Fixation

1
Fixation

• Introduction Shortly after death or removal from
  the body, cells and tissue begin to undergo
  changes, which results in their breakdown and
  ultimate destruction. These are referred to as
  post- mortem changes, which may be either
  putrefactive or autolytic in nature.
• Putrefaction is due to invasion of cells or
  tissue by bacteria/fungal which generally
  disseminate from alimentary tract and spread
  quickly into surrounding organs.
• Autolysis is due to the action of enzymes from
  the dead cell. This phenomenon occurs chiefly in
  the central nervous system and the endocrine
  system.
• These changes may be retarded by low temperatures
  or prevented by the use of chemical fixatives.

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Fixation contd.

• Fixation is the basis of histological technique
  and the results of all subsequent procedures
  depend upon the correct selection and use of the
  fixative employed. It is therefore essential to
  understand the action which different fixatives
  have upon the cell and tissue constituents.
• Definition A fixative may be described as a
  substance that will preserve after death the
  shape, structure, relationship and chemical
  constituents of tissues and cells.

3
Characteristics of a good fixative

• It must kill the cell quickly and in so doing
  should produce the minimum of distortion.
• It must penetrate the tissues and cells rapidly
  and evenly.
• It must render insoluble the substances of cells
  and give food optical differentiation.
• It must inhibit bacterial decay and autolysis.
• It must harden the tissue and render it
  insensitive to subsequent treatment.
• It must permit at a later date the application of
  numerous staining procedures in order to render
  the constituents of the tissues and cells more
  readily visible.

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• Action The action of fixatives is mainly on the
  protein elements of the cells and tissue. The
  preservation should be such that the fixed tissue
  resembles as closely as possible the form which
  it had during life. In addition to preserving the
  tissues and cells, the fixing fluid or vapor must
  also render them insensitive to such subsequent
  treatment which may be necessary for the
  production of the final slide or specimen.
• Temperature has an important effect upon the
  action of fixatives. A low temperature will
  retard the fixation but will also reduce the
  autolytic action of enzymes released after death
  a high temperature will decrease the time
  required in the fixation but will also increase
  autolysis.
• In cases where fixation is not possible until
  some time after death, storage at a low
  temperature (2-5C) is essential.

5
Types of fixatives

• It is necessary to mix together several
  substances, each of which has its own particular
  effect upon the cells and tissue constituents, in
  order to obtain the combined effect of their
  individual actions.
• These individual substances are known as simple
  fixatives, and the solution resulting from a
  mixture of two or more of them are referred to as
  compound fixatives.
• Compound fixatives may thus be described as
  product of two or more simple fixatives mixed
  together in order to obtain the combined effect
  of their individual actions upon the cell and
  tissue components.

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• Fixatives are usually grouped under headings
  according to their action upon the cell and
  tissue constituents. Those which preserve the
  tissue in a manner which permits the general
  microscopical study of the tissue structures and
  allows the various layers of tissues and cells to
  retain their former relationship with each other
  are termed micro-anatomical fixatives.
• Those which are employed for their specific
  action upon a specific part of the cell
  structures are termed cytological fixatives which
  are further subdivided into nuclear and
  cytoplasmic fixatives, depending upon which of
  the cell inclusions they act on.
• Generally speaking those cytological fixatives
  which contain glacial acetic acid or have a
  reaction of pH 4.6 or less are nuclear fixatives,
  while those which do not include glacial acetic
  acid as a constituent and have a pH at the
  critical level of 4.6 are cytoplasmic fixatives.

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• Some simple fixatives are used to preserve
  certain cell products for histochemical
  demonstrations. Chief among these are cold
  acetone (0-5C) and formol-saline buffered to a
  pH of 4.6. The cold acetone is used when it is
  required to demonstrate phosphatases buffered
  formol-saline permits the majority of
  histochemical procedures to be performed.
  Absolute ethanol may also be used as a
  histochemical fixative, usually on sections cut
  from freeze-dried material or which were prepared
  by a use of a cryostat.
• To preserve an accurate picture of the cell, its
  necessary to fix the tissue as soon as possible
  after it is removed from the body. The specimen
  is immersed in a large volume of the fixative
  (50-100 times). In the case of chrome-osmium
  fixatives, e.g. Flemmings fluid, the volume need
  only be ten times that of the tissue.

