LECTURE 2: Fixation (Part 2)

Fixatives

ALDEHYDE FIXATIVES

They are satisfactory for routine paraffin sections, electron microscopy, and when histochemical and enzyme studies are indicated.

1. Formaldehyde (Formalin)

2. 10% Formol-Saline

3. 10% Neutral Buffered Formaldehyde

4. Formol-Corrosive

5. Alcoholic Formalin

6. Glutaraldehyde

ALDEHYDE FIXATIVES:

1. Mercuric Chloride

2. Zenker’s Fluid

3. Zenker-Formol

4. Heidenhain’s Susa Solution

5. B-5 Fixative

METALLIC FIXATIVES:

10% formalin

One of the most widely used fixative is the ____: Gas produced by oxidation of methanol

formaldehyde (40%)

Readily-made pure stock solution of _____ is not ideal because it over hardens the outer layer of tissue and affects staining adversely.

24 hours

Fixation time for Formaldehyde (Formalin)

FORMALDEHYDE (FORMALIN)

Usually buffered to pH 7.0 with a phosphate buffer

FORMALDEHYDE (FORMALIN)

It is recommended for color tissue photography

FORMALDEHYDE (FORMALIN)

Also known as the tolerant fixative because it is used for mailing specimens, or sending specimens out, because it can be left in formalin indefinitely

Prolonged storage of formaldehyde, especially at very low temperature

This may induce precipitation of a white paraformaldehyde deposit and produce turbidity, although this in itself does not impair the fixing property of the solution.

10% methanol

Precipitates of a white paraformaldehyde deposit can still be removed by filtration, or by the addition of ______.

Methanol

Is a preservative added to formaldehyde to prevent its decomposition to formic acid that will lead to the precipitation of formaldehyde.

FORMALDEHYDE (FORMALIN)

It is a drawback because it denatures protein, making it unsuitable for electron microscopy

• Reduces basophilic and eosinophilic staining of cells, thus reducing the quality of routine cytologic staining

• Abundant brown pigment granules on blood containing tissues can form

Disadvantages of Formaldehyde if unbuffered:

1. Bleaching of specimen and loss of natural tissue color

2. Dispersal of fat from the tissue into the fluid

3. Dissolution or loss of glycogen, biurate of sodium crystals and uric acid.

Prolonged fixation with the use of formaldehyde may lead to:

Cadmium and Cobalt

Prolonged fixation with the use of formaldehyde may lead to:

Dispersal of fat from the tissue into the fluid

• Can be remedied using _____.

Cadmium and Cobalt salts

Prolonged fixation with the use of formaldehyde may lead to:

Dispersal of fat from the tissue into the fluid

• _______ are added to prevent dispersion of fat into the fluid

10% methanol

Precautions when dealing with Formaldehyde:

Induce precipitation at very low temperature.

• Precipitants that may be removed by filtration or the addition of _____

Changing the fluid fixative every 3 months

Precautions when dealing with Formaldehyde:

Bleaching of tissues may be prevented by?

alcoholic Picric acid and 1% solution of Potassium hydroxide in 80% alcohol

Precautions when dealing with Formaldehyde:

Brown or black crystalline precipitate formed can be removed prior staining by treatment of?

Magnesium carbonate or Calcium carbonate

Precautions when dealing with Formaldehyde:

_______ may be used to neutralize acid reaction due to Formic acid formation

5mm

Precautions when dealing with Formaldehyde:

Tissue blocks should not exceed ____ in thickness

neutralized

Precautions when dealing with Formaldehyde:

Concentrated solutions must never be ____ since it might precipitate violent explosions

10% FORMOL-SALINE

A diluted Formaldehyde with Sodium chloride

10% FORMOL-SALINE

Recommended for central nervous tissues and general post mortem tissues for histochemical examination

10% FORMOL-SALINE

Penetrates tissues evenly but is a slow fixative (takes 24 hours or more)

10% FORMOL-SALINE

Ideal for most staining techniques including silver impregnation

1. Fixed tissues shrink during alcohol dehydration, but can be reduced by submitting the specimen to secondary fixation

2. Metachromatic reaction of amyloid is reduced

Disadvantages of 10% Formol-saline:

secondary fixation

Disadvantages of 10% Formol-saline:

Fixed tissues shrink during alcohol dehydration, but can be reduced by submitting the specimen to _____

10% NEUTRAL BUFFERED FORMALDEHYDE

Also known as Phosphate-Buffered Formalin

Phosphate-Buffered Formalin

10% Neutral Buffered Formaldehyde is also known as?

