histopath techniques 2

7/29/2019 Histopath Techniques 2

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TCA

4-8 days very slow actingGood nuclear stainingWeak


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Sulfurous

WeakFor minute pieces of bone


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Chromic Acid (Flemmings fluid)

Chromic acid, osmium tetroxide, glacialHAc

Fixative and decalcifier Nuclear staining with hematoxylin isinhibitedForms precipitate at the bottom*carcinogenic, corrosive to skin


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Citric Acid citrate buffer pH4.5

With zinc sulfate and chloroform6 days slowGood nuclear and cytoplasmic staining


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HNO3 - summary

Most common, rapid (1-3 days)Yellow color imparted prevented by 5%

sodium sulfate, wash in running water x 12hours or + 0.1% ureaFormol Nitric (1-3 days)Perenyis w/ chromic acid + abs. EtOH

Decalcifies & softens; slow (2-7 days)

Phloroglucin Nitric most rapid (12-24h); poor nuclear staining


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HCl slower than HNO3

Formic fixative/decalcifier, slowFormic Acid-Sodium Citrate solution - slow

TCA good nuclear staining, no washingout; weak & slow actingSulfurous very weakChromic (Flemmings) fixative/decalcifier Citric Acid Citrate buffer pH4.5 - slowVon Ebners NaCl, HCl


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Chelating Agents

EDTA (versene)Excellent stainingVery slowSlight tissue hardening produced


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Ion Exchange Resins

* artifacts produced, usually caused byCO2 bubbles

slow


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Electrophoresis(Electrical ionization)

Formic acid, conc HCl, distilled water


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Tests for Completeness of Decal:

Physical / MechanicalX ray / RadiologicalChemical litmus paper red if due toacidity, add NH3 drop by drop bluelitumus; if cloudy still w/ calcium; if clear:+ammonium oxalate, 30 mins cloudy if incomplete


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Tissue softeners

Perenyis 12 24 hours4% aqueous phenol 1 3 daysMolliflex (swollen & soapy appearance)2 % HCl1 % HCl in 70 % alcohol


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*Post decalcification

Remove acid by saturated lithiumcarbonate solution or 5-10 % aqueous

NaHCO3 for several hoursRunning tap water If EDTA is used use 70 % alcohol


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*Rate of decalcification

More concentrated acid solutions morerapid but more harmful to tissue

Heat hastens decalcification, butincreases damaging effect of acids totissues

Mechanical agitation, sonicationIdeal time 24 to 48 hours


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Dehydration most critical stageof processing difficult to correct

Ideal Dehydrating Solution:Rapid acting w/ no tissue shrinkage or

distortionShould not evaporate fastCan dehydrate even fatty tissues

Non toxicNot fire hazardShould not harden tissues excessively


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Dehydrating Agents:

Alcohol most common AcetoneDioxaneCellosolveTriethyl phosphateTHF


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Alcohol progressively increasing

Ethyl best, fast, mixes with water andpenetrates easily, not poisonous, cheap

Methyl toxic, for blood filmsButyl slow, for Plant and Animalmicrotechnique


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* Alcohol

Prolonged storage in 70 % alcohol macerates tissues

Directly placed in high grade alcohol shrinkage & hardening of tissues


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Acetone

CheapRapid actingVolatileTissue shrinkage


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Dioxane (Diethylene Dioxide)

Excellent dehydrating & clearing agentLess tissue shrinkage

Tissues can be left here for a long timeRibbons poorlyExpensive

Dangerous vapor cumulativeGraupners method (dioxane, paraffin wax) Weisebergers method (gauze, Ca oxide)


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Cellosolve

Ethylene Glycol Monoethyl Ether RapidStorage without producing hardening or distortion


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Triethyl Phosphate

Minimum shrinkageMinimum distortion and hardening of tissue


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Tetrahydrofuran - THF

Dehydrates and clearsLess shrinkageEasier cutting w/ fewer artifactsNon toxic; 6 months exposure conjunctival irritationOffensive odor (use well ventilated room)


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*additives to dehydrating agents

4 % phenol added to 95 % ethanol softens hard tissues

Hard tissues immerse in glycerol/alcoholmixture or in Molliflex


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Clearing / Dealcoholization

Substance will dissolve wax with which thetissue is to be impregnated

After staining transparent tissuesHigh refractive index


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Clearing Agents:

Miscible with alcohol and paraffin &/or mounting medium

Should not produce excessive tissueshrinkage & hardeningShould not evaporate quickly in a

waterbathShould make tissues transparent


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Clearing Agents:

XyleneToluene

BenzeneChloroformCedarwood oil

Aniline oilClove oilCCl4


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* Clearing Agents (cont)

* methyl benzoate and methyl salicylateslow

`THFDehydrates and clearsNon toxic

Offensive odor


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Xylene (Xylol)

Most common, colorlessMost rapidCheapMilky if incomplete dehydrationHardens/shrinks tissues not for nervoussystem & lymph nodesHard/brittle tissues if > 3 hours


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Toluene

Not carcinogenic; but will emit fumes thatare toxic upon prolonged exposure

Rapid actingTissues do not become excessively hardand brittle even if left here for 24 hours

Slower than xylene & benzeneexpensive


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Benzene

Rapid actingVolatilizes rapidly in paraffin ovenTissue shrinkage if left for a long timeCarcinogenic; aplastic anemia

histopath techniques 2

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