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Simple Fixatives (Action and properties)

• Formaldehyde (HCHO) It is a gas produced by the
  oxidation of methyl alcohol and is soluble in
  water to the extent of 40 by weight. Fats and
  mucin are preserved but not precipitated by HCHO.
  It is a powerful reducing agent, but is often
  used irrationally in conjunction with certain
  oxidizing agents e.g. Zenker-formol. After
  prolonged storage, HCHO often develops a white
  deposit of para-formaldehyde which can be avoided
  by storage at low temperatures. Its presence does
  not impair the fixing properties of HCHO. The
  solution is usually acid in reaction due to the
  formation of formic acid. Though not harmful,
  this acid can be neutralized by the addition of
  small quantity of magnesium carbonate or a few
  drops of sodium hydroxide. Care should be taken
  when mixing magnesium carbonate, as carbon
  dioxide may be released suddenly.

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• Mercuric chloride (HgCl2)
• Mercuric chloride is included in many
  fixatives and is frequently used as saturated
  aqueous solution (at RT its solubility in water
  is 7 approx.). It precipitates all protein but
  does not combine with them and penetrates and
  hardens the tissues rapidly. Fixatives
  containing HgCl2 leave black ppt in the tissue,
  and this must be removed by Lugols iodine and
  sodium thiosulphate after sectioning the
  processed tissue.
• Osmium tetroxide(OsO4)
• Osmium tetroxide, commonly known as osmic acid,
  is a yellow powder which dissolves in water up to
  6 at RT. It is strong oxidizing agent, volatile
  and easily reduced in day light. It should be
  therefore kept in a dark, chemically clean
  bottle. Prolonged exposure to this acid should be
  avoided since the black oxide OsO2, can become
  deposited in the cornea, resulting in blindness.

10
OsO4 contd.

• Although an expensive reagent, Osmium tetroxide
  is widely used in cytological fixatives. it is
  the only substance that permanently fixes the
  fat, rendering it insoluble during subsequent
  treatment with alcohol and xylene. The Golgi
  element and mitochondria are also preserved. OsO4
  is seldom alone used as a fixative, its usually
  combined with a chromium salt. After fixation in
  these solutions, tissue should be washed in
  running tap water. It is a poor penetrating
  agent, suitable only for small pieces of tissue.
  The vapour of OsO4 may be used to fix some
  tissue, such as the adrenal. The vapour
  penetrates better than solution, washing out is
  unnecessary and production of artifacts is
  minimized.

11
Picric acid and Chromic acid

• Picric acid (C6H2(NO2)3OH)
• It is normally used as saturated aqueous
  solution (i.e. approx 1), It precipitates all
  proteins and combines with them to form picrates.
  These picrates are soluble in water, and the
  tissue must not come in contact with water until
  the picrates have been rendered insoluble by
  treatment with alcohol.
• Chromic acid
• It is prepared by dissolving crystals of
  anhydride CrO3 in distilled water, and is
  conveniently stored as a 2 stock solution. It is
  a strong oxidizing agent and should not be
  combined with reducing agents, such as alcohol
  and formalin. It is a strong protein precipitant,
  and preserves carbohydrate. Tissue fixed in
  chromic acid should be thoroughly washed in
  running tap water before dehydration, to avoid
  the formation of a precipitate of the insoluble
  sub-oxide.

12
Potassium dichromate

• Potassium dichromate (K2Cr2O7) is one of the
  oldest and most widely used of the simple
  fixatives. Two entirely different forms of
  fixation can be produced, depending on pH of the
  solution. At a more acid reaction than pH 4.6 the
  results are similar to those of chromic acid (
  i.e. Protein precipitant and carbohydrate
  preserver). At a more alkaline reaction than pH
  4.6, the cytoplasm is homogenously preserved and
  the mitochondria fixed.
• One of the most important properties of pot.
  dichromate is its strong fixative action on
  certain lipids. It is ideal fixative in the study
  of myelinated nerve fibres. Tissue preserved in
  this solution must be thoroughly washed in
  running tap water prior to dehydration.

