FIXATION

SIMPLE FIXATIVES

I. Adelhyde Fixatives

They are satisfactory for the routine paraffin sections, frozen sections and for electron microscopy.

A. Formaldehyde (HCHO)

Formaldehyde is a gas produced by the oxidation of methyl alcohol and is soluble in water to the extent of 40% by weight. It is a powerful reducing agent and used as such in the make-up for Helly’s fluid. For purposes of designating concentrations of strength of the solution consider the purest solution that of the 40% by weight gas as 100%. To make the routine formalin solution which is 10% in concentration, mix 10mL of the strong formaldehyde with 90mL of water or saline to make 10% solution. The 10% formalin is at the ratio of 1:10 dilution of the saturated solution of 40% w.v. solution of the gas. Usual fixation time is 12-24 hours.

COMMERCIAL FORMALDEHYDE often becomes turbid after prolonged storage because of the formation of paraformaldehyde which is caused by the polarization of HCHO. Its presence does not impair the fixing qualities of the formaldehyde.

Advantages:1. It is cheap, readily available, easy to prepare and relatively stable.2. It penetrates tissue well.3. It does not overhardens tissues.4. It preserves fat and mucin.5. It is the best fixative for the nervous tissue.

Disadvantages:1. The solution is irritating to the skin causing allergic dermatitis.2. Fumes are irritating to eyes and nostrils causing allergic rhinitis and simusitis.3. It may produce considerable shrinkage of tissues.4. Formalin reduces both the basophilic and eosinophilic staining of cells.5. It forms abundant brown artifacts, pigments and granules.

Removal of Formalin Pigments:1. Karadeswitsch’s Method

a. Bring sections down to waterb. Place the section for 5 minutes to 2 hours, depending on the amount of pigments in a

mixute of 70% alcohol – 50mL; 28% ammonia water – 1-2mL c. Wash thoroughly in water

2. Lillies’s Methoda. Bring section down to water.b. Place the sections for 1-5 minutes in a mixture of Acetone – 50mL; 3 vol. hydrogen peroxide

– 50mL; 28% ammonia waterc. Rinse in 70% alcohol.d. Wash in running water.

3. Picric Acid Methoda. Bring the sections down to water.b. Place the sections in a saturated solution of picric acid for five minutes to 2 hours.c. Wash in running water for 11-15 minutes.

B. Glutaraldehylde

It is an aldehyde M.W. 100 or 2 formaldehyde residues linked by a straight 3 carbon chains.

A 2.5% solution is best used for small fragments and small needle biopsies, fixed adequately for 2-4 hours in room temperature. A 4% solution is recommended for large tissue not exceeding 4mm. in thickness for 12-24 hours at room temperature.

Advantages:1. It has a stable nature, stable effect on tissues, giving a firmer texture and color.2. It preserves cellular and plasma protein better.

Disadvantages:1. It is more expensive.2. It is less stable.

C. Paraformaldehyde

It is a polymer of formaldehyde and it is a white powder. It is used in a 4% strength at 4 degree Celsius.

Advantages:1. It is an excellent fixative for routine paraffin sections.2. It is used for thin and ultrathin sections for plastic embedding.

Disadvantages:1. It is expensive.2. It is unstable.

II. Metallic Fixatives

A. Mercuric Chloride

Frequently used in saturated solution. At room temperature, its solubility in water is 7%.

Advantages:1. It penetrates and hardens tissues rapidly.2. It gives better staining of nuclei and connective tissue.3. It is recommended for renal, fibrin, and consecutive tissues.

Disadvantages:1. It causes marked shrinkage of cells.2. It is less stable.3. It corrodes metals except nickel.

Removal of Mercuric Chloride Granules:1. Bring sections to water.

2. Place sections in alcoholic iodine (0.5mL of iodine in 100mL of 70% alcohol) for 1-2 minutes.3. Rinse in water and place in 5% sodium thiosulfate (hypo) solution for 1-2 minutes to remove

iodine.4. Wash in running water for 2-5 minutes.

B. Chromate Fixatives

These are used in 1-2% aqueous solution.Chromic fixatives are strong oxidizing agents and should not combine with reducing agents such as alcohol and formalin.

