Describe isoelectric point (IEP) | when protein has equal number of pos and neg charges (therefore no migration)
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| if a protein has a ph below 6 it will become? | positively charged and attract Eosin
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| if a protenin has a ph above 6 it will become? | negatively charged and will not attract eosin
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| The optimal ph range for a protein is | 4.6 - 5.0
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| If the ph is too low what occurs | -COO group of eosn combines with hydrogen becoming a free acid (uncharged eosin)
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| Metachromatic means | tissue is stained a different color than the dye
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| Orthochromatic means | tissue is stained the same color as the dye
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| Polychromatic means | Single dye that stains tissues different colors
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| Name two classes of dyes | Natural and Synthetic
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| List the natural dyes (4) | hematoxylin, carmine, orcein, and saffron
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| Synthetic dyes are most common and are derivitives of ________ and __________ | Coal tar or benzene
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| Basic dyes are what _____philic and have what charge | basophilic, positive (cationic)
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| Basic dyes are attracted to what proteins | acid (nuclei acids)
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| Acid dyes are what ______philic and have what charge | acidophilic, negative (anionic)
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| acid dyes are attracted to what proteins? | cytoplasm, muscle, connective tissue
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| give 3 examples of acidophilic dyes | eosin, orange G, picric acid
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| give 3 examples of basophilic dyes | hematoxylin, crystal violet, saffron
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| A chromophore in artificial dye gives what | color, the intensity depends on the #'s (can be reduced due to affinity for hydrogen)
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| A Chromagen in artificial dyes is a what | benzene derivative w/ chromophoric groups
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| An Auxochrome in artificial dyes does what | links dye to tissue (help chromagens link dye to tissue)
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| trinitrobenzene and picric acid are both what and what | chromophores and auxochromes
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| Name 5 factors that affect dye binding | PH, Temp, Dye concentration, salt and dye competition, fixatives
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| How does ph affect dye binding | establishes charge on tissue and dye molecule
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| How does temp affect dye binding | Increases rate of diffusion and swells tissue
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| How does dye concentration affect dye binding | an increase in conc. will increase the binding
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| How does salt and dye competition affect dye binding | salt and dy will compete for the same binding site
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| How doe s a fixative affect dye binding | alters staining characteristics by reacting with chemical groups in tissues, therefore making them unavailable for dye
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| explain how formalin affects dye binding | it reacts with amino (NH2) group therefore there is less eosin bound since NH2 is the primary eosin binder
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| explain how potassium dichromate affects dye binding | it reacts with carboxyl (COOH) and hydroxyl (OH) group therefore will uptake less hematoxylin and more eosin
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| explain how zenkers, bouins, and unbuffered formalin (acidic fixatives) affect dye binding | decreases nuclear staining (lose basophilic nuclear staining properties)
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| explain how picric acid affects dye binding | increased anionic/negatively charged dye binding
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| explain progressive staining | staining to desired intensity
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| explain regressive staining | tissue is overstained and a weak acid or weak alkalin is used to differentiate the tissue
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| What is a mordant | substance or metal that links dye and tissue (can be progressive or regressive)
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| give 4 examples of a mordant | aluminum, tungsten, iron, chromium
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| what is a "lake" | combination of a mordant and a dye
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| name 4 types of differentiation methods for mordants used regressively (one uncommon) | Basic (cationic), excess mordant, oxidizers (bleaching), Buffers
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| Explain basic and acidic differientiation | Basic dyes are diff'd by weak acid, acidic dyes are diff'd by weak alkaline (ex hematox diff'd with HCL and Eosin diff'd with ammonium hydroxide
