SEMINAR 2 Bullet Notes in Histopath –Fixation-Dehydration-Clearing-Infiltration-Embedding-Decalcification
FIXATION Osmium Tetroxide
Non-additive fixatives Glutaraldehyde
Fixation temp for EM & histochemistry paraformaldehyde
Fixation temp using autotechnicon Fixation method that Involves thermal coagulation of proteins
Fixative & dehydrating agent
Temperature for Manual Fixation
For phosphatases & lipases
Penetration rate of formalin
Presence of mucus, blood, fatty tissues, cold temperature
Fixative volume for maximu effective fixation
Temperature range that will accelerate fixation
Carnoy’s ; Bouin’s; Newcomer’s Heidenhain’s Susa;
Flemming’s with HAc Fixative volume for museum preparations
Fixatives for Enzyme 4% formaldehyde & formol saline
Regaud’s / Moller’s ; Orth’s ; Formalin with post chroming
histochemistry
Flemming’s without HAc
100 ml of 37-40% formalin + 900 ml distilled water Smallest aldehyde
Added to prevent precipitation of formaldehyde to white Bouin’s Brasil’s & Hollande’s
paraformaldehyde Can be used for myelin & peripheral nerves
FORMALDEHYDE 10% formol saline _______________________________________ Proposed as mercuric chloride substitute
/FORMALIN FIXATIVES 10% Nuetral Buffered formalin ______________________________ Used to wash out excess mercuric fixatives
Formol corrosive/formol sublimate ___________________________ Used to wash out excess amount of picric acid fixatives
Alcoholic formalin/Gendres_________________________________ Can be used as fixative, decalcifying agent & stain
Mercuric chloride + formaldehyde Often used in conjunction with other fixatives to produce a compound
Glutaraldehdye – for EM ___________ for small tissue fragments/needle biopsies fixative
___________ for larger tissues less than 4 mm thick DECALCIFICATION
Most common metallic fixative ; Potent anticoagulant but weak decalcifying agent
Excellent for Trichromoe staining & Tissue photography Binds with calcium to form weakly dissociated complexes
MERCURIC CHLORIDE _______________ liver, spleen, CT & nuclei Decalcification method that involves attraction of positively charged
FIXATIVE _______________ bone marrow calcium ions with negatively charged electrodes
Heidenhain’s Susa _____________________________________ Decalcifying acid used for surface decalcification of blocks
___________________ pituitary gland, BM, spleen, liver Both a decalcifying agent & a tissue softener
a.k.a helly’s fluid Most commonly used decalcifying agent especially recommended for
CHROMATE FIXATIVES _____________________ for carbohydrates urgent biopsies, for needle & small biopsies
Potassium dichromate 3% - for lipids, mitochondria Temperature range for decalcification
____________________ mitochondria, RBC & colloid containing tissues Ratio of decalcifying agent to tissue volume
______________________ Rickettsiae & other bacteria, tissue necrosis Most reliable method of testing for the completeness of decalcification
Acid mucopolysaccharide Method of testing for the completeness of decalcification that uses
1.______________________ For brain tissues for the diagnosis of rabies calcium carbonate
2.______________________ Duration of decalcification thur ion exchange resin
Excellent for Glycogen demonstration Method of testing for the completeness of decalcification done by
For embryos ; pituitary biopsies & endometrial curettings probing tissue with a needle
Not for kidney structures Most rapid nitric containing decalcifying agent
Not compatible with feulgen’s An excellent bone decalcifer for EM and enzyme histochemistry
For touch preparations Remedy for Inactivation ofalkaline phosphatase by EDTA
Solidifies at 17 degC
Must be used at ice cold temperature DEHYDRATION
Most rapid tissue fixative, for fixing chromosomes & lymph glands Important requirement when using alcohol as dehydrating agent
For mucopolysaccharides & nuclear protein For dehydrating sections and smears
Prepared by: Ma. Cristina Liwanag Page 1
SEMINAR 2 Bullet Notes in Histopath –Fixation-Dehydration-Clearing-Infiltration-Embedding-Decalcification
Toxic by ingestion, inhalation & by skin contact INFILTRATION / EMBEDDING
Highly corrosive on skin Celloidin infiltration method recommended for whole eye specimens
