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Fixatives & Ingredients

Acetic acid facts Fast, swells, soft tissue, good nucleoproteins, lyse rbc
Formaldehyde facts Hardens, less shrinkage, fast penetration, slow fixation b/c cross-linking NH2 groups, preserves lipids-lose over time, traps glycogen, fixes proteins
10% formalin solution 1 part stock (37%-40% formaldehyde) to 9 parts water hypotonic
Paraformaldehyde facts Good for EM b/c no additives, heat to depolymerize, yields pure formaldehyde
Formalin pigment facts aka black acid hematein, forms when pH < 6.0, may react during staining or mask
Two ways to remove formalin pigment? Treat w/ 100% sat'd w/ picric acid, wash; or treat w/ 70% w/ NH4OH, wash, 1% acetic acid, wash
When removing formalin pigment can substitute what for NH4OH? NaOH or KOH
10% formalin saline solution NaCl formaldehyde 37% to 40% water isotonic
Calcium formalin solution + fact CaCl formaldehyde 37% to 40% water Good for phospholipids
Formalin ammonium bromide solution NH4Br formaldehye 37% to 40% water
Acetate formalin solution sodium acetate formaldehyde 37% to 40% water good if no 10% NBF
Pseudocalcification of tissue may be caused by? Calcium acetate if sub'd for sodium acetate in acetate formalin
10% neutralized formalin solution + fact CaCo3 or MgCO3 formaldehyde 37% to 40% water Becomes acidic after opening
10% NBF solution + pH sodium phosphate monobasic sodium phosphate dibasic formaldehyde 37% to 40% water pH 6.8, somewhat hypotonic
Modified Millonig formalin solution + pH + fact sodium phosphate monobasic NaOH formaldehyde 37% to 40% water pH 7.2 to 7.4, dual-purpose fixative, isotonic
A dual purpose fixative means it is? Good for EM & light microscopy; e.g. modified Millonig
Alcoholic formalin solution 100% ethanol formaldehyde 37% to 40% water dehydrates & fixes, can use on processor
Glutaraldehyde facts Slow, hardens, poor penetration, only thin tissue, good for EM/ultrastructure, bad IHC, breaks down in O2, 2-4 hr time limit, free aldehyde group gives false pos. w/ Schiff
Glutaraldehyde phosphate-buffered solution + pH sodium phosphate monobasic NaOH glutaraldehyde water pH 7.2 to 7.3 , drops over time
Mercuric chloride facts Shrinks, hardens, enhances staining, inhibits freezing, mercury pigment, v. toxic
How to remove mercury pigment? Treat w/ Gram or Lugol iodine, wash, 5% sodium thiosulfate, wash
Lugol Iodine Solution iodine potassium iodide water
OsO4 facts Post-fix tissue for EM, makes lipids insoluble, bad antigenicity, fix small amount fat for paraffin, enhances staining w/ basic dyes, expensive, hazardous, TWA 0.002 ppm
Picric acid facts Extreme shrinkage, good for glycogen, bad for DNA, RNA (hydrolyzes), must rinse out, is a stain & fixative, decal small calcium deposits (e.g. in breast tissue), incr. tissue affinity anionic dyes, explosive
What is used to wash picric acid out of tissue before processing? 50% alcohol or 50% alcohol w/ LiCO3
Potassium dichromate facts Shrinks, soft tissue, good for mitochondria, chromaffin granules, can make fat insoluble, dissolves DNA, incr. tissue affinity for eosin (anionic), chromate pigment, v toxic, corrosive skin & mucosa
How to prevent chromate pigment? Wash out chromate before in alcohol, pigment considered insoluble
How to try & reduce chromate pigment after it has formed? Treat w/ 70% w/ 1% HCl or treat w/ acidic alcohol
Zinc salts facts Preserve antigenicity, poor ultrastructure, may precipitate in processor
What can be used as a replacement for mercury? Zinc salts
B-5 fixative solution HgCl sodium acetate water B-5 stock formaldehyde 37% to 40% mix before use
B-5 facts Good nuclear detail, paraffin antigen demo, good hematopoietic & lymphoreticular tissue, bad some silver stains, after fix store in 70%
Bouin solution acetic acid, picric acid, formaldehyde 37% to 40%
Bouin facts Good for GI, trichrome stain, delicate structures, conn. tissue; bad for EM, nucleic acid demo; yses rbc, 24 hr max, wash w/ 50% to 70%, or 70% w/ LiCO3
Gendre solution 95% sat'd w/ picric acid acetic acid formaldehye 37% to 70%
Gendre facts good for glycogen wash out picric w/ 80%
Hollande solution copper acetate picric acid acetic acid formaldehyde 37% to 40% water
Hollande facts Good for GI, stabilizes rbc, decal bone biopsies, wash out to avoid PO4 precipitate
Zenker/Helly Stock Solution HgCl potassium dichromate sodium sulfate (opt.) water
Zenker solution Zenker-Helly stock acetic acid
Zenker facts Good nuclear, Mallory PTAH, lyse rbc, bad for silver stains, max time 24 hr, wash & store in 70% to 80%
Helly solution Zenker-Helly stock formaldehyde 37% to 40%