13
Acetic Acid (CH3COOH)

• It is a colourless solution with a pungent
  smell. At about 17ºC it solidifies, which
  accounts for its name glacial acetic acid.
• It is included in a number of histological
  fixatives. It is a powerful precipitant of
  nucleoprotein. When used alone it causes
  considerable swelling of the tissue, and this
  property is used in certain compound fixatives to
  counteract the shrinkage produced by other
  components. In Heidenhains susa for example
  the shrinkage produced by mercuric chloride is
  reduced by the addition of acetic acid. It is
  often used by cytologist in studying chromosomes,
  and its chromatin-precipitating properties make
  it useful in nuclear studies. It destroys
  mitochondria and Golgi elements, and when used in
  conjunction with potassium dichromate destroys
  the lipid fixing properties of that reagent.

14
Ethyl Alcohol (C3H5OH)

• Ethyl alcohol (C3H5OH) is a colourless liquid
  that is readily miscible with water. It was
  extensively used by early histologists, but today
  its use as a simple fixative is confined to
  histochemical methods and it is also used in
  compound fixatives. It is a reducing agent, and
  should not be mixed with chromic acid, pot.
  dichromate and osmium tetroxide. As a simple
  fixative, it is used at a concentration of
  70-100. It causes considerable hardening and
  shrinkage of the tissue.
• Trichloracetic Acid (CCl3COOH) is sometimes
  incorporated into compound fixatives. It is a
  general protein precipitant but has marked
  swelling effect on many tissues, a property made
  use of to counter the shrinkage produced by other
  simple fixatives. It is also used as a slow
  decalcifying agent and the softening effect which
  it has on the dense fibrous tissue facilitates
  the preparation of sections during section
  cutting.

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Compound Fixatives

• Compound fixatives may be consider under two
  headings
• Micro-anatomical and Cytological.
• Micro-anatomical fixatives are used to preserve
  the various layers of tissue and cells in
  relation to one another, so that general
  structure may be studied.
• Cytological fixatives are subdivided into two
  groups Nuclear and cytoplasmic. They are used
  for preservation of the constituent element of
  the cells, though this often entails the loss of
  the properties of the micro-anatomical fixatives.

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Micro-anatomical Fixatives

• 10 formol-saline
• Recommended for the fixation of central nervous
  system and general
• post-mortem tissue. The period required for
  fixation is more than 24 hrs.
• Composition
• 40 formaldehyde 100 ml
• sodium chloride 9 g
• Dist. water 900 ml
• Advantages
• Excellent for post-mortem samples/ causes little
  shrinkage/indefinite period of sample
  storage/basis of all museum fixatives/restores
  the natural color of specimen/does not interfere
  with most staining procedures.
• Disadvantages
• Slow fixative / tissue shrinkage during
  dehydration / irritant vapour injures the nasal
  mucosa / causes dermatitis with prolonged use /
  pigment is always formed when dealing with blood
  containing sample / metachromatic reaction of
  amyloid is reduced / needs periodic changes of
  new solution by 3 months.

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• 10 Neutral buffered formalin
• Recommended for the preservation and storage of
  surgical, post-mortem and research specimens. The
  period of fixation is 24 hrs or longer.
• Constituents
• Sod. dihydrogen phosphate (anhydrous) 3.5 g
• Disodium hydrogen phosphate (anhydrous) 6.5 g
• 40 formaldehyde 100 ml
• Dist. water 900 ml
• Advantages
• Has the same advantages as formal-saline, but in
  addition it prevents formation of acid formalin
  pigment. No need of periodic changes of solution.
• Disadvantages
• Disadvantages are similar to those listed in
  formol saline, and also take longer time on the
  preparation of this fixatives.

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Heidenhains susa

• This is recommended mainly for biopsies. The
  period of fixation
• required is from 3-24 hrs.
• Composition
• Mercuric chloride 45 g
• sodium chloride 5 g
• Trichloracetic acid 20 g
• Glacial acetic acid 40 ml
• Formaldehyde 40 200 ml
• Dist. water 800 ml
• Advantages Penetrates rapidly, producing good
  and even fixation/causes minimum shrinkage and
  hardening/allows brilliant staining result with
  sharp nuclear details/ large blocks of fibrous
  tissue may sectioned more easily after this
  fixation than after any other/ tissue can
  transfer directly from the fixative to 95 or
  absolute alcohol.
• Disadvantages Slice of tissue should not exceed
  1 cm in thickness for processing of sample. Red
  blood cells are poorly preserved. Some
  cytoplasmic granules are dissolved.