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| When diffing cationic and anionic dyes it is best to use _________ instead of ___________ in the solutions | diff'd solutions are controlled better with alcohol than H20
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| Excess mordant can Diff a stain by? | breaking the tissue/mordant dye complex
ie: increased mordant V. bound dye will dissolve dye (this will leave the background colorless)
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| Oxidizers can Diff a stain by? | oxidizers bleach out the dye (it is slow process)
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| name two oxidizing differs' | potassium permanganate and chromium trioxide
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| Name an uncommon method for diffing stains | using buffers (not used in histology), is used in wright stain for blood smears
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| Hematoxylin can be ripened in what two ways | natural (via O2) and chemical oxidation (sodium iodate, mercuric oxide, potassium permanganate
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| name a drawback to natural and chem oxidation of hematoxylin | natural takes a long time, and chemical shortens the life span (although it is quick)
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| ripened hematoxylin must have a _______ to bind to tissue | Mordant
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| The mordant in Alum Hematoxylin can be (2) | Aluminum potassium sulfate or aluminum ammonium sulfate
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| Describe "bluing" | red stained nuclei converted to blue black in weak alkaline solution
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| give 3 examples of bluing agents | ammonium water, lithium carbonate, scotts tap water substitute
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| what occurs during "bluing" | PH chnage of bluing agent changes solubility of dye lake (alum heme is red and soluble below ph 5), bluing converts red soluble compolex to blue insoluble "lake"
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| Harris heme's mordant and oxidizer are | aluminum and sodium iodate
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| harris heme can be used progressively and regressively how? | prog - due to intensity
regress - uses acid/alcohol rinse
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| Harris heme formula | hematox, absolute ethanol, ammonium aluminum sulfate, distilled h2o, mercuric oxide
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| Mayers heme mordant and oxidizer | aluminum and sodium iodate
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| what can be used to adjust ph of mayers heme | citric acid
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| what does the citric acid and chloral hydrate in mayers heme do | prevent scum and precipitates
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| what is mayers heme recommended for? | immunoperoxidase tech because it doesn't contain alcohol
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| give 2 drawbacks to mayers heme | 2-3 month shelf life and when used for long time it will have dye exhaustion and produce pale nuclei
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| mayers heme is used pro or regress? | progressively but is very slow (but doesn't overstain)
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| Mayers heme formula | hematox, distilled h20, soduim iodate, amonium or potassium aluminum sulfate, citric acid, chloral hydrate
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| Delafield mordant and oxidizer | aluminum and natural oxidizer
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| alcohol in delefield heme serves as a? glycerol in delefield heme serves as a? | preservative, stabilizer (prevents overoxidation)
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| how is delafield heme typically used? | regressively
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| give 3 ways to test if delafield is ready for use | 1. smells winelike 2. add drops of h20, if blue black then it is good 3. drop on filter paper, should give maroon spot with purple border.
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| Ehrlich heme formula | hematox, 95% alcohol, distilled h20, glycerol, ammonium or potassium aluminum sulfate, glacial acetic acid
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| Ehrlich hem mordant and oxidizer | aluminum, can be done natural or chemical (natural takes 2 months)
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| Which staining method is ehrlich used for | regressive staining
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| Ehrlich heme is good for what two tissues | bone and cartilage
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| What is ehrlich heme especially useful for | sections exposed to acids such as acid decalcifiers or tissue stored for a long time in formalin
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| Gills heme formula | distilled water, ethylene glycol, anhydrous hematoxylin, sodium iodate, aluminum sulfate, glacial acetic acid
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| Gills heme mordant and oxidizer | aluminum, and sodium oxidate (best if ripened for 1 week at 37C)
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| what is used as a solvent for gills heme and why | ethylene glycol, prevents surface precipitates
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| How many strengths of conc. does gills heme have and which is used for histology | 3 strengths of conc (1x, 2x, 3x), 2x and 3x are used in histology
