FIXATIVES Flashcards by Sharmaine Madjid

for routine paraffin sections, em, histochemical and enzymes

Aldehyde Fixatives

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commonly used Aldehyde

10% Formalin

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Fixation time of Aldehyde Fixatives

24 hours

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Advantage of Aldehyde Fixative

cheap, readily available, easy to prepare, stable especially if stored in buffered solutions

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Compatible w/ many stains
Does not overharden tissues
Penetrates tissues well
Preserve fat and mucin
Preserves glycogen
Does not precipitate proteins, for nervous tissue
For frozen tissue, does nor require washing out
Tolerant fixative

Advantage of Formaldehyde

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Irritating fumes
Shrinkage of tissue
Soft fixative-does not harden some cytoplasmic structures
If unbuffered: reduces basophilic and eosinophilic stains; produce brown pigment granules on bld-contng tissue( spleen)
Prolonged: bleaching, fat dispersal, glycogen and biurate of sodium crystals dissolution

Disadvantage of Formaldehyde

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Made up of saturated formaldehyde (40% by weight volume) diluted to 10% with NaCl

10% Formol-Saline

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10% Formol-Saline is used for..

For CNS and general post mortem tis for histochemical exams

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Fixation time of 10% Formol-Saline

Fixation time:

24 hrs at 35oC (95oF)
48 hrs at 20-25oC (65-77oF)

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Preserves enzymes and nucleopoteins
Demonstrates fats and mucin

advantage of 10% Formol-Saline

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Disadvantages are similar to formaldehyde with the ff addition:

It is slow fixative
Metachromatic reaction of amyloid is reduced
Acid dye stains less brightly than when fixed with Mercuric chloride

disavantage of 10% Formol-Saline

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used for preservation and storage of surgical, post mortem and research specimens

10% Neutral Buffered Formalin or Phosphate-Buffered Formalin (pH 7)

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Prevent pption of acid pigments on post mortem tissue and Best fixative for Iron pigments and elastic fibers

advantages of 10% Neutral Buffered Formalin or Phosphate-Buffered Formalin (pH 7)

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fixation time of 10% Neutral Buffered Formalin or Phosphate-Buffered Formalin (pH 7)

4 to 24 hours

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Time consuming
Positivity of mucin to PAS is reduced
Raecativty to Weigert’s Fe hematoxylin stain is reduced
Inert to lipids

disadvantage of 10% Neutral Buffered Formalin or Phosphate-Buffered Formalin (pH 7)

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used For routine post mortem tissues

Formol-Corrosive (formol-sublimate)

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Formol-Corrosive (formol-sublimate) fixation time

3 to 24 hours

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Excelelnt for silver reticulin
Cytologic structures and blood cells are well preserved
NO need for washing out, tissues can be directly transferred to alcohol
Fixes lipids, neutral fats and phospholipids

advantage of Formol-Corrosive (formol-sublimate)

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Penetration is slow, (not more than 1 cm)
Forms Mercuric chloride deposits
Does not allow frozen tissue sections
Inhibits tissue decalcification

disadvantage of Formol-Corrosive (formol-sublimate)

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In alcoholic formalin, the post-fixation with …

Post fixation with phenol-formalin for 6 hrs or more can enhance immunoperoxidase studies on the tissues

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Fixation is faster
Fixes and dehydrates
Preserves glycogen and for micro-incineration technique
Fix sputum

advantage of alcoholic formalin

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Cause partial Lysis of RBC

disadvantage of alcoholic formalin

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used For enzyme histochem and EM

Glutaraldehyde

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fixation time in glutaraldehyde

2.5% (small tissue fragments)-2-4 hrs
4% Large- 6-8 to 24 hrs (4mm)

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More pleasant and less irritating to the nose Specimen vial should be refrigerated Does not cause dermatitis More expensive, less stable

Glutaraldehyde.

5-7% included in compound fixatives Penetrates and hardens tissue rapidly Routine fixative of choice in Tx photography

Metallic Fixatives

common metallic fixative

Mercuric Chloride

Metallic fixatives May produce black granular deposits except

Heidenhens susa

in metallic fixatives, Removal can be done by addition of ____

saturated iodine solution of 96% Alcohol

fluids/solutions under mercuric chloride

Zenker's Zenker-Formol Heidenhain's Susa B5 Fixative

Contains Glacial Acetic acid For small pieces of liver, spleen, CT fibers and Nuclei Recommended for trichrome staining