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Formol-sublimate

• This is recommended for routine post-mortem
  samples. The period of fixation required is from
  3-24 hrs, depending on the thickness of the
  tissue.
• Composition
• Saturated aqueous mercuric chloride 90 ml
• Formaldehyde 40 10 ml
• Advantage
• Excellent routine fixatives, and produces no
  hardening or shrinkage of the tissue. Can be
  followed by most staining procedure, including
  silver reticulum methods with excellent result.
  Cytological details and red blood cells well
  preserved/ tissue can be transferred directly
  from fixative to 70 alcohol.
• Disadvantage
• Slices of tissue should not exceed 1 cm in
  thickness.

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Zenkers Solution

• This is recommended for the fixation of small
  pieces liver and spleen. The period of fixation
  required is from 12-24 hours.
• Constituents
• Mercuric chloride 5.0 g
• Potassium dichromate 2.5 g
• Sodium sulphate (Optional) 1.0 g
• Dist. Water 100 ml
• Add 5 ml of glacial acetic acid just before use.
• Advantages Zenkers solution permits excellent
  staining of nuclei and connective tissue fibres
  and is recommended particularly for tissues
  which are to be stained by trichrome techniques.
• Disadvantages poor penetration / tissue pieces
  should not exceed 0.5 cm in thickness / makes
  brittle if the tissue kept more than 24 hrs and
  must be wash for several hrs in running tap
  water/ not recommended for frozen sections /
  solution does not keep well after the addition of
  acetic acid.

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Zenker-formol (Hellys)

• This is recommended for the fixation of pituitary
  tissue and bone marrow.
• Constituents
• Mercuric chloride 5.0 g
• Potassium dichromate 2.5 g
• Sodium sulphate (Optional) 1.0 g
• Dist. Water 100 ml
• Add 5 ml of 40 formaldehyde just before use.
• Advantages Although the fixative contain both
  oxidizing and reducing agents, it produces
  excellent nuclear fixation. Staining of nuclei is
  even more intense than after fixation with
  Zenkers solution. Cytoplasmic granules are well
  preserved.
• Disadvantages same as listed in zenkers
  solution. If material is allowed to remain in the
  fixative for longer than 24 hours, a brown scum
  is produced on the tissue.

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Bouins solution

• This is recommended for the fixation of embryos.
  The period of fixation required is from 6-24
  hours.
• Constituents
• Saturated aqueous picric acid 75 ml
• Formaldehyde 40 25 ml
• Glacial acetic acid 5 ml
• Advantages
• Produces very little micro-anatomical distortion
  and permits brilliant staining results. Tissue
  should not be washed in water, but transferred
  directly from fixative to 70 alcohol. It is good
  preservatives of glycogen.
• Disadvantages poor penetration so not
  preferable for big biopsy.

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Gendres Fluid

• A general micro- anatomical fixatives which is
  also widely used for the preservation of
  glycogen.
• Constituents
• Glacial acetic acid 5 ml
• Picric acid saturated solution in 95 alcohol 80
  ml
• Formaldehyde 40 15 ml
• Advantages Similar to Bouins solution, but the
  combined action of both alcohol and high picric
  acid content make it an excellent fixative for
  glycogen. When fixation is complete the tissue is
  washed several changes of 80 alcohol.
• Disadvantages
• These are similar to those listed under Bouins
  fluid.

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Cytological fixatives

• Cytological fixatives are usually divided into
  two groups
• Nuclear fixatives Cytoplasmic fixatives
• 1.Flemmings fluid 1. Flemmings fluid without
  acetic acid
• 2.Carnoys fluid 2. Hellys fluid
• 3. Formalin with post chroming

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Nuclear fixatives

• Flemmings fluid
• This fixative is recommended for the preservation
  of nuclear structure. The period of fixation is
  from 24-48 hours.
• Constituents
• Chromic acid (1) 15 ml
• Aqueous osmium tetroxide (2) 4 ml
• Glacial acetic acid 1 ml
• Advantages Excellent fixative for nuclear
  elements, especially for chromosomal study and it
  is the only fixative which permanently preserves
  the fat / Only 10 times greater amount of
  fixative sufficient than the tissue volume.
• Disadvantages poor penetration power / costly /
  used for small pieces of tissue/ the solution
  deteriorates rapidly, and must be prepared before
  only / tissue should be washed for 24 hrs prior
  to dehydration.