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| What is gills heme (strength 3x) used for | Glycol methacrylate sections
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| How is 2x gills heme conc. prepared versus 1x | 2x hematox and sodium iodate and 4x aluminum sulfate
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| Name 2 disadvantages of gills hematox | will stain adhesives and glass slides, will stain goblet cells
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| What must be done to hematox before use | filtering or it will get a metallic sheen if allowed to stand (a result of oxidation which produces blue black preciptate)
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| Mayers heme is less likely to produce the metallic sheen because of what two formula additives | citric acid and chloral hydrate
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| Acetic acid in hematox will reduce what? | oxidation
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| over oxidation of heme will result in what | blue goes to red then to brown and will therefore stain nuclei brown
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| what happens to stains if they are repeated with an alum hematox | decolorization
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| aluminum mordant will decolorize if (2) | staining is too long or is quite acidic
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| what two acids can remove hematox and give two alternative stain to use | picric acid/acid fuchsins, use weigerts hematox or clestine blue
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| weigert hematox formula (2) and why seperate | A. ferric chloride, distilled water, hcl (aka iron solution)
B. hematox, 95% alcohol (aka hematox soln)
(mixed before use to prevent overoxidation)
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| Weigerts hematox mordant and oxidant | Ferric chloride (aka iron salt)
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| Weigerts hematox demonstrates what well | nuclei, muscle striations, protozoa, mitotic figures
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| Celestine Blue formula | celestine blue, ferric ammonium sulfate, distilled water
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| Celestine blue can be used as a substitute in what two situations | H&E prodecure and weigerts
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| celestine blue mordant and used as a _____ type of stain | iron, progressive
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| celestine blue is resistant to what? | acid
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| Tungsten hematox formula (PTAH aka phosphtungsten acid hematox) | hematox, phosphotungstic acid, distilled water
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| mordant and oxidizer of for PTAH stain | phosphotungstic acid, and the oxidizer is variable based on two varied ingredients
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| In PTAH if hematein is used what is the oxidizer, what if hematoxylin is used | if hematein it is not needed, if hematoxylin then permanganate or naturally ripened
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| give 7 variables affecting stain times | hematox type, age of stain, usages, prog v. regress, tissue/section pretreatment, post treatment of section, personal affinity
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| hematox type will affect stain time how | some take longer than others
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| hematox age will will affect stain how | the older the stain the longer it will take
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| if hematox is over used this will affect stain how? | overusage will cause stain to lose staining power
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| how can overfixed tissue be fixed to improve stain times | using dilute nitric acid (for 5 min) and then washed
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| What is the purpose of the Feulgen stain | to demonstrate DNA
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| what is the principle behind feulgen reaction | hydrolysis of dna by Hcl with removes purine bases. This will generate an aldehyde group than can be demonstrated w/ schiffs reagent
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| what fixative cannot be used if fuelgen reaction will be used later and why | bouins (it causes excess hydrolysis)
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| describe in detail the feulgen procedure (2 reactions at same time) | 1) warm hcl, followed by schiff reagent. The hcl removes purine and forms aldehyde groups which react with schiff reagent. 2. If hydrolysis continues too long it will predominate and cause neg. feulgen reaction.
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| Following schiff reagent in feulgen reaction what needs to be done (per staining) | rinse in sulfurous acid and then counterstain with lt. green stain
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| what are the results (colorwise) to the DNA and Cytoplasm after the feulgen process | DNA is reddish purple, and the cytoplasm is lt green
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| What types of structures is the methyl green pyronin y stain for | plasma cells and immmunoblasts, and will differentiate between DNA and RNA
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| Methyl green demonstrates what and pyronin demonstrates what? | methyl green for DNA, pyronin for RNA
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| What is the preferred fixative for methyl grn pyr Y | formalin preferred, but B5, helly's and zenkers are ok.