Zenker

Dezenkeration is removal of ___ deposits using ____ and _____

Mercury ; Lugol's Iodine and Na Thiosulfate

Zenker-Formol composition

Potassium Dichromate and Formalin

Zenker-Formol is used for

For pituitary gland, BM and Bld containing Organs

Zenker Formol preserves

Preserves cytoplasmic granules

Zenker-formol produce brown pigments which can be removed

remove using picric acid or NaOH

Has TCA, Glacial acetic acid and Formaldehyde

Heidenhain’s Susa

Heidenhain’s Susa is used for

For skin tumor biopsies

Heidenhain’s Susa is excellent ___

cytologic ficative

in Heidenhain’s Susa, the RBC preservation is ___

poor

has Anhydrous Na acetate and used for bone marrow biopsies

B5 Fixative

Time in B5 Fixative

1/2

the chromate fixatives

Chromic Acid Potassium Dichromate

1-2% aqueous solution Strong oxidizing agents

Chromic Acid

Chromic Acid preserves

Preserves CHONs and CHO

in chromic acid, a ___ must be added before used in order to prevent _____ of solution on prolonged standing

Strong reducing agent must be added before use in order to prevent counteracting effects and decomposition of solution on prolonged standing

used in a 3% aqueous solution and it Fixes but does not ppt cytoplasmic structures

Potassium Dichromate

K Dichromate preserves

Preserves lipids, mitochondria (pH 4.5-5.2)

if K dichromate is acidified:

fixes cytoplasm, chromatin bodies and chromosomes

2 fluids under potassium dichromate

Regard's (Muller) Orth

For chromatin, mitochondria, mitotic figure, Golgi apparatus, RBC and colloid-containing tissues

Regard's (Muller's Fluid)

For early degenerative processes and tissue necrosis and demonstrate rickettsia and other bacteria

Orth's Fluid

Preserves myelin better than buffered formalin

Orth's Fluid

Used in 4% aqueous solution of the basic lead acetate

Lead Fixatives

Used for acid mucopolysaccharides

Lead Fixatives

Lead Fixatives fixes ___

tissue mucin

In lead fixative, CO2 is took up to form insoluble lead carbonate on prolonged standing this maybe removed by

maybe removed by adding acetic acid drop by drop to lower pH and dissolves residue

Usually used in strong or saturated solutions 2,4,6 trinitrophenol Explosive when dry

Picric Acid

Picric Acid is used for

Glycogen

Picric Acid can be used as ___ and _____ can be seen

Can be used as stain (yellow); small tissue fragments can be seen

2 fluids under Picric Acid

Bouin's and Brasil

embryo and pituitary biopsies Excellent for soft and delicate sructures For tissue stained with Masson’s trichrome NOT for kidney, lipid and mucus

Bouin's

Has TCA Better and less messy than Bouin’s Excellent for glycogen

Brasil’s Alcoholic Picroformol

Usually incorporated in compound fixatives Solidies at 17 C Precipitates nuceloCHONS, chromatin materials NOT for cytoplasmic fixation

GLACIAL ACETIC ACID

Fixes and removes water Rapidly denatures and precipitates CHONs

Alcohol Fixatives

Alcohol Fixatives is excellent for..

glycogen

Alcohol Fixatives dissolves

fats and lipids

Alcohol Fixatives preserves

nuclear stains

Fixes chromosomes, lymph glands and urgent biopsies Fixes and dehydrates

Carnoy's Fluid

For nissl granules and cytoplasmic granules

Carnoy's Fluid

Acts as both nuclear and biochemical fixative

Newcomer's

For mucopolysaccharides and nuclear CHONs

Newcomer's

Preserves mitochondria and golgi bodies Fixation for ultrathin sectioning in EM Very expensive Inhibits hematoxylin, volatile Pale yellow powder dissolves in water Fats are stained black

Osmium Tetroxide

it Fixes conjugated fats and lipids Preserves mitochondria and golgi bodies

Osmium Tetroxide

Osmium Tetroxide causes

Causes conjunctivitis, blindness

Osmium Tetroxide inhibits

Inhibits hematoxylin, volatile

Most common chrome-osmium acetic acid fixative

Flemming's Solution

is used for Nuclear preapration (chromosomes)

Flemming's Solution

Permanently fixes fats Requires less than 10x the volume of the tissues to be fixed, very expensive

Flemming's Solution

Made up of chromic and osmic acid Cytoplasmic structures (mitochondria) Time: 24-48 hrs

Flemming’s solution w/o Acetic acid

Ppts CHONs Weak decalcifying agent Has softening effect on dense fibrous tissues facilitates preparation of such sections Small pieces of tissues or bones

Tricloroacetic Acid

Used at ice cold temp ranging from -5 C to 4 C For water diffusible enzymes (phosphatases and lipases) For brain tissues (rabies) Solvent for metallic salts Dissolves fats, preserves glycogen poorly, evaporates rapidly

Acetone