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UND 362 Fixative gen
UND 362 Fixatives general knowledge
| Question | Answer |
|---|---|
| Additive Fixative (10) hint (3 aldehydes, 2 acids, 2 salts) + MOP | Formaldehyde, Glutaraldehyde, Glyoxal (3 aldehydes), Osmium Tetroxide, Potassium Dichromate, Mercuric Chloride, Chromic Acid, Picric Acid, Zinc Salts, Cupric Salts |
| Non-Additive Fixatives (3) | Alcohols, Acetone, Acetic Acid |
| Non Coagulant Fixative (5) | Formaldehyde, Glutaraldehyde, glyoxal, potassuim dichromate, Osmioum Tetroxide |
| Coagulant Fixative (8) | Mercuric, Chloride, Chromic Acid, Picric Acid, Zinc Salts, Cupric Salts, Alcohols, Acetone, Acetic Acid |
| Formalin Pigment AKA | Black Acid Hematin |
| Additive Def. | Combines with tissue |
| Non-Additive Def. | Doesn't combine with tissue |
| Coagulant Def. | Creates network allowing penetration (tea ball) |
| Non-Coagulent Def. | Forms gel preventing penetration (Jello) |
| 9 Factors affecting fixation | Temperature, size, tissue/fixative ratio, time, fixative choice, penetration, tissue storage, PH, Osmolality |
| Proper fixation temp | *typically room temp* LOWER than 45 deg. C = slow reaction and limited effect, ABOVE 60 deg. C = tissue distortion |
| Proper specimen size for fixation | 3-4 mm thick (nickel thick), approx 20mm (postage stamp size) |
| Proper specimen to fixative volume ratio | 1 to (15-20) |
| Ideal condition for getting specimen into fixative (name 2 types) | *Quicker is better* 1. Immersion (most common) 2. Perfusion (fixative delivered directly through circulatory system) |
| Minimum time requirements for 3mm section in fixative | 6-8 hrs (thinner is better) |
| Zenkers is good for what tissue/stain (2) | muscle/PTAH stain (phosphotungstic acid-hematoxylin) bone marrow/Giemsa stain |
| Bouins is good for what stain | Trichrome stain |
| Zenkers has what effect on RBCs | Lyses due to acetic acid |
| Bouins has what effect on RBCs | Lyses due to acetic acid |
| Helly's does what to RBCs and therefore is good for what tissue in particular | Preserves RBCs - Bone Marrow with RBCs |
| Orths is good for what spec | chromaffin cells of adrenal medulla |
| Absolute alcohol is best for what spec and why | Urate crystals, because they are soluble in water and formalin |
| Millonig's fixative had dual advantage for | Electron Microscopy and Light Microscopy |
| Carnoy's fixative effects RBCs and Glycogen | Lyses RBCs and preserves Glycogen |
| List fixatives from fastest to slowest acetic acid, picric acid, methanol, mercuric chloride, formaldehyde, osmium tetroxide | Formaldehyde, acetic acid, mercuric chloride, methanol, osmium tetroxide, picric acid |
| How does heat affect the penetration of fixatives? | increased heat = increases fixation speed |
| What effect does fixative concentration have on penetration rates | Concentration of fixative does not affect the penetration rate. |
| What is the typical fixative PH for routine specimens | 4-9%, and is not critical |
| What is the typical fixative PH for Electron Microscopy | 7.2-7.4 PH, and is very important |
| What happens to fixative molecules over time and what effect to they have in regards to volume to spec ration. | They become depleted and change the ph of the solution |
| Define Osmolality | Numbers of particles in a solution |
| Explain the hypotonic effect | Too little (salt) in surrounding solution, therefore the water enters the cell and diffuses salt out lysing the cell |
| Explain the hypertonic effect | Too much (salt) in surrounding solution therefore the water in cell diffuses out causing the cell to shrink |
| Explain an isotonic solution | (salt) and H20 ratio is equal so water flows in and out. |
| Name three aldehydes | Formaldehyde, glyoxal( is a dialdehyde), gluteraldehyde |
| What charge do aldehydes have and what do they link to? | Negative charge, link with positive amino acids |
| At what PH does black acid hematin (formalin pigment) occur | less than 6 |