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| What are the results of Methyl grn pyr Y | DNA blue green, RNA red, Goblet Cells, mint, Plasma/immunoblasts intense red, Nuclei blue green
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| Eosin is a _____________ for routine staining | Counterstain
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| Eosin has what charge and therefore combines with what charge of tissue | eosin is anionic (neg charge) and combines with cationic tissue
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| Name the four types of Eosin | Eosin Y, Eosin B, Ethyl Eosin, eosin phloxine B
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| Eosiy Y is used for what and has what 3 characterics | pap stain, water/ethanol soluble, pink acid dye, displays yellow green flourescence
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| What is eosin B soluble in | Alcohol
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| Ethyl Eosin is soluble in what and used in what stain | water and ethanol, romanowsky stain
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| What does eosin demonstrate | RBCs, Collagen, muscle (strength of stain will vary with tissue)
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| After using Methylene blue what % soln of eosin should be used | 5%
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| Post Zenkers how should eosin be used | diluted or at a shorter time with normal strength (zenkers stains intensly w/ eosin
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| What is the optimum ph for Eosin staining | 4.6 - 5
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| Eosine phloxine B has what positive and negative characteristics | produces vivid colors, but can overstain
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| List steps in order for H&E staining | Deparaffinization, hydration, nuclear staining, Bluing, Dehydration, Clearing, mounting
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| The slide must be ______ in order for comlete deparaffinization | dry (ie water and xylene don't mix)
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| What is typically used to deparrafin and how many change (and time per) should be used | xylene with 3 changes at 3 min. per
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| What are three outcomes if a slide is not dry prior to deparaffinization with xylene | imcomplete deparaffin, staining issues, loss of sections
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| what does the hydration step of H/E stain do? | readies tissue for aqueous dyes
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| How is h20 introduced into sections duiring H/E stain | water is introduced through successive gradients of alchol (abolute 1-2 changes, 95% 1-2 chng, 80-70 1 chg, 50-60 1 chg
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| What can be done additionally during the hydration process of H/E | Removal of formalin and mercury pigment and the yellow color of picric acid
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| what can be used to remove formalin pigment in the hydration process of H/E | using alcoholic picric acid or alkaline alcohol
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| What can be used to remove mercury pigment in the hydration process of H/E | Grams iodine and then sodium thiosulfate
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| what can be used to remove the yellow color of picric acid during the H/E staining process | 70% alcohol with saturated lithium carbonate
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| During the Nuclear staining of H/E it is done with what | hematoxylin
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| progressive nuclear staining is done with what 4 stains | Gills, Mayers, harris, delafield
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| Regressive nuclear staining is done with what stains (3) | Delafield, harris, ehrlich
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| explain why timing is crucial during an h2o based acid rinse for regressive staining | the tissue rapidly destains (it is recommended to use 70 or 95% alcoholic acid rinse since it can be done slow and repeatedly)
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| What two acids can be used when regressive staining | acetic acid and HCL (the acetic needs a higher conc. than the hcl though) and tissue must be rinsed or differentiation will continue and cause poor nuclear stain.
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| What is the function of the bluing agent | To blue the Nuclei
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| which of the two types of staining methods is bluing used in. | both progressive and regressive
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| name 4 bluing agents | ammonium h20, lithium carbonate, scotts solution, tap h20 above ph of 8
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| what will happen if the slide is left in the bluing agent? | nothing, it cannot be overdone
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| what is a crucial mistake when working with bluing agents | if the tissue is agitated in the soln they may fall off.