10% NEUTRAL BUFFERED FORMALDEHYDE

Recommended for preservation and storage of surgical, post mortem, and research specimens.

10% NEUTRAL BUFFERED FORMALDEHYDE

Has a pH maintained at 7.0

10% NEUTRAL BUFFERED FORMALDEHYDE

Advantages is similar to Formol-saline, but is considered as the best fixative for tissues containing iron pigments and for elastic fibers, which do not stain well after Susa, Zenker, or Chromate fixation

10% NEUTRAL BUFFERED FORMALDEHYDE

Does not require post treatment after fixation, therefore, it goes directly into 80% alcohol for processing

1. Making of this fixative is longer and time consuming

2. It produces gradual loss in basophilic staining of cells

3. It is inert towards lipids, especially neutral fats and phospholipids

Disadvantages of 10% Neutral Buffered Formaldehyde:

FORMOL-CORROSIVE

Also known as “Formol-sublimate”

Formol-sublimate

Formol-Corrosive is also known as?

Formol-mercuric chloride solution

Is recommended for routine post-mortem tissues

FORMOL-CORROSIVE

Penetrates small pieces of tissues rapidly

FORMOL-CORROSIVE

Excellent for many staining procedures including silver reticulum methods

3-24 hours

Fixation time for Formol-Corrosive:

FORMOL-CORROSIVE

There is no need for “washing-out”, therefore, tissues can go directly into 80% alcohol

1. It forms Mercuric chloride deposits

2. It does not allow frozen sections to be made

3. It inhibits the determination of the extent of tissue decalcification

Disadvantages of Formol-corrosive:

ALCOHOLIC FORMALIN

Also known as “Gendre’s fixative”

Gendre’s fixative

Alcoholic Formalin is also known as?

ALCOHOLIC FORMALIN

Used for faster diagnosis because it fixes and dehydrates at the same time

Phenol-formalin

Post fixation with ____ for 6 hours or more can enhance immunoperoxidase studies on the tissue

ALCOHOLIC FORMALIN

Good for preservation of glycogen and micro-incineration technique

ALCOHOLIC FORMALIN

Used to fix sputum, since it coagulates mucus

ALCOHOLIC FORMALIN

Disadvantage: Causes partial lysis of RBC

GLUTARALDEHYDE

Made up of two formaldehyde residues, linked by three carbon chains

GLUTARALDEHYDE

It acts similar to that of formaldehyde and sometimes utilize for routine light microscopic work

Buffered Glutaraldehyde followed by secondary fixation in Osmium tetroxide

GLUTARALDEHYDE:

Satisfactory for electron microscopy: _____

2.5% solution (Glutaradehyde)

A _____ is used for small tissue fragments.

4% of Glutaraldehyde

_____ is used for larger tissues but less than 4mm thick, fixed in 6-8 hours up to 24 hours

less than 4mm; 6-8 hours up to 24 hours

4% of Glutaraldehyde is used for larger tissues but ____ thick, fixed in ____.

1. It has a more stable effect on tissues and less irritating, giving a firmer texture with better tissue sections especially on central nervous system

2. Preserves plasma proteins better

3. Preserves cellular structures, hence it is recommended for enzyme histochemistry and electron microscopy

4. Fixation time: 30 minutes to 2 hours

5. Specimen vial must be kept refrigerated during fixation process

Advantages of Glutaraldehyde:

30 minutes to 2 hours

Fixation Time for Glutaraldehyde:

MERCURIC CHLORIDE

The most common metallic fixative

MERCURIC CHLORIDE

Used in saturated aqueous solutions of 5-7%

MERCURIC CHLORIDE

Widely used as a secondary fixative

MERCURIC CHLORIDE

Tissues fixed may contain black precipitates of mercury except Heidenhain’s Susa solution