Helly facts Preserves rbc, good for blood-forming tissues (marrow, liver, spleen), bad for silver stains, mix just before use, turns dark, turbid, 24 hr max, wash & store in 70% to 80%
Orth solution potassium dichromate sodium sulfate formaldehyde 37% to 40% water
Orth facts Good for chromaffin granules, diagnose phenchromocytoma, wash & store in 70% to 80%
Zamboni solution (buffered PAF) + pH paraformaldehyde picric acid NaPO4 monobasic monohydrate NaPO4 monobasic anhydrous NaOH water pH 7.3
Zamboni facts Good general purpose fix, good for secondary OsO4 fix, preferred for EM
Alcoholic zinc formalin solution ZnCl isopropanol 99% formaldehyde 37% to 40% DI water
Alcoholic zinc formalin facts Post-fix after NBF for better antigenicity & nuclear detail, fix 6-8 hr, better for fatty tissue than aq. Zn
Unbuffered aq. zinc formalin solution zinc sulfate formaldehyde 37% to 40% dH2O Precipitates in 70% on processor & in specimen
Unbuffered aq. zinc formalin facts biopsy 4-6 hr, other 6-8 hr wash before phosphate-buffered soln. to avoid PO4 precipitate
Acetone fixation facts Very fast, extreme shrinkage, hardening, good demo enzymes, fix brain for rabies diagnosis, toxic
Ethanol fixation facts Fast, hardens, shrinks, preserve water-soluble components, pigments, dissolves fat, begins dehydration
Methanol fixation facts Fix touch prep, blood smears, toxic, flammable
The nonaqueous fixatives include_ Acetone, methanol, ethanol, non-additive, coagulating, to preserve special components
What can help preserve glycogen & urate crystals? Ethanol
Carnoy solution Abs. ethanol chloroform acetic acid
Carnoy facts Fast, shrinks, hardens, good for glycogen, mucus, nucleus, Congo red, bad acid-fast bacilli, lyses rbc, dissolves lipids, 4 hr max
Carnoy variation w/ less shrinkage & hardening? Methacarn; uses methanol instead of ethanol
Michel Transport Medium solution + pH + fact anhydrous citric acid N-ethylmaleimide -- slows proteolytic activity magnesium sulfate ammonium sulfate -- fixes immunoglobin water pH 7.0 to 7.2, v. important maintain pH for good staining
Michel Transport Medium use Hold unfixed tissue for days, transport before freezing; hold tissue ~2 weeks before immunofluorescent or immunoperoxidase studies, transport kidney biopsy
PBS Buffer stock solution + pH NaPO4 monobasic potassium phosphate dibasic NaCl dH2O pH 7.4
PBS-10% Sucrose solution PBS Buffer stock sucrose water
PBS 10% sucrose used to? Wash Michel's Medium from tissue before freezing
Calcium/magnesium carbonate help to__ "Neutralize" formalin
Fixative additives that help adjust pH? Ammonium bromide & NaOH
Fixative additives to buffer pH close to physiological pH? Sodium phosphate monobasic & dibasic
Fixative additive to reduce shrinkage? NaCl
Fixative additive to preserve membrane phospholipids? CaCl
Fixative additive to prevent lysis of rbc? Copper acetate
What fixative is best for preservation of Nissl substance? Carnoy
Best pH for formaldehyde cross-linkage? pH 7.5 to 8.0,
Alcoholic formalin facts Fix & dehydrate, good when processor starts @ 70%, phosphate precipitation
PBS stands for Phosphate-buffered saline
PAF stands for Picric acid-formaldehyde
Formalin Ammonium Bromide facts Good for CNS, esp. Cajal, lyses rbc, causes nuclei to give pos. Schiff rxn, good when have no buffer
Glacial acetic acid is Conc'd form of acetic acid, freezes at 16.6 °C, incr'd protein swelling pH <4, collagen dramatic swelling pH <2.5
Clarke's Fluid Abs. ethanol acetic acid
Glyoxal facts Fast, less toxic, smallest dialdehyde, good nuclear detail, okay PAS, bad H. pylori & iron stains, lyse rbc, reacts w/ carbs & proteins at 2 different pH's
Clarke's facts Fast, 3-4 hr, good nucleic acid, bloody cytology smears, bad lipids, mix just before use
At what pH does potassium dichromate change from behaving like a non-coagulant to behaving like chromic acid? Non-coagulant above pH 3.4-3.8, coagulant below pH 3.4-3.8
Glutaraldehyde can also be in a __ solution Cacodylate-buffered solution, v. toxic, arsenic
Color of formalin pigment? Color of mercury pigment? Dark brown Black
Phosphate-buffered glutaraldehyde fixation is followed by_ Phosphate buffer wash, post-fixation in OsO4
What groups does chromium interact with? Carboxyl & hydroxyl groups, breaks up protein links to free amine groups
An aqueous fixative includes: One fixative plus water & other reagents
A compound fixative includes: Multiple fixatives, counterbalance each other
Phosphate-buffered paraformaldehyde solution sodium phosphate, monobasic NaOH paraformaldehyde adj. pH to 7.2-7.4
Formaldehyde-glut (4CF-1G) solution sodium phosphate, monobasic NaOH dH2O formaldehyde glutaraldehyde Good for EM & histo, bad IHC
Created by: CCF