Histopath LAB - Fixation

Fixation

First and most critical step in histotechnology

Fixation

Classically defined as the killing, penetration, and hardening of tissues

swell, shrink

Leaving the tissue in water causes the cell to (?)

Strong salt causes the cell to (?)

handling

Fixation's second aim: To harden and protect the tissue from the trauma of (?)

Additive fixation

The chemical constituent of the fixative is taken in and becomes part of the tissue by forming cross-links or molecular complexes, and giving stability to protein

cross-links

Additive fixation

The chemical constituent of the fixative is taken in and becomes part of the tissue by forming (?) or molecular complexes, and giving stability to protein

Non-additive fixation

Wherein the fixing agent is not incorporated into the tissue, but alters the tissue composition and stabilizes the tissue by removing the bound water attached to H-bonds

H-bonds

Non-additive fixation

Wherein the fixing agent is not incorporated into the tissue, but alters the tissue composition and stabilizes the tissue by removing the bound water attached to (?)

6 - 8

Satisfactory fixation occurs between pH (?)

room

Fixation is traditionally carried out at (?) temperature

40

Many laboratories use tissue processors that work at (?)°C

0-4C

Electron microscopy and some histochemistry - ideal

temperature

60C

Formalin heated to (?) is sometimes used for the rapid fixation of very urgent biopsy specimens- increased risk of tissue distortion

tuberculosis

Formalin at 100°C can be used to fix tissues with (?)

100C

Formalin at (?) can be used to fix tissues with tuberculosis

Brain

(?) is usually suspended whole in 10% buffered formalin for 2-3 weeks

10% buffered formalin

Brain is usually suspended whole in (?) for 2-3 weeks

slightly hypertonic solutions (400-500 mOsm)

OSMOLALITY

Best results are usually obtained using (?)

glutaraldehyde

Low concentrations of (?) have been found to be an ideal concentration for immunoelectron microscopy

3 hours

For electron microscopy, tissues should be fixed for (?) and then placed in holding buffer

1mm

Formalin diffuses into the tissue at the rate of (?) per hour

10-25

The amount of fixative used has been (?) times the volume of tissue to be fixed

20x

Maximum effectiveness of fixation: (?) the tissue volume

2 square cm, 4mm

To maintain an adequate fixation time of 4-6 hours, the recommended size of the tissue is (?), and no more than (?) thick

brain

Most tissue can be cut and trimmed without prior fixation, except for the (?)

Refrigeration

(?) is used to slow down decomposition if the tissue needs to be photographed and cannot be fixed immediately

30 minutes

Bone marrow continues to undergo mitosis up to (?) after death when refrigerated

False

True or False:

A good fixative must kill cells slowly to avoid damage to the tissue

Simple fixatives

Aremade up of only one component substance (only one component is present ex. Aldehyde, metallic fixative, mercury chromium)

Compound fixatives

Are those that are made up of

two or more fixates

Microanatomical fixatives

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

Cytological fixatives

Are those that preserve specific parts and particular microscopic elements of the cell itself

Nuclear fixatives

Fixatives with glacial acetic acid as their main component

4.6 or less

Nuclear fixative pH

Flemming's fluid

Carnoy's fluid

Bouin's fluid

Newcomer's fluid

Heidenhain's susa

5 nuclear fixatives

Cytoplasmic fixatives

This type of fixative must never contain glacial acetic acid

mitochondria, golgi bodies

Cytoplasmic fixatives must never contain glacial acetic acid as they destroy (?) and (?)

4.6 or more

Cytoplasmic fixative pH

Histochemical fixatives

Are those that preserve the chemical constituents of cells and tissues

Heat fixation

Simplest form of fixation

Microwave heating

Speeds fixation and can reduce times for fixation for some gross specimens and histological sections from more than 12 hours to less than 20 minutes

liquid nitrogen, -40C

Freeze-drying fixation

Tissues are cut into thin sections, immersed in (?), and the water is removed in a vacuum chamber at (?)

Chemical method

Used by most methods of fixation in the processing of tissue for histopathological diagnosis

Chemical method

Utilizes organic or non-organic solutions to maintain adequate morphological preservation

Coagulant Fixatives

Compound Fixatives

Cross-linking Fixatives

3 chemical fixatives

Alcohol and acetone

Most commonly used dehydrant coagulant fixatives

80%

Methanol fixation begins at a concentration of (?) or more

True

True or False:

Methanol has a closer structure with water

- Fixation begins at a concentration of 80% or

more

Ethanol competes more strongly in interaction with hydrophobic areas of molecules

- Begins at a concentration of 50%-60%

Acid coagulants

Coagulant fixatives that change the charges

Cross-linking fixatives

Has a potential actions forming cross-links between proteins and nucleic acids pr oteins

Covalent additive fixatives

AKA Cross-linking fixatives

mercuric chloride, potassium dichromate

Fixatives containing (?) or (?) can be effective for the preservation of lipids in

cryostat

aldehydes

Phospholipids are fixed by (?)

imidazole, osmium hydroxide

Ultrastructural demonstration of lipids is achieved by post-fixing in (?) and (?)

digitonin

Cholesterol may be fixed with (?) for ultrastructural demonstration

Neutral buffered formol saline

Most commonly used fixative for amino acid histochemistry

Alcoholic fixatives

Generally recommended for glycogen fixation

Rossman's fluid

Most useful fixative for preserving glycogen

Karnovsky's Paraformaldehyde-Glutaraldehyde

Two aldehyde fixative mixtures useful for electron cytochemistry

Formalin

Most widely used fixative

Gas formed by oxidation of methyl alcohol

True

True or False:

Formaldehyde preserves fats and mucin

Formaldehyde

A "tolerant" fixative, used for mailing specimens because they can be left in here indefinitely

False

True or False:

Concentrated formaldehyde may be neutralized to prevent explosion

10% methanol

Turbidity caused by formaldehyde may be removed by filtration or addition of (?)

magnesium carbonate, calcium carbonate

Acid reaction due to formic acid formation can be buffered or neutralized by adding (?) or (?) to 10-15% formalin

Dark brown

Unbuffered formalin leads to formation of what colored granules?