2. Fixation and Simple Fixatives

Flashcards covering foundational tissue fixation principles, physical and chemical parameters, tissue handling protocols, and simple fixative classifications from the histology lecture.

What is putrefaction in tissue processing?

The breakdown and dispersal of cells caused by microbial action, typically from normal flora or microorganisms.

Can fixation undo tissue damage caused by autolysis or putrefaction once it has begun?

No, fixation halts both putrefaction and autolysis, but it cannot reverse or repair damage that has already occurred.

How does lower temperature affect autolysis and putrefaction?

Lower temperatures slow down both processes, but do not completely stop them.

What are the microscopic hallmarks of improperly fixed tissue?

Nuclei appear broken up or absent, and cytoplasm becomes cloudy, loses its staining ability, and shifts out of place.

Why is tissue hardening considered a desirable effect of fixation?

Hardening firms the tissue, making it significantly easier to cut sections compared to soft, squishy tissue.

How does fixation improve optical contrast under microscopy?

It increases the refractive index of the tissue, making cellular structures easier to view.

By what physical process does chemical fixative penetrate tissue?

Passive diffusion.

What is the standard temperature for routine tissue fixation?

Room temperature, typically around $22^\circ\text{C}$.

At what temperature should fixation occur when preserving enzymes, antigens, or ultrastructure?

$4^\circ\text{C}$.

How does fluid agitation benefit the fixation process?

It accelerates diffusion and penetration of the fixative without causing tissue damage.

What is the recommended volume ratio of fixative fluid to tissue specimen?

Between $15$ and $20$ times the volume of the tissue.

What is the recommended thickness for tissue placed in a processing cassette?

$3\text{ to }4\,\text{mm}$ thick.

What is cold ischemic time, and what is its target threshold?

The time elapsed from when tissue is excised from the body to when it is submerged in fixative; it should be less than half an hour ($< 30\,\text{minutes}$).

Why must fixative solutions maintain an isotonic osmolality?

Hypertonic solutions draw water out and make tissue brittle, while hypotonic solutions cause tissue swelling by drawing water in.

Why are dedicated histology microwaves used instead of domestic commercial microwaves?

Histology microwaves provide uniform heating to eliminate hot spots, preventing uneven fixation rates and unintentional tissue cooking.

What is the typical time frame required for routine chemical fixation?

Between $12$ and $72\,\text{hours}$ (commonly $24\text{ to }72\,\text{hours}$).

Why was the whole brain suspended by the brainstem during fixation?

To prevent it from resting on the container bottom and flattening against the sides, ensuring the fixative diffuses evenly from all surfaces.

What differentiates a simple fixative from a compound fixative?

A simple fixative consists of a single chemical, whereas a compound fixative contains two or more chemicals.

How do coagulant fixatives act on tissue proteins?

They compete for and remove water bound to protein groups, causing the tertiary structure to collapse, clump, and slightly shrink the tissue.

How do additive fixatives stabilize tissue structure?

They chemically combine with proteins (predominantly via ionic bonds) to cross-link them into a scaffold, typically producing slight tissue swelling.

What is the operational difference between a tolerant and an intolerant fixative?

Tissues can remain in tolerant fixatives indefinitely without harm, whereas intolerant fixatives rapidly cause damage if tissues are exposed past the required duration.

What is a histological artifact?

Any distortion or alteration introduced into a tissue during processing (from initial excision to coverslipping) that is not present in life.

What is the chemical difference between formaldehyde and formalin?

Formaldehyde is a pure gas, whereas formalin is an aqueous solution saturated with formaldehyde at $37\%$ to $40\%$.

What is the actual formaldehyde concentration in a standard $10\%$ formalin solution?

Approximately $4\%$ formaldehyde.

Why is methanol (up to $10\%$) added to concentrated commercial formalin?

To stabilize the solution and prevent the formaldehyde molecules from polymerizing.

What are the penetration and fixation rates of formalin?

It penetrates rapidly at approximately $4\,\text{mm/hour}$ ($0.4\,\text{cm/hour}$), but fixes slowly over $8\text{ to }12\,\text{hours}$, completing up to $72\,\text{hours}$.

Why is glutaraldehyde typically restricted to electron microscopy rather than routine histology?

Being a dialdehyde, only one aldehyde binds to tissue while the free second aldehyde reacts with downstream staining reagents to produce false positives.

Which amino acid residue does mercuric chloride react with, and what artifact does it produce?

It reacts specifically with histidine residues and leaves a distinct black pigment artifact.

What are the classification properties of ethyl alcohol (ethanol) as a simple fixative?

It is non-additive, strongly coagulant, and intolerant.

What safety hazard and tissue artifact are associated with picric acid?

It is a high explosive when dry (must always be kept wet) and deposits a yellow pigment artifact that can be washed out with water.

What are the principal cellular effects of acetic acid in fixation?

It lyses red blood cells, produces exceptional nuclear preservation, and causes tissue swelling that counteracts shrinkage from other agents.

How does pH determine the fixation mechanism of potassium dichromate?

At a low pH it functions as a coagulative fixative, whereas at a pH above $3.4$ it acts as a non-coagulative fixative.

Which fixative is used by $99.9\%$ of routine pathology laboratories for light microscopy and immunohistochemistry?

$10\%$ neutral buffered formalin (NBF).