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Fixatives
Lab
| Question | Answer |
|---|---|
| Acetic acid facts | Fast, swells, soft tissue, good nucleoproteins, lyse rbc |
| Formaldehyde facts | Hardens, less shrinkage, fast penetration, slow fixation b/c cross-links NH2 groups, preserves lipids-lose over time, traps glycogen, fixes proteins |
| 10% formalin solution | 1 part 37%-40% formaldehyde 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? | 100% sat'd w/ picric acid, wash; or 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, thin samples, good for EM/ultrastructure, bad IHC, breaks down in O2, 2-4 hr max, aldehyde 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? | Gram or Lugol iodine, wash, 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, hydrolyzes DNA, RNA, must rinse out, decal small calcium deposits, 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 insol., dissolves DNA, incr. tissue affinity for anionic dyes, 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? | 70% w/ 1% HCl or other 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 formaldehyde 37% to 40% mix before use |
| B-5 facts | Good nuclear detail, paraffin antigen demo, 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, delicate structures, bad for EM, nucleic acids, lyse 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 to avoid 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 24 hr, wash & store in 70% to 80% |
| Helly solution | Zenker-Helly stock formaldehyde 37% to 40% |
| Helly facts | Preserves rbc - good for 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, 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 (days), transport before freezing; hold tissue ~2 weeks for immunofluorescent or immunoperoxidase, kidney biopsies |
| 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 @ 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 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 |
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CCF
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