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| What is the function of cytoplasmic staining | it functions as a counterstain
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| what agents used for counterstaining | eosin b, eosin y, and eosin phloxine B
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| what are three variables to account for when using eosin | time in eosin needs to be sufficient, the conc. of alcohol (that will remove excess), and time in the alcohols
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| Eosin is soluble in what soln' and therefore it is best to use dilute alcohol | eosin soluble in water (ex if anhydrous isopropol is used no eosin would be removed due to lack of h20
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| what is the function of dehydration in the H/E stain process | removal of excess eosin
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| what is the typical alcohol tech. for removing water and excess eosin in the H/E stain | 2 changes of 95% and 3 changes of Absolute alcohol
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| what must be done before going to the clearent step in the H/E stain process | H20 MUST be removed before clearant (ie xylene)
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| What is the function of clearing in H/E | de-alcoholizing and rendering tissue transparent
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| what agents are typically used for a clearant in H/E and what do they have to be miscible with? | Xylene and Toluene, they must be miscible with the mounting media (they have high refractive index)
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| What is the typical clearing tech. in H/E staining | 3 changes of (xylene) at 1 min. per change
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| Mounting blue can be what two types | aqueous or resinous (preferred)
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| What are 4 requirements of mounting | refractive index close to tissue, provide protection, miscible with clearing agent, stable over time
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| what is the refractive index of tissue typically? | 1.53 to 1.54
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| give 3 issues mounting media shouldn't do | change tissue via crystals, leach out stain, shrink tissue
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| Resinous mounting media is _______ phobic and therefore? | hydrophobic and must be dri via alcohol and cleared with a compatible solvent
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| what two types of resins exist | natural or synthetic
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| name three drawbacks to natural resins | acidic which causes fading, dries slowly, yellows over time
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| name three positives to synthetic resins | harden quickly, do not fade or yellow, can be stored long term
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| give 3 requirements for resinous media usage | must be viscous and flow between tissue space and coverslip, displace air bubbles quick, dissolve in (xylene/toluene)
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| What is a drawback to using toluene during mounting | it has a tendancy to bubble more
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| If the romanowski stain is used what will eventually happen and what can be done to prevent it | gradual fading, use mineral oil to mount and seal the edges shut
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| Once mounted and the slide appears cloudy what is the issue and what should be done | incomplete dehydration. remove coverlip, xylene, rehydrate, clear and remount
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| aqueous mounting media is _________ philic and when is it commonly used | hydrophilic, used when dehydrating and clearing will dissolve the stain
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| name a few aqueous mount media | gum arabic, glycerol gelatin, syrups
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| when are aqueous mount media used | used for IHC, Enzymes, lipis
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| what is the refractive index of aqueous mount media and the 2 drawback | R. index is 1.41-1.43 therefore the tissue won't be as transparent and it will be hard to view above 45x
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| what is the most common coverslip size used? | #1 size (150mm thick)
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| if the thickness of the coverslip is increased what will occur | there will be a decrease in transparency
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| what should be avoided when coverslipping | don't use an excess of mounting media or attempt to remove it (may get on cover)
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| What should the final color results of the tissue components be after H/E | nuclie - blue, RBC and eosin granules - pink to red, cytoplasm - shades of pink
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| name 11 staining hints (5 pt 1) | check microscopically, prevent drying, cover containers to prevent evaporation, filter hematox, have protocols for soln changes
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| name 11 staining hints (4 pt 2) | don't dehydrate too quick since it will diff' eosin, long term fixation increased stain time requirements, fix type will alter stain time, xylene substitutes REQUIRE ABSOLUTE alc.
