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UND 362 Fixative gen

UND 362 Fixatives general knowledge

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
Created by: mustangvxd



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