Advantages:1. It is a strong protein precipitant.2. It preserves carbohydrates.

Disadvantages:1. It forms a precipitate of insoluble suboxide.2. It is poor for glycogen fixation.

Potassium Dichromate – used in 3% aqueous solution. It is a strong fixative for certain lipids.

Advantages:1. It fixes but does not precipitate cytoplasmic contents.2. It preserves mitochondria.

Disadvantage:1. It penetrates tissues slowly.

C. Lead FixativesUsed in 4% aqueous solution.

Advantages:1. It is used mainly for mucopolysaccharides.2. It precipitates protein.

Disadvantages:1. It penetrates tissues slowly.2. It is expensive.

III. Picric Acid Fixatives

It is usually used in strong or saturated solution. The solubility of picric acid is that 1.7 grams will just dissolve in 100mL of distilled water at 15 degree Celsius. Chemically, picric acid is 2.4,6-trinitrophenol.

Advantages:1. It is the best fixative for glycogen.2. It penetrates tissues rapidly.

Disadvantages:1. It lyses red blood cells.2. It causes considerate shrinkage of tissues.

Removal of Picric Acid Yellow Color:

Bring sections to saturated solution of lithium carbonate in 70% alcohol and then wash in water.

The tissue is placed in 70% ethyl alcohol followed by 5% sodium thiosulfate and then washed in running water.

IV. Acetic Acid Fixative

Used at ice-cold temperature (-40 degree Celsius)

Advantages:1. It fixes and precipitates nucleoprotein.2. It precipitates chromosomes and chromatin materials, which makes it useful in nuclear studies.

Disadvantages:1. It causes considerable swelling of tissues.2. It destroys mitochondria and golgi elements.

V. Acetone

Used at ice-cold temperature (-40 degree Celsius)Used only in enzyme studies.

Advantages:1. It is recommended for phosphatases and lipases studies.2. It is used for fixing brain tissues for rabies.

Disadvantages:1. It evaporates rapidly.2. It dissolves fats.

VI. Alcohol Fixatives

It is used for rapid denaturing and precipitation of proteins by destroying hydrogen and other bonds. It must be used in concentration from 20-100% because lesser concentrations lyse the cells.

Advantages:1. Ideal for smaller tissues fragments.2. Excellent for glycogen preservation.

Disadvantages:1. It causes lysis of red blood cells.2. It dissolves lipids and fats.3. It causes polarization of glycogen.

VII. Osmium Tetroxide Fixatives

Commonly known as osmic acid. It is pale yellow. It dissolves in water (up to abous 6% at 20 degree Celsius) forming a solution which is a strong oxidizing agent.

Advantages:1. It fixes conjugate fats, and lipids permanently by making them insoluble during subsequent

treatment with alcohol and xylem.2. It produces excellent nuclear staining.

Disadvantages:1. It is very expensive.2. It is a slow fixing agent suitable only for small tissues.

Compount Fixatives

I. Micro-anatomical Fixatives

A. 10% Formol-saline

Recommended for materials from the nervous system and general post mortem materials.Fixation time: 12-24 hours

Preparation:37-40% formaldehyde – 100mL; Sodium chloride – 8.5 g; distilled water – 900mL

B. 10% Buffered Formalin

Recommended for preservation of post mortem surgical research specimens.Fixation time: 24 hours or more

Preparation:Sodium hydrogen phosphate (anhydrous) – 3.5 gDisodium hydrogen phosphate (anhydrous) – 6.5 g37-40% formaldehyde – 100mLDistilled water – 900mL

Advantages:1. Prevents the formation of the troublesome post mortem precipitated acid formalin pigment.2. It fixes tissues evenly.

Disadvantages:It takes longer time to prepare.

C. Heidenhain’s Susa Solution

Recommended for biopsies of the skin.Fixation time: 3-12 hours

Preparation:Mercuric chloride – 45 gSodium chloride – 5 gTrichloroacetic acid – 20 gGlacial acetic acid – 40mL37-40% formaldehyde – 200mL

Distilled water – 800mL

Advantages:1. It penetrates rapidly.2. It fixes tissue evenly.