Toxic to reproductive, fetal, urinary system Will require use of Gilson’s mixture to store blocks
Dehydrating agent whose main disadvantage is its being toxic Melting point of paraffin wax commonly used
Decomposes when expose to sunlight Substitute for paraffin wax recommended for bones and brain specimens
Temperature that will hasten dehydration time Substitute for paraffin wax that is water soluble & is recommended for
Dehydrating agent that is both a skin & eye irritant enzyme histochemical studies
Has an offensive odor Temperature range of paraffin oven when in used
Rapid acting dehydrant suited for urgent biopsies but not for routine Approximate temperature of paraffin oven when in used
tissue processing
Infiltration under negative atmospheric pressure inside the oven
Most commonly used alcohol for dehydration process
Infiltration method not suited for fatty tissues
Alcohol for plant & animal microtechniques Substitute for paraffin wax used for eye specimens
Purpose of phenol in alcohol Substitute for paraffin wax that will require heavy duty type of microtome
bath
Melting point of paraplast
CLEARING/DEALCOHOLIZATION Melting point of ester wax
Clearing agent that can damage the bone marrow & may cause aplastic Infiltration method for enzyme & histochemical studies
anemia Celloidin infiltration method for bones, brain & teeth specimens
Toxic to liver; recommended for skin, fibroid & decalcified tissues Will require 70-80% alcohol to store blocks
For tough tissues , nervous tissues, lymph nodes, embryos Combination of Chloroform & cedarwood oil
Routinely used clearing agent but not for nervous tissues & lymph nodes Infiltration method recommended for specimens with large and hollow
Can become milky because of prolonged storage cavities that tends to collapse
Can become milky after placing an incompletely dehydrated tissue in it Plasticizers to prevent cracking tissues when infiltrated using LVN
For clearing insects, embryos & other delicate specimens Infiltration method that will require tissues not to be more than 2 -3 mm
When used, tissues become adulterated thick
Same property as chloroform A disposable Embedding mold that will require smearing of inner mold
Recommended for eye specimens with glycerine
Clearing agent obtain from citrus fruit An embedding mold consisting of a series of interlocking plates resting
With faint pleasant odor on a flat metal base
Clearing agents that can be used for dense tissues like uterus Disposable mold that gives perfect blocks without trimming
Clearing agent for CNS tissues & cytological studies , an extremely slow Other term for infiltration
clearing agent Other term for embedding 1. ______________________________________
can be used as xylene & benzene substitute 2. _______________________________________
Clearing agents for double 1. _____________________________________ Process of infiltrating tissue with celloidin and embedding with paraffin
embedding which are slow 2. _____________________________________ Embedding mold consisting of L shaped strips made of heavy brass
acting agents 3. _____________________________________ Method of paraffin infiltration that will require 2-3 changes of wax
a.k.a. diethylene dioxide Use of phenol in gelatin
a.k.a. ethylene glycol monoethyl ether infiltration
Clearing agent that will not make tissues transparent Celloidin Infiltration Thin _______________
Clearing agents difficult to remove from tissues Medium____________
Clearing agent easily removed from tissues Thick ______________
Clearing agent that can attack rubber seal used in vacuum impregnation Spurr Fastest Epoxy resin
Steps in tissue processing 1. ________________________________ Polyester Plastic resin originally introduced for EM
that will require complete 2. ________________________________ Polyglycl methacrylate/GMA Extremely hydrophilic
clearing of tissues Methylmethacrylate/MMA Plastic resin considered ideal for undecalcified bones & hard tissues
Prepared by: Ma. Cristina Liwanag Page 2
SEMINAR 2 Bullet Notes in Histopath –Fixation-Dehydration-Clearing-Infiltration-Embedding-Decalcification
Prepared by: Ma. Cristina Liwanag Page 3
