Histology - Module 2, Unit 3

What factors determine the use of different fixatives?

Convenience, economics, effectiveness and efficiency, habit, history, practicality and integrity, preference, safety.

What are the characteristics of an optimal or ideal fixative?

Does not distort or dissolve tissue components, renders enzymes inactive, kills and fixes microorganisms, does not shrink or swell tissue, maintains integrity of tissue architecture, penetrates tissue rapidly, permits or enhances subsequent staining, is inexpensive, stable, non-toxic and safe to handle.

What is a simple fixative?

• one chemical/reagent

• one active component

• ex. ethanol, formaldehyde, acetone.

What is a compound fixative?

• more than one chemical/reagent

• more than one active ingredient

• ex. Bouins, Zenker, Carnoy.

What is the chemical formula for glutaraldehyde?

C5H8O2 - dialdehyde

What is the appearance of glutaraldehyde?

Colourless liquid.

How is glutaraldehyde classified as a fixative?

Additive, non-coagulant, microanatomical.

What is glutaraldehyde mainly used as in histology?

A primary fixative for electron microscopy (EM) because it preserves ultrastructure the best of any aldehydes.

What concentration of glutaraldehyde is used?

2-4%.

What is the fixation time for glutaraldehyde?

2-4 hours.

At what temperature is glutaraldehyde usually used?

4°C.

At what pH is glutaraldehyde buffered?

7.4

How does glutaraldehyde react with proteins?

non-coagulant and additive, making crosslinks like formalin.

What is unique about glutaraldehyde's extra aldehyde group?

It is not involved in most cross linkages and is left to react with any method that uses Schiff's reagent (detection of aldehydes).

What is the effect of glutaraldehyde on lipids?

Lipids are preserved but not made insoluble, like formaldehyde.

What is the effect of glutaraldehyde on carbohydrates?

Carbohydrates are not fixed.

What are the disadvantages of glutaraldehyde?

• Overhardens tissue

• shrinkage

• poor penetration (thin pieces needed, 1.0mm3)

• unstable: breaks down on exposure to O2

• drop in pH, needs buffering like formaldehyde

• can produce glutaric acid and form artefact similar to formalin pigment.

What are the hazards of glutaraldehyde?

Similar to formaldehyde: sensitizer, irritant, toxic, corrosive, carcinogen; incompatible with oxidizers and alkalis.

What is the chemical formula for osmium tetroxide?

OSO4

What is the appearance of osmium tetroxide?

Pale yellow crystals supplied in brown ampoules.

Why is osmium tetroxide supplied in brown ampoules?

Because it is easily reduced to OsO2 by light and organic material, forming a black precipitate.

How is osmium tetroxide classified as a fixative?

Non-coagulant, additive (crosslinks proteins), microanatomical

What concentration of osmium tetroxide is used in histology?

1% buffered solution, pH 7.2.

At what temperature is osmium tetroxide usually used?

4°C

When is osmium tetroxide used as a vapour?

If sections are very thin.

Is osmium tetroxide ever used routinely?

No - secondary fixative

What is osmium tetroxide used as for EM?

A secondary fixative after glutaraldehyde, ensuring preservation of lipids.

How does osmium tetroxide react with lipids?

• It fixes and demonstrates lipids

• reacts with (oxidizes) unsaturated lipids forming complexes that are insoluble in the usual fat solvents (xylol, alcohol).

what fixative is best for lipids?

OSO4 - osmium tetroxide

• all fatty acids and phospholipids will stand out

What is osmium tetroxide reduced to?

OsO2 = a black precipitate = electron dense

What are the disadvantages of osmium tetroxide?

• Poor penetration - thin sections

• causes layer fixation

• intolerant

what are the hazards of osmium tetroxide?

• vaporizes readily

• vapor will easily fixes nasal mucosa or conjunctiva of the eye = neurotoxin

• must use goggles, fume hood, tight stoppers, great care when mincing OsO4 fixed tissue

What is the post fixation treatment for osmium tetroxide?

Wash tissue well with water before dehydration to remove excess OsO4

Why must excess OsO4 be removed before dehydration?

excess OsO4 will react with alcohol to form OsO2 and ethanoic acid (acetic acid).

What is the reaction between osmium tetroxide and ethyl alcohol?

Osmium tetroxide + ethyl alcohol = non-specific black deposit + acetic acid + water.

What is the chemical formula for acetic acid?

CH3COOH

At what temperature does glacial acetic acid solidify?

17°C

• therefore RT before use

At what concentration is acetic acid used in histology?

5%.

What is the appearance of acetic acid?

Water-based colourless liquid

How is acetic acid classified as a fixative?

Non-coagulant and non-additive

What is acetic acid used for in histology?

Cytological fixative for nuclei

What is the effect of acetic acid on cytoplasm components?

Poor for cytoplasm components; mitochondria and lipids dissolve

What is the effect of acetic acid on collagen?

Terrible for collagen; swells as protein bonds are broken and water is absorbed, eventually dissolves collagen.

What is the effect of acetic acid on calcium?

Dissolves out calcium (some decalcification).

What is the effect of acetic acid on red cells?

Red cells are lysed = poor preservation

How does acetic acid react with proteins?

• non-coagulant but does precipitate nuclear proteins

• disrupts linkages between adjacent proteins

• H2O adds to hydrophilic groups = swelling and softening of tissue.

What is the penetration rate of acetic acid?

Quick penetration.