1. Trichome staining is excellent

2. Routine fixative for preservation of cell detail in tissue photography

3. Permits brilliant metachromatic staining of cells

4. Recommended for renal tissues, fibrin, connective tissues, and muscles

5. Precipitates all kinds of proteins

Advantages of using Mercuric Chloride:

ZENKER’S FLUID

Made up of Mercuric chloride stock solution for which glacial Acetic acid has been added

ZENKER’S FLUID

Good general fixative and gives excellent staining results

ZENKER’S FLUID

Recommended for fixing small pieces of liver, spleen, connective tissue fiber and nuclei

ZENKER’S FLUID

Recommended for trichome staining

ZENKER’S FLUID

It is not stable after addition of Acetic acid

1. It is not stable after the addition of Acetic acid

2. It can cause lysis of red cells and removes Iron from hemosiderin

3. Does not permit cutting of frozen sections and tissues must be washed in running water for several hours, or even overnight, before processing.

4. Insufficient washing can inhibit or interfere with good cellular staining.

5. Is not that ideal as a fixative

Disadvantages of using Zenker’s fluid:

dezenkerization

ZENKER’S FLUID:

Mercury deposits may be removed by “_____” because it contains Mercuric chloride

1. Bring the slides to water

2. Immerse in Lugol’s iodine (5 mins)

3. Wash in running water (5 mins)

4. Immerse in 5% Sodium thiosulfate (5 mins)

5. Wash in running water (5 mins)

6. Proceed with required water soluble stain

Process of Dezenkerization:

ZENKER-FORMOL

Also known as “Helly’s solution”

Helly’s solution

Zenker-Formol is also known as?

ZENKER-FORMOL

Excellent microanatomic fixative for pituitary gland, bone marrow, and blood containing organs such as spleen and liver.

ZENKER-FORMOL

Nuclear fixation and staining is better than Zenker’s fluid

ZENKER-FORMOL

Preserves cytoplasmic granules well

HEIDENHAIN’S SUSA SOLUTION

Recommended mainly for tumor biopsies especially of the skin and an excellent cytologic fixative

Weigert’s method

HEIDENHAIN’S SUSA SOLUTION:

_____ of staining elastic fibers is not possible in Susa-fixed tissues

HEIDENHAIN’S SUSA SOLUTION

After fixation, tissues should be transferred to a high grade of alcohol (96% or absolute alcohol) to avoid undue swelling of the tissue.

3 to 12 hours

Fixation time for Heidenhain’s Susa Solution

B-5 FIXATIVE

Commonly used for bone marrow biopsies

B-5 FIXATIVE

Very fast acting fixative wherein fixation is achieved in 1 and a half hour to 2 hours

1 cc of 40% formalin for 10 cc of B-5

B-5 FIXATIVE:

Prior usage, add _____

1. Chromic acid

2. Potassium dichromate

3. Regard’s fluid

4. Orth’s fluid

CHROMATE FIXATIVES:

CHROMIC ACID

Precipitates all proteins and adequately preserves carbohydrates

POTASSIUM DICHROMATE

Preserves lipids

POTASSIUM DICHROMATE

Preserves mitochondria (pH 4.5 – 5.2)

REGARD’S FLUID

Also known as “Muller’s fluid”

Muller’s fluid

Regard’s Fluid is also known as?

REGARD’S FLUID

Recommended for the demonstration of chromatin, mitochondria, mitotic figures, Golgi bodies, RBC and colloid containing tissues

REGARD’S FLUID

It deteriorates and darkens on standing due to acidity

ORTH’S FLUID

Recommended for study of early degenerative processes and tissue necrosis

ORTH’S FLUID

Demonstrates rickettsia and other bacteria

LEAD FIXATIVES

Used in 4% aqueous solution of basic lead acetate

LEAD FIXATIVES

Recommended for acid mucopolysaccharide

1. Bouin’s Solution

2. Brasil’s alcoholic picroformol fixative

PICRIC ACID FIXATIVES:

PICRIC ACID

Also dyes tissues:

• May give yellow color to the tissue

PICRIC ACID

Preserves glycogen but can cause shrinkage of tissue

PICRIC ACID

Suitable for aniline stains (Mallory’s, Masson’s and Haidenhain’s methods)