| What can be done to PREVENT black acid hematin | Keep ph near neutral, and keeping the proper volume/spec ratio |
| How is black acid hematin removed (2 options) | treat tissue with Alcoholic picric acid or alkaline alcohol |
| What 2 problems does black acid hematin create | simulates microorganisms, reduces silver solutions used in staining certain items |
| How can black acid hematin be monitored | it is birefringent and can be monitored by polarization |
| If formalin isn't removed before HCL decalcification it will cause | Bis-chloromethyl ether (a carcinogen) |
| Formalin will preserve __________ but they will not be __________ | lipids, insoluble |
| Formalin will NOT fix __________ but will stabilize the protien and trap __________ | carbohydrates, glycogen |
| Formalin creates less __________ than other fixatives but __________ tissue more than all but (ethanol and acetone) | shrinkage, hardens |
| 10% aqueous formalin has what osmolality | Hypotonic |
| 10% formalin saline has what osmolality | Isotonic |
| Calcium Formalins (Bakers & Lillies) are best for preservation of what | Phospholipids |
| Lillies fixative can create what drawback | pseudocalcification |
| Formalin Ammonium Bromide is recommeneded for what tissue | Central Nervous System (for Cajal Astrocyte procedure) |
| What PH does Formalin Ammoniom Bromide have | PH 1.5 (which lyses RBCs) |
| Formalin Ammonium Bromide causes what with Nuclei | direct positive Schiff reaction due to Feulgen Hydrolosis |
| What ph is Modified millonig formalin | 7.2 - 7.4 (good for E/M and L/M) |
| Alcoholic formalin has what four positives | penetrates/fixes fast, removes fat (to allow formalin to fix), no formalin pigment, indefinite storage |
| What fixative will FDA not allow to be used on breast tissue (Her2Neu) | Alcoholic Formalin, MUST use 10% formalin instead |
| 10% Neutral Buffered Formalin has what ph and osmolality | ph 6.8 and hypotonic |
| Glyoxal doesn't react with which stain | Periodic Acid-Schiff |
| Glyoxal has which drawbacks (3) | Leaches out iron, dissolves eosinophil granules,can't be used for H. Phylori stain |
| Glyoxal has what effect on RBCs | Lyses |
| Gluteraldehyde and glyoxal are both what type of aldehydes | Di-aldehyded |
| Gluteraldehydes (di-aldehyde) reacts with Schiff reagent causing what | False positive with reaction to the extra aldeyde |
| What is the penetration rate of Gluteraldehyde | Fixes at the same rate as penetration (slow and poorly) |
| Due to Gluteraldehydes slow fixation/penetration rate it is best to | have thin sections of tissue |
| Gluteraldehyde is best for what microscopy | Electron Microscopy (because it is best of aldehydes at preserving ultrastructure) |
| Gluteraldehyde will do what to tissue if left to fix more than 2 hours | overharden (tissue should be moved to buffer solution for holding) |
| Name 3 metal salts in fixation | Mercuric Chloride, Zinc sulfate (or zinc chloride), cupric acetate (or cupric sulfate) |
| Cupric Acetate is also know as what | Copper Acetate |
| What negative do metal salts cause and describe it | radiopacity, opaque to x-rays |
| Mercuric chloride cannot be used with metal objects because | it is corrosive to metal |
| Mercuric Chloride has what effect on staining | it enhances staining by leaving tissue receptive to dyes |
| Mercuric Chloride cannot be use with frozen sections because | it prevents tissue from freezing when present |
| Mercuric Chloride causes what main issue | Mercuric pigment (cannot be prevented but can be removed) |
| Give details of Mercuric pigment | Crystalline or amorphous brown precipitate, polarizes light |
| Removal of Mercuric Pigment requires | Iodine (which oxidises mercury to soluble mercury iodide), then sodium thiosulfate to remove excess iodide |
| B-5 Fixative has what potential artifacts | Mercuric pigment and formalin pigment (due to sodium acetate raising PH) |
| What to tissue types does B-5 work well with | Hematopoetic and lymphoreticular tissue (gives good nuclear detail) |
