Table of Contents

Chemical or Physical Agents which are used to prevent and preserve the tissue are called fixatives.

Types of Fixatives:

Physical Agents:

  1. Heat
  2. Ultra Violet Rays
  3. Microwaving
  4. freeze-drying

Chemical Agents:

  1. Coagulants
  2. Non Coagulants

Classification of Fixative:

On the basis of working condition

  • 95% ethyl alcohol best fixative for cytology
  • Formalin best fixative for histopathology

Hopwood Classification of Fixatives:

1.  Aldehyde Fixatives:

  • Formaldehyde Fixatives
  • Gluteraldehyde Fixatives
  • Acrolein
  • Glyoxal

2.  Oxidizing Agents:

  • Osmium tetroxide
  • Potassium permanganate
  • Potassium dichromate

3.  Protein Denaturing Agents:

  • Acetic Acid
  • Methyl Alcohol
  • Ethyl Alcohol

4.  Cross Linking Agents:

  • Carbodiimides
  • Dimethyl Subrimidate

5.  Unknown Mechanism/Miscellaneous:

  • Mercuric Chloride
  • Picric Acid (Explosive when dry or when complexes with metal and metallic salts)


  • Aldehydes include formaldehyde (formalin) and gluteraldehyde.
  • Tissue is fixed by cross-linkages formed between the proteins particularly between lysine residues.
  • Only those lysine residues which are on the exterior of protein molecule react.
  • The reaction with formaldehyde is largely reversible by excess of water especially over the first 24 hour of reaction time.
  • With gluteraldehyde the reaction is rapid and irreversible.
  • Reaction between aldehydes and proteins is pH dependent proceeding more rapidly at high pH.
  • Fixation denatures proteins to some extent which is acceptable and satisfactory for routine pathology work.
  • For enzyme histochemistry, immunocytochemistry and electron microscopy this issue should be considered.
  • Enzyme molecules and antigenic sites must not be altered or destroyed by chemical fixation.
  • Shapes of DNA and RNA should not be changed.
  • Gluteraldehyde causes loss of up to 30% of αhelix structure of protein and formaldehyde markedly less.
  • Formaldehyde does not give as good morphological picture as gluteraldehyde.
  • Cross linkage does not damage the arrangement of proteins significantly therefore antigenicity is not vanished.
  • Therefore, formaldehyde is good for immunoperoxidase techniques. Formalin penetrates tissue well, but is relatively slow.
  • Normal solution is about ten (10%) percent neutral with buffered formalin solution.
  • A buffer stops acidity which stimulate autolysis and reason for precipitation of formol haem pigment in the tissues.
  • Formaldehyde in its 10% neutral buffered form.
  • Neutral Buffered Formalin (NBF) is the best fixative in diagnostic pathology labs.
  • Pure formaldehyde is a vapor which when completely dissolved in water forms a solution containing 37–40% formaldehyde; this aqueous solution is known as ‘formalin’.
  • The typical 10% formalin used in the fixation of different histopathological. This may consists of four (4%) percent weight/volume of formal.

Formaldehyde Fixatives:

  • 37% Formaldehyde is available commercially and should be stored at room temperature in close and air tight container
  • Clear, colorless and no precipitates
  • Storage should not be more than 1 year
  • Already opened bottles should not be used more than six months
  • 10% formalin should not be used more than 3 months.

Other variants of formaldehyde containing fixatives:

  • Mostly in lab formaldehyde buffered to pH 7 with phosphate buffer is used.
  • Use of neutral buffered formalin stops formation of formalin pigment which forms in non-buffered formaldehyde solutions.
  • For lipids formol calcium is used.
  • Alcoholic formalin has the advantage of rapid fixation and especially recommended for glycogen.
  • They are carcinogenic.
  • PPE should be wear during working with formalin.


  • Gluteraldehyde causes deformation of alpha-helix structure in proteins so it is not good for immunoperoxidase staining.
  • Though, it fixes very rapidly and excellent for electron microscopy.
  • It infiltrates/penetrate very poorly and unwell, but provides finest cytoplasmic and nuclear features.
  • The standard solution is a 2% buffered Gluteraldehyde
  • Rapid irreversible reaction have led to its use as a sterilizing agents of fiberoptic endoscope

Mercuric Chloride Containing Fixative:

  • These fix tissue by an unknown mechanism.
  • Examples include fixatives as Zenker, Susa and Helly’s fluid.
  • These fixatives penetrate poorly and cause some tissue shrinkage so mostly used in combination of other fixatives or as secondary fixatives.
  • Their best application is for fixation of hematopoietic and reticuloendothelial tissues and fetal brain
  • They comprise mercury so should be dispose of sensibly.
  • Mercuric chloride is significantly preferred for its abilities of improving the staining qualities of tissues, mostly for trichrome stains.
  • Tissues fixed with mercuric chloride fixatives (except susa) contain black precipitate
  • This is removed by treating in alcoholic iodine soln. and then decolorizing in sodium thiosulfate.

Alcohol Containing Fixative:

  • Alcohols, as well as methanol and ethanol, are protein denaturants and are not suitable for daily basis for tissues because they create too much brittleness/fragility and hardness/rigidity in the tissues.
  • They are excellent for cytological smears because their action is rapid and provide maximum nuclear details and features.
  • Spray cans of alcohol fixatives are promoted to labs perform PAP smears but inexpensive hairsprays do fair as well.
  • Recommended for preservation of glycogen
  • Example include carnoy’s fixative

Oxidizing Agents Fixatives:

  • Oxidizing mediators/agents contain permanganate fixatives for example potassium permanganate, dichromate fixatives such as potassium dichromate, and osmium tetroxide.
  • Few of them have specific applications but are used very rarely.
  • Some of them are identified according to their appliance of action but osmium tetroxide make crosslinking with proteins.

Picric Acid Containing Fixative:

  • They have an unknown mechanism of action.
  • Examples include Bouin’s, gendre’s and rossman’s fluid.
  • Picric acid has ability of burst/explosive danger in dry its form.
  • Yellow color may be removed but another acid dye or lithium carbonate or washing with changes of 50-70% alcohol

Reaction of fixatives of proteins:

  • In fixation of tissues for routine histopathology the most important reactions are those which stabilize proteins
  • Proteins comprise more than 50% of dry body weight
  • The principle involved is that the fixative have the property of forming crosslinks with proteins forming a gel which provides strength and keeping everything as close to their in vivo state as possible
  • Soluble(globular) proteins are fixed to structural(fibrous) proteins and rendered insoluble and whole structure is given mechanical strength allowing subsequent preparations to take place
  • Fixation normally takes place in aqueous solution. Occasionally vapors can be used.

Reaction of fixatives with Nucleic Acids:

  • Fixation bring about changes in physical and chemical state of DNA & RNA
  • In their native state DNA & RNA do not react with formaldehyde at room temperature
  • Reaction mixture are heated 450C for RNA and 650C for DNA
  • High temperature relates to the uncoiling of DNA & RNA
  • Up to 30% of nucleic acids are lost during fixation
  • Alcoholic fixatives like ethanol, methanol and carnoy’s fixative are commonly used
  • DNA will collapse at 65% conc. Of ethanol

Reaction of fixatives with Lipids:

  • They are largely removed during tissue preparation
  • If lipid are to be demonstrated specifically frozen sections or cryostat should be used
  • Formol calcium is the choice of fixative

Reaction of fixatives with Carbohydrates:

  • Alcoholic fixatives are the fixative of choice for glycogen.



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