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| name 11 staing hints (2 pt 3) | use caution with tap water - work for rinses but may alter stain via iron, sulfer, chlorine (weaken hematox stain), some stain problems are result of fixation or processing
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| what would be the cause of white spot/ under stained areas (1 of 2) | incomplete drying (paraffin in section or sections missing)
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| if incomplete drying results in white spots in an H/E what is the proper remedy | remove h20 with alcohol, remove paraffin with xlyene
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| what can cause white spots/under staining in H/E | insufficient time in xylene (paraffin removal)
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| if a tissue has white spots due to lack of time in xylene what is the appropriate fix | return to xylene then decolorize and restain
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| What is the cause of milky slides following rehydration | xylene on slide (in residual paraffin)
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| what is the remedy for milky slides following rehydration | change alcohols, and bring slide back to absolute through to xylene (to remove paraffin)
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| what are 3 reasons that cause pale nuclei | insufficient time in hematox, hematox is overoxidized, over differientated (too long)
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| what can remedy pale nuclei that haven't been in hematox long enough (2 options) | increase time if using acid fixatives - or use weigerts hematox (which is resistant to decolorization via acids)
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| if pale nuclei are present due to over differentiation what can remedy this | back up to hematox, and restain (then check microscopically)
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| What is a fourth reason that pale nuclei can exist and what is the fix | over decalcification, restain with a tech that will restore basophilia
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| if basophilia is gone due to bouins overexposure what is the proper method to fix | deparaffinize then put in 5% lithium carbonate for 1hr, wash in water 10 min., stain
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| give 2 methods for restoring basophilia | 1. 5% aqueous soduim bicarbonate 3 hr (4hr if due to zenkers overexposure), water 5 min, stain. 2. 5% periodic acid for 30min, rinse 3 chg of distilled h20, stain
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| what are 3 causes for overstained nuclei/diffuse cytoplasm hematox | too long in hematox, sections are too thick, differientation too short
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| give 2 remedies for overstained nuclei/diffuse cytoplasm | recut if section is too thick, decolorize and adjust either the stain time or differientation time.
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| what are the 2 causes of red/red brown nuclei? | over oxidized hematox and insufficient bluing
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| what can remedy red/red brown stained nuclei | check to see if hematox is overoxidized or increase time in bluing
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| If Eosin is pale what are 3 possible reasons | The PH is too high, the sections may be too thin, or the sections were in dehydration too long
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| If pale eosin is caused by the ph being too high how can this be corrected | adjust with acetic acid (the ph must be 4.6-5 to give proteins a + charge)
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| pale eosin has been caused by sections being in dehydration too long what can remedy this | keep the sections out of the lower conc. % alcohols (the lower alcohols have higher h20 content that bleaches out eosin)
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| What are 3 causes of overstained cytoplasm/poor differentiation? | too high eosin %, staining too long, to short of time in dehydration
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| what are 4 remedies for overstained cytoplasm/poor differentiation | dilute eosin solution, decrease stain time, increase time in dehydration soln' to allow good diff'n, check thickness
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| If a stained section has a blue black precipitate what is the cause and remedy | cause-picked up metallic sheen from unfiltered hematox. remedy - filter hematox daily
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| what can cause microscopic water bubbles on a section, what are the steps to remedy | improper dehydration before xylene. remove coverslip via xylene, use fresh absolute, clear with fresh xylene
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| What can cause difficulty in focusing after a slide has been coverslipped | mounting media has gotten on top of coverslip
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| what causes retracted coverslips (2) | warped coverslips or too little mounting media
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| what is the cause of milky slide following rehydration and remedy | xylene still present, change alcohol, bring slide back to absolute, dehydrate and coverslip
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| what caused hazy/milky slides prior to coverslipping and steps to fix | incomplete remove of water before xlyene. change absolute, rehydrate, clear and coverslip
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| if sections lack transparency what is the likely cause | thick mounting media (xylene and remount)
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| what can cause hazy blue nuclei (3) | too much heat in processor, too long in paraffin, short fixation followed by high % alcohol
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| if nuclei have a hazy blue appearance what are the possible remedies (3) | be careful about heat exposure, don't hold sample in paraffin too long, proper fixation and start in lower alcohol % (60-70%)
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| What are 3 likely causes of uneven staining/poor chromatin detail(3) | water or fixative in paraffin, contaminated reagents, environmental absorption
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| if uneven staining/poor chromatin detail is present what can rememdy this (2) | use toluene (over xylene) for increased humidity with an open processor, check closed processor for malfunctions
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| what causes electrocautery artifact and what can remedy it | heat (during time of collection) which shows up as dark basophilic stain of nuclei and cytoplasm. There is NO remedy.
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| what can cause gloss black nuclei/brown stippling and the remedy | sections were allowed to dry before coverslipping. remove coverslip, rehydrate with h20 for several min., dehydrate, clear, and mount.
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