| After using B-5 as a fixative what must wet tissue be stored in | 70% alcohol (prolonged storage will lead to brittle tissues) |
| B-5 works well for what but not | tissue antigens, but not some silver stains |
| Zenkers and Helly's must be treated for what issue after fixation | mercuric pigment |
| Zenkers can get what pigment in addition to mercuric pigment | chromium pigment |
| Hellys can get what pigment in addition to mercuric pigment | Formalin pigment |
| Tissue should not be in Zenkers or Hellys fixative longer than | 24hrs (then stored in alcohol), or it will become overhard |
| Zenkers/hellys decrease nuclear basophilia and increase cytoplasmic acidophilia and therefore | may require increased staining time with hematoxylin and decreased eosin time |
| Zenkers and hellys are not good for what stain | Silver stains |
| Zenkers does what with RBCs and is a good fixative for what | lyses RBCs, good nuclear fixative (both via acetic acid) |
| Zenkers can be used as a fixate and decal for what spec | Bone marrows (although it may dissolve iron) |
| Hellys if dark or turbid is? | unusable if formaldehyde not added immediately to hellys (indicates that solution is not stable) |
| Hellys does what for RBC's | preserves RBCs |
| Zinc Salts can be a replacement for what | Mercury replacement |
| Zinc Salt has what actions as a fixative___________ and a ___________ | Coagulent and additive |
| Zinc Salts are poor for what Microscopy | E/M due to poor ultra structure preservation |
| The penetration rate for Zinc salts are | slow penetration rate for this fixative |
| Cross linking with Zinc salts take how much longer than the mercury it can be used to replace | 1.5 - 2 times longer |
| Zinc chloride can be used instead of zinc sulfate but it can cause what problem | precipitation in processors (and itssue) if the initial alcohol % is above 70 |
| What can be used to remove zinc cloride precipitate from processors | dilute acetic acid (5-20%) |
| Copper salt fixatives have what uses | Decalcify small bone specs, stabilizes (RBC membranes, eosinophils and endocrine cells) to reduce lysis via the copper acetate |
| What must be done to a spec fixed in copper salt before it is put in a processor | must be washed out |
| Osmium Tetroxide are used in what microscopy after being post fixed with O.T. | E/M |
| How far will osmium tetroxide penetrate | penetrates only a few cell layers therefore sections must be very thin |
| Osmium tet. fixes what but not what | lipids, but not proteins |
| name 3 dis adv to osmuim tet. | Inhibits enzymes so it cannot be used for immunogold labeling, expensive, toxic .0002ppm (used under hood) |
| picric acid has what charge and an affinity for what | neg. charge and affinity for eosin |
| name 4 fixatives with picric acid in the formula | bouins, gendre, zamboni, hollandes |
| what must be done to tissue after using picric acid (Picric acid fixatives) | tissue must be washed in changes of 50& 70% ethanol to remove yellow color |
| name 3 advantages to picric acid | fixes glycogen, preserves nuclei, good for GI's and testicular tissue |
| name 4 disadvantages for picric acid | explosive when dry, removes calcuim and iron, causes shrinkage (countered by acetic acid), lyses RBC's |
| what is bouins fixative good for (and not good for) | used on GIs, CT stains, but not good for nucleic acids |
| What is the Gendre fixative good for | good for preserving glycogen |
| what is Zamboni (PAF) fixative good for | (it is pure formaldehyde) - Good for EM |
| what is the hollandes fixative good for | GI's |
| if picric acid remains in tissue what will happen | staining characteristics will change over time |
| potassium dichromate has a dual use that is dependant on ph when does the changeover occur | 3.4 - 3.8 Ph (approx) UND lists it as 3.5 ph |
| what will pot. dichro. behave similar to if the ph is below 3.5 | chromic acid |
| at below 3.5 ph how does pot. dic. react with proteins, DNA, Lipids, Carbs | acts as a coagulant, coagulates aldehydes, oxidizes unsaturated, oxidises to aldehydes |