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Nuclear fixatives

• Carnyos fluid
• This is recommended for fixing chromosomes, lymph
  glands, and urgent biopsies. The period of
  fixation required is from 30 minutes to 3 hours.
• Constituents
• Absolute alcohol 60 ml
• Chloroform 30 ml
• Glacial acetic acid 10 ml
• Advantages permits good nuclear stain / fixes
  rapidly and dehydrates, and is there useful for
  biopsy material / glycogen is preserved /
  following tissue transferred directly to absolute
  alcohol.
• Disadvantages causes excessive shrinkage by this
  solution and is only suitable for small pieces of
  tissue. Red cells are haemolysed.

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Cytoplasmic fixatives

• Flemmings fluid without acetic acid
• This is recommended for mitochondria and the
  period of fixation required is from 24-48 hours.
• Constituents Flemmings fluid but omitting the
  acetic acid.
• Advantages
• Excellent fixative for nuclear elements,
  especially for chromosomal study and it is the
  only fixative which permanently preserves the
  fat. Only 10 times greater amount of fixative
  sufficient than the tissue volume.
• Disadvantages
• Has poor penetration power / costly / used for
  small pieces of tissue/ the solution
  deteriorates rapidly, and must be prepared before
  only / tissue should be washed for 24 hrs prior
  to dehydration.

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Cytoplasmic fixatives

• Hellys fluid
• This is synonymous with zenkers- formal and
  can be used as a cytoplasmic fixatives.
• 10 Formol saline
• Fixation in 10 formol- saline, followed by the
  post chroming of tissue in 3 potassium
  dichromate for 3-5 days, permits good cytoplasmic
  staining , particularly of mitochondria.

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The fixation of gross specimen

• It is often necessary to fix specimens of entire
  organs. This may be done with 10 formol-saline,
  or with one of the museum fixatives consisting of
  formaldehyde in conjunction various acetates,
  such as wentworths solution.
• Constituents
• sodium acetate 40 g
• Formaldehyde 100 ml
• Dist. water 900 ml
• The techniques varies with the organ to be
  preserved. But this brief notes will act as
  guide. Further details can be found in
  histological museum Techniques.

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Secondary fixation

• Following fixation with formol-saline it is
  sometimes advantageous to refix the tissue for
  further 4 hours in a second fixative. The
  fixatives usually selected for this purpose are
  formol-sublimate, Zenkers-formol, and
  Heidenhains susa. This procedure, which is
  known as secondary fixation. It is the advantage
  of imparting a firmer texture to the tissue and
  in many condition it facilitate the processing of
  tissue and improve the qualities of stain.
• Post- Chromatization In order to facilitate
  certain staining procedures fixed tissue or
  section can be immersed in 3 potassium
  dichromate for several hours prior to staining
  This procedure is known as Post- Chromatization
  or post-chroming, and is used with tissue fixed
  mainly in formol-saline.
• The purpose is to mordant the tissue.

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Washing out

• Reference has been made to the washing of tissue
  in running tap water, after certain fixatives
  have been used. This may be done in several way,
  but whatever the method, it is important to
  ensure that the specimen is bathed in constant
  stream of fresh water. It is important that
  neither the tissue or the accompanying label are
  washed out of the container.
• It is important to make sure that the surrounding
  the tissue is constantly being changed,
  preferably by means of syphon system. Failure to
  observe this point may result in the fixative
  being insufficiently removed from the tissue.
• The purpose of washing tissue in running water is
  to remove oxidizing agent such as potassium
  dichromate and osmium tetroxide to prevent
  reduction when coming to contact with the
  alcohol.
• It is also important to remove all traces of
  formaldehyde from tissue to be embedded in
  gelatin.
• The end