Why is acetic acid excellent in compound fixatives?

To counteract the shrinkage effect of another fixative.

Does acetic acid fix lipids or carbohydrates?

No

What are the advantages of acetic acid?

Fixes nuclei well - precipitates and preserves nucleoproteins and precipitates DNA

• Counteracts shrinkage and hardening and enhances nuclear staining

what are the disadvantages of acetic acid?

• swelling

• softening

• not used alone

What are the hazards of acetic acid?

Corrosive; transported in acid carriers and used under fume hood.

What happens to RBCs in acetic acid?

RBCs lyse.

What is the chemical formula for potassium dichromate?

K2Cr2O7

What is the appearance of potassium dichromate?

Bright orange crystals.

How is potassium dichromate used in histology?

compound fixatives in a 2.5-5% solution; rarely used alone.

How does potassium dichromate act as a fixative if pH >3.8?

Acts as a non-coagulant; good for cytoplasm and mitochondria, but not good for nuclei.

Is potassium dichromate additive?

Yes - forms cross links

How does chromium in potassium dichromate affect tissue charge?

Chromium attaches to COO- and OH+ groups = increases number of reactive basic groups (NH2) which increasing overall positive charge, therefore enhancing attraction for coloured anions (-)

How does potassium dichromate act as a fixative if pH <3.4?

Acts as a coagulant fixative like chromic acid; good for nuclei.

What is the penetration like for potassium dichromate?

Fairly good.

What is the effect of potassium dichromate on tissue swelling?

Tends to swell the tissue, leaves it unstable, but allows for more shrinkage after paraffin processing than other fixatives

How should potassium dichromate best be used?

In compound fixatives or as a secondary fixative.

What pigment does potassium dichromate yield?

A fixation pigment that once formed is insoluble.

How can potassium dichromate pigment be reduced?

Treating with 1% HCl in 70% alcohol

What is potassium dichromate used to help demonstrate?

Myelin, phospholipids, and enhancing some anionic stains.

What is the effect of potassium dichromate on carbohydrates?

Not recommended; they are oxidized (therefore not preserved).

What is the effect of potassium dichromate on lipids?

Attaches to some unsaturated lipids, rendering them insoluble in lipid solvents, especially phospholipids of mitochondria membranes.

What are the hazards of potassium dichromate?

Highly toxic by inhalation and ingestion; carcinogenic and corrosive to skin and mucous membranes; chromium must be tracked and collected for disposal in the same way as mercury.

What is the chemical formula for ethanol?

CH3CH2OH or C2H5OH.

What is the appearance of ethanol?

Colourless liquid.

How is ethanol used in histology?

As absolute 100% and as a cytology fixative.

How is ethanol classified as a fixative?

Coagulant, non-additive, cytological.

How does ethanol react with proteins?

Reacts by dehydration but does not precipitate nuclear proteins; breaks H+ and ionic bonds, followed by removal of H2O.

What is the effect of ethanol on tissue?

Causes extreme shrinkage and hardening; tissue is brittle (intolerant fixative).

When is ethanol used alone?

Only for smears of blood or tissue (e.g., PAP smear).

What is the penetration rate of ethanol when used alone?

Slow penetration.

What concentration of ethanol is used for PAP smears?

95%.

What concentration of ethanol is used for tissue?

100%.

Why is ethanol used in compound fixatives?

To counteract swelling and softening caused by acetic acid and K2Cr2O7.

What is the effect of ethanol on glycogen?

Precipitates glycogen rendering it less soluble in other reagents, therefore good for preservation, but causes glycogen streaming which becomes granular in appearance.

What is the effect of ethanol on lipids?

Dissolves lipids, especially neutral lipids.

What are the advantages of ethanol?

Counteracts other fixatives, dehydrates while fixing, good fixative for natural pigments (hemosiderin, bile pigments, melanin), good for histochemical procedures (usually mixed with formalin and used at 4°C, enzymes retain some activity).

What are the hazards of ethanol?

Flammable.

What is the chemical formula for picric acid?

C6H2(NO2)3OH.

What is another name for picric acid?

Trinitrophenol.

What is the appearance of picric acid?

Bright yellow crystals.

How is picric acid used in histology besides as a fixative?

May be used as a stain.

What concentration of picric acid is used as a saturated solution in H2O?

1%.

What is the solubility of picric acid in alcohol?

Up to 5%.

How is picric acid classified as a fixative?

Coagulant, additive.

How do picrates attach to proteins?

Picrates are negatively charged and attach to positive groups; not cross linked but additive.

What is the penetration rate of picric acid?

Fairly slow.

What is the effect of picric acid on tissue shrinkage?

Causes a lot of shrinkage, but not hardening like alcohol.

What is the texture of tissue fixed in picric acid?

Turns the tissue rubbery rather than brittle like alcohol.

What does picric acid create in tissue that aids processing?

A spongy network good for penetration of processing agents.

What is the effect of picric acid on lipids?

No effect on lipids.

What is the effect of picric acid on glycogen?

Fairly good for glycogen; traps glycogen in protein mesh but can still see glycogen streaming.

What are the advantages of picric acid?

Good precipitant of nucleoprotein (good cytological for nuclei).

What are other uses of picric acid in histology besides fixation?

As an acid or cytoplasmic dye, as a differentiator (removes excess colour of one dye by replacing it with another), used to remove formalin pigment, will decalcify small amounts of Ca++ deposits, as a pseudo mordant (helps combine tissue dye to tissue).