Disadvantages:1. It preserves poorly the RBC.2. Prolonged fixation of large specimens.

D. Formol Sublimate

Recommended for routine post mortem materials.Fixation time: 3-24 hours

Preparation:Saturated aqueous mercuric chloride – 90mL37-40% formaldehyde – 10mL

Advantages:1. It is excellent as routine fixative.2. It produces little or no shrinkage of tissues.

Disadvantage:It does not preserve thick slices of tissues thus not to be used for more than 1 cm thick specimen sections.

E. Formol Saline Sublimate

Recommended for post mortem materials.Fixation time: 3-24 hours

Preparation:Saturated aqueous mercuric chloride – 90mL37-40% formaldehyde – 10mLSodium chloride – 4 g

Advantages and disadvantages similar to formol sublimate.

F. Zenker’s Solution

Recommended for post mortem materials.Fixation time: 3-24 hours

Preparation:Mercuric chloride – 5 gPotassium dichromate – 2.5 gSodium sulfate (optional) – 1 gDistilled water – 100mLAdd 5mL glacial acetic acid before use.

Advantages:1. It permits excellent staining of nuclei.2. It is recommended for tissues to be stained by one of the trichrome technique.

Disadvantages:1. It penetrates tissue poorly.2. It is not stable after the addition of acetic acid.

G. Zenker’s Formol (Helly’s Fluid)

Recommended for pituitary tissues and bone.Fixation time: 12-24 hours

Preparation:Mercuric chloride – 5 gPotassium dichromate – 2. 5 gDistilled water – 100mLAdd 5mL of 40% formaldehyde just before use.

Advantages:1. It gives excellent nuclear fixation.2. It preserves cytoplasmic granules well.3. Excellent fixatives for pituitary and bone marrow tissue.

Disadvantages:1. It causes lysis of RBC.2. Prolonged fixation of tissue produces brown scum.

H. Bouin’s Solution

Recommended for embryos.Fixation time: 6-24 hours

Preparation:Saturated aqueous solution of picric acid – 75mL37-40% formaldehyde – 25mLGlacial acetic acid – 5mL

Advantages:1. The yellow stain is useful when handling fragments of tissues.2. It preserves glycogen.

Disadvantages:1. It penetrates slowly.2. It should never be used for preserving kidneys because of extreme distortion.

II. Cytological Fixative- Nuclear Fixative

A. Flemming’s Fluid

Recommended for nuclear structures.Fixation time: 24-48 hours

Preparation:Chromic Acid – 15mLAqueous Osmium tetroxide 2% - 4mLGlacial acetic acid – 1mL

Advantages:1. It is excellent for nuclear elements (chromosomes).2. It permanently preserves fats.

Disadvantages:1. It penetrates poorly.2. It deteriorates rapidly thereby it must be prepared immediately before its use.

B. Carnoy’s Fluid

Recommended for chromosomes study, lymph nodes and urgent studies for glycogen.Fixation time: ½ -3 hours

Preparation:Absolute alcohol – 60mLChloroform – 30mLGlacial acetic acid – 10mL

Advantages:1. It preserves glycogen.2. It fixes and dehydrates simultaneously.3. It is extremely rapid, probably, the most rapid of all fixatives.

Disadvantages:1. It causes excessive shrinkage and hemolysis of RBC.2. Only small pieces of tissues may be used.

C. Bouin’s Fluid

Recommended for embryos, and glycogen.Fixation time: 6-24 hours

Preparation:Saturated aqueous picric acid40% formaldehydeGlacial acetic acid

Advantages:1. It gives better nuclear fixation than Flemming’s fluid.2. It is useful for minute fragments of tissue as the yellow color given to the tissue makes it easier

to locate the specimen.Disadvantages:

1. Kidney is badly distorted and should never be fixed in Bouin’s fluid.2. Cytoplasmic structures such as mitochondria are distorted or dissolved.

D. Newcomer’s Fluid

Recommended for mucopoplysaccharides, nuclear protein and chromosomes.Fixation Time: 2-3 hours

Preparation:Isopropyl alcoholPropionic acidPetroleum etherAcetoneDioxane

Advantages:1. It is very rapid.2. Tissue require only one charge of absolute alcohol before clearing.

