Fixation 1

Fixation

it is the process of preserving cells and tissue constituents in a condition identical to that existing during life. It is used to prevent autolysis

Fixation

2 important goals of fixation

Preserve the morphological and chemical energy of the cell

Harden and preserve tissue for further handling

Methods of Fixation

Heat fixation

Perfusion

Immersion

It is where the fixative becomes part of the tissue by formation of crosslinks, it stabilizes the tissue proteins.

Ex. Formalin, Mecury, Osmium Tetroxide

Additive Mechanism

Mechanism where the fixative is NOT incorporated into the tissue. It alters the tissue composition and stabilizes the tissye by removing water

Non-additive

Temperature of Fixation

Routinely at:

Autotech:

EM and HIstochem at

Rapid fixation at:

Tx with TB:

Routinely at: 22-25

Autotech: 40C

EM and HIstochem at: 0-4C

Rapid fixation at: 60C

Tx with TB: 100C

Tissues should not be over <5mm except for

Edematous Lung Tissue

Brain tissue is suspended in whole _______________________ for how long?

10% NBF for 2-3 weeks

Osmolality of tissues should be slightly hypertonic at around _______ mOsm

400-450

Concentration for normal tissues:

____ Formalin

____ Glutaraldehyde

10% Formalin

3% Glutaraldehyde

Concentration of Glutaraldehyde ideal for Immunoelectron Microscopy

0.25% Glutaraldehyde

Time of duration of Primary Fixation in buffered formalin:

Time of EM fixation:

2-6 hrs for Primary Fixation

EM: 3 HRS

Practical Considerations for Fixation:

Speed of <1hr is to prevent ______________

Rate of Penetration of Formalin: __mm/hr

Volume of fixative is ___ to ___ that of the tissue, ___ is ideal

Prevents autolysis

1mm/hr

10x to 20x that of tissue, 20x is ideal

Volume of more expensive fixatives like Osmium Tetroxide is around ___ to ___

For Museum preparations: at least ___ times

5x to 10x

50 times

Duration of Fixation can be hastened through these 3 methods:

Heat, Agitation

Microwave

Vacuum

In the three methods of hastening Fixation, this is the fastest

Vacuum fixation

Considerations for Fixations

Autopsy materials are kept ata mortuary ref at a temperature of:

Considerations for Fixations

Brain:

Hollow Organs:

Air-filled Lungs:

Brain should be fixed before grossing

Hollow organs aree filled with Moist cotton with Formalin

Air-filled Lungs are to be surrounded with gauze to prevent floating

Considerations for Fixations

Eyes:

Hard tissues:

Eyes are injected with Formol Alcohol before immersing in the fixative (fix before gross)

Hard tissues are washed out overnight in running water and immersed in 4% aqueous phenol solution for 1-3 days (Lendrum’s Method)

Fixatives according to composition

Simple Fixatives

Compound Fixatives

It is fixatives that are made up of only one component substance

Simple Fixatives

Simple Fixatives (7)

Aldehyde

Metallic fixatives

Picric Acid

Acetic Acid

Acetone

Alcohol

Osmium Tetroxide

Fixative that are made up of two or more fixatives which have been added together to obtain the optimal combined effect of their individual actions upn the cells and tissue consitiuents

Compound Fixatives

Types of Fixatives according to action:

Microanatomical Fixatives

Cytological Fixatives

Type of fixative that permit the general microscopic study of tissue structures without altering the structural pattern and normal intracellular relationship of the tissues in question

Microanatomical Fixatives

Cytological Fixatives are those that preserve specific parts and particular microscopic elements of the cell itself

Cytological Fixatives

Fixative that preserve the nuclear structures (e.g., chromosomes) in particular. They usually contain glacial acetic acid as their primary component due to its affinity for nuclear chromatin. They have a pH of 4.6 or less.

Nuclear Fixatives

Fixative that preserve cytoplasmic structures in particular. They must never contain glacial acetic acid which destroys mitochondria and Golgi bodies of the cytoplasm. They have a pH of more than 4.6.

Cytoplasmic Fixatives