| at above 3.5 ph how does pot. dic. react with proteins, DNA, Lipids, Carbs | noncoagulant, dissolves DNA, makes lipids insoluble, no reaction to carbs |
| what is the penetration rate of pot. dic. below/above 3.5ph | below = slow above = faster |
| what is a good use of pot. dic. below 3.5, how about above | preserves organelles (below 3.5), used in Orth fixative for chromaffin granules |
| name 4 problems associated with pot. dic (above and below 3.5ph) | inhibits enzymes, chrome pigment, toxic, soft tissue which shrinks post paraffin embedding |
| name the 3 fixatives with pot. dichromate | Orths, Zenkers, Hellys, |
| what is the Orth solution used for | chromaffin granules |
| what is added just before use with the orth solution | formalin |
| How do zenkers and hellys formulas differ in their working solutions (stock contains pot. dic.) | zenkers has glacial acetic acid, hellys, had formaldeHYDE |
| potassium dicromate can cause a pigment(what does it look like) how is it prevented or removed | fine yellow deposit, prevented by washing in H20 before processing, removed by 1% acid alcohol then h20 ( |
| name the 3 non-aqueous fixatives | acetone, methanol, ethanol |
| what are some of the uses for non-aqueous fixatives | gout (urate) crystals (which dissolved in water), enzymes, antigens, glycogen cytology |
| what does acetone demonstrate | alkaline and acid phosphates, rabies in the brain, FS for surface antigens |
| give 4 properties of acetone | clear/colorless, rapid acting, causes shrinkage/over hardening of tissue, defats skin |
| what are methanol and ethanol good for | methanol - touch preps and blood smears ethanol - cytology/glycogen and urate (gout) crystals, preserves pigments, dissolves fat, but over hardens tissue |
| give to alcohol fixative mixtures and what they are good for | Clarke - preserves DNA Carnoy - cytology (bloody specs), but not good for acid fast bacteria stains |
| what is the formula for methacarn and what is better than between clarke and carnoy, why? | acetic acid, methanol, chloroform - less hardening than carnoy |
| give some factors about acetic acid | splits amino acid chain and increased exposed R groups therefore water gets bound, tissue swells, and RBC's are lysed |
| what are some options for transport media | saline dampened gauze (short time period) Michels transport (medium to long distanc) PBS (10% sucros solution) |
| what is the formula and ph for michels transport media | anhydrous citric acid, ammonium sulfate, N ethylmalimide, magnesuim sulfate, distilled h20 ph best at 7.0-7.2 |
| what is the PBS 10% sucrose transport media good for | tissue for immune complex deposit studies |
| why are some formulas a mixture | trying to counteract certain issues shrinkage (picric acid) V swelling (acetic acid) or hardening (alcohol) V softening (picric acid), fixes nuclei (zinc) V cytoplasm (formalin) |
| how is formalin pigment removed | alcoholic picric acid (wash with h20), alcoholic ammonium OR potass. hydroxide OR soduim hydroxide (wash, rinse with 1%acetic acid, wash) |
| how is mercury pigment removed | gram or lugol iodine, wash, then 5% soduim thiosulfate (hypo), wash |
| how is pot dichromate pigment removed | 1% acid alcohol |
| what causes auto lysis and what can correct it | delayed fixation resulting in degenerated nuclei, FIX- immediate fixation, open large specs, resection large specs |
| what can correct incomplete fixation | increase time, use another fixative, maintain proper section size, use alcholic formalin in processor |
| AAA is an anagram for what? | no additives alcohol, acetone, acetic acid |
| apdot is an anagram for what | 5 non coagulents (create gel so nothing can penetrate) aldehydes (formal, gluter, glyoxyl), pot. dic, osmium tetrox. |
| paczacam is an anagram for what | the 8 coagulents (tea ball) picric acid,alcohol, chromic acid, zinc salt, acetone, chromic acid, acetic acid, mercuric chloride |
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mustangvxd
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