Disadvantages:1. It causes excessive shrinkage.2. Only small pieces of tissues should be used.

- Cytoplasmic Fixatives

A. Flemming’s Fluid (minus acetic acid)

Recommended for cytoplasmic structures.Fixation time: 26-48 hoursAdvantages and disadvantages similar to Flemming’s nuclear fixative.

B. Champy’s Fluid

Recommended for mitochondria, Golgi elements and fats.Fixation time: 24-48 hours

Preparation:3% Potassium dichromate – 7mL1% chromic acid – 7mL2% osmium tetroxide – 4mL

Advantage:Only a small amount of fixative is required, 5-10 times the bulk of the tissue.Disadvantages similar to Flemming’s fluid.

C. Regaud’s Fluid (Moller)

Recommended for mitochondria and yolk.Fixation time: 12 hours

Preparation:3% potassium dichromate40% formaldehyde

Advantage:It is more penetrating than chromium fixatives.Disadvantages:

1. Fat is preserved.2. It needs post-chroming.

D. Orth’s Fluid

Recommended for study of early degenerative process and tissue necrosis.Fixation time: 36-72 hours

Preparation:2.5% potassium dichromateSodium sulfate (optional)Strong 40% formaldehyde (to be added prior to use)

Advantages:1. It demonstrates Rickettsia and other bacteria.2. It preserves myelin better than buffered formalin.

Disadvantages similar to Regaud’s Fluid.

DEHYDRATION

Dehydration is the process of removing extracellular and intracellular water from tissue following fixation and prior to wax infiltration.

Characteristics of an Ideal Dehydrating Agent:1. It must dehydrate tissue rapidly without producing considerable shrinkage or distortion.2. It should not evaporate very fast.3. It should be able to dehydrate even fatty tissues.4. It should not harden the tissue excessively.5. It should not be toxic to the handler.

As a general rule, whatever dehydrating agent is used, the amount should not be less than 10 times the volume of the specimen in order to ensure complete penetration of the tissue.

The three solutions commonly used for this purpose are alcohol, acetone and dioxane.

1. Alcohol Method – the use of ethyl alcohol, methyl alcohol, butyl and isopropyl alcohol are recommended for routine dehydration of tissues. Butyl alcohol and isopropyl alcohol maybe utilized too but is a slow dehydrating agent.

The alcohol method consists of passing the tissue through a series of progressively more concentrated alcohol baths. The more delicate the tissues, the lower is the grade of alcohol

suitable for commencing dehydration and the smaller the intervals should be between the strengths of the ascending alcohols.

2. Acetone Method – this is used for the most urgent biopsies. Only small pieces of tissues should be treated and dehydration takes from ½ to 2 hours. Considerable shrinkage is produced during the process, rendering it unsuitable for routine work.

3. Dioxane Method (Diethylene Dioxide) – this is a unique reagent, which has the property of being miscible with both water and molten paraffin wax. It produces very little shrinkage and is simple to use. These advantages offset by the highly toxic vapor, the high cost of the reagent and the fact that sections are prone to fall out of the surrounding wax. Dioxane should be used only in a well-ventilated laboratory and any residue should be washed down the sink. Dioxane is an excellent dehydrating and clearing agent.

4. Tetrahydrofuran (THF) – this is used as both dehydrating and clearing agent of tissues since it is miscible both in water and in paraffin. It can dissolve many substances including fats and is in itself miscible with lower alcohols, ether, chloroform, acetone, benzene and xylene.

It causes less shrinkage and easier cutting of sections with fewer artifacts. It does not dissolve out amiline dyes. In fact, most staining procedure gives improved results with tetrahydrofuran.

5. Cellulose (Ethylyne Glycol Monoethylether) – this is used as a dehydrating agent because of its rapid action without producing overhardening and distortion of tissues.

6. Tri-ethyl Phosphate – this is used as dehydrating agent as it removes water very rapidly and produces very little distortion and hardening of the tissue. It is soluble in alcohol, ther, benzene, chloroform, acetone and xylene. It produces minimum tissue shrinkage.