Killing and Fixation (Fixatives in Histopathology)

Learning objectives: General account of Killing and Fixing of Plant Specimens for Anatomical and Histological Studies; Agents used for killing and fixation – Alcohol, Formalin, Acetic Acid; Common fixatives: Formalin – Acetic – Alcohol (FAA) mixture; Carnoy’s fluid; Farmer’s Fluid; Chromic acid – Acetic acid – Formation (CRAF), Fixatives in Histopathology.

What is killing and fixation?

Ø  Killing is the sudden stopping of all living process in all the cells of a collected biological specimen such as a piece of stem, leaf, flower bud etc.

Ø  Killing agent is the chemical reagent used for killing the plant specimens.

Ø  Fixation is the preservation of all structural and cellular elements in a biological specimen in as near their original state as possible.

Ø  Fixing agent is the chemical reagent used for fixing the plant specimens.

Ø  Usually the killing and fixing are done by a single fluid called Fixative.

Ø  The fixative may be a single chemical reagent of a combination of many reagents.

Ø  “A good fixative is one that changes the cell chemistry the least and preserves the cell structures the best”

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The goal of killing and fixation is to

$. Preserve the cell structures and contents in as natural form as possible.

$. Modify the refractive index of some cellular elements to make them better distinguishable under the microscope.

$. Make the materials resistant and hard to reactions during further treatment in the processing.

$. Prepare the materials to improve upon effects of certain stains.

Significance of killing and fixation

$. Killing and fixation help to harden the tissue and give them consistency.

$. Increases visibility of cell components under the microscope.

$. Prevents the post-mortem changes (bacterial decay and autolysis) in the specimen.

$. Killing and fixation make the plasma membrane insensitive to the changes in the osmotic potential of the extracellular fluids.

$. It also makes the cell contents insoluble in water.

$. Also reduce the shrinkage and distortion of cells.

Reagents in Killing and Fixation

Ø  There is NO single reagent to meet all the requirements of killing and fixation.

Ø  Thus, a combination of many reagents is used.

Ø  Combination of reagents should balance all properties of the reagents involved.

Ø  Two reagents with same the disadvantages should never be combined, like:

(1). Substance which tends to shrink the cytoplasm must be mixed with one which swells the cytoplasm.

(2). Tissue hardening reagents must be combined tissue softening reagents.

(3). Oxidizing reagents should never be combined with reducing reagents.

Fixatives in Histopathology

Ø  Single chemical reagents used in Killing and Fixation

  1.   Ethyl alcohol (ethanol)
  2.   Formalin
  3.   Acetic acid
  4.   Picric acid crystals
  5.   Chromic acid crystals
  6.   Potassium dichromate
  7.   Propionic acid
  8.   Mercuric chloride
  9.   Iodine

(1). Ethyl alcohol (Ethanol)

Ø  Ethyl alcohol is miscible with water.

Ø  The boiling point is comparatively low (78.37oC).

Ø  It is highly inflammable.

Ø  Alcohol is a reducing agent.

Ø  It dissolves fats and lipids of the cells.

Ø  Had very rapid penetration capacity.

Ø  Alcohol shrinks the tissue and hardens very much.

Ø  Precipitate the proteins and nucleic acids in the cell.

Ø  It makes tissue difficult to stain.

(2). Formalin

Ø  Formalin is the trade name of ~40% aqueous solution of formaldehyde.

Ø  It is miscible with water.

Ø  Formalin is a reducing agent.

Ø  It has no effects on lipids.

Ø  Shows slow penetrability.

Ø  Formalin does not cause shrinkage, but cause shrinkage with alcohol.

Ø  Has a very great hardening effect.

Ø  Formalin makes the tissue difficult to stain, except with acid dyes.

Ø  The fumes are irritating to the mucous membranes and the eyes.

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(3). Acetic acid

Ø  Acetic acid is miscible with water.

Ø  Had very rapid penetration capacity.

Ø  It has no hardening effect on cells.

Ø  Acetic acid does not fix the cytoplasm.

Ø  It precipitates the nucleoproteins.

Ø  Acetic acid makes the tissue soft and incapable of being hardened by alcohol.

Ø  It does not affect the staining reaction.

Combination reagents for killing and fixation (Fixatives)

Ø  Different types of fixatives are there according to their ingredients.

Ø  Some formulations are stable, some are unstable.

Ø  Stable combinations can be prepared, stored and can use at any time.

Ø  The unstable formulations have to prepare fresh every time.

Ø  Some can be stored at room temperature while others have to be stored in the refrigerator.

Ø  Some formulations should be protected from light (should be stored in amber coloured bottle).

Ø  The names of fixatives are usually based on:

$.  Names of investigators who first developed it

$.  Major ingredients in the combination

Examples for Fixatives in Histopathology

(I). Acetic acid – Alcohol mixtures

(a).    Carnoy’s Fluid

(b).    Farmer’s Fluid

(II). Formalin – Acetic acid – Alcohol mixtures (FAA)

(a).    Rawlin’s formula (FAA)

(III). Chromo-Acetic acid-Formalin mixtures

(a).    Navaschin’s formula (CRAF)

(I). Acetic acid – Alcohol mixtures

(a). Carnoy’s Fluid

Preparation of Carnoy’s Fluid (for 30 ml)

Absolute alcohol       :           10 ml

Chloroform                :           15 ml

Glacial acetic acid      :           5 ml

Ø  Carnoy’s fluid is an acetic acid – alcohol mixture.

Ø  It is the best preparation for cytological preparations such as for mitosis and meiosis study (root tips, anthers etc.).

Ø  Nuclear and chromosomal features will be best preserved.

Ø  The fixation time 10 to 15 minutes.

Ø  After fixation, wash in 85% alcohol, Store in 85% alcohol solution.

(b). Farmer’s Fluid

Preparation (20 ml)

      Glacial acetic acid            :           5 ml

      Absolute alcohol              :           15 ml

Ø  A good fixative for cytological studies

Ø  Fixation time is 15 minutes for root tips and 1 hour for anthers

Ø  After fixing wash and store in 70% alcohol

(II). Formalin-Acetic Acid-Alcohol Mixture (FAA)

Rawlin’s Formula of FAA

Preparation: (for 100 ml)

95% Ethyl alcohol     :           50 ml

Glacial acetic acid     :           5 ml

Formalin                     :           10 ml

Water                          :           35 ml

Ø  Rawlin’s FAA is best for the materials for histological studies.

Ø  Good for algae, bryophytes and such delicate materials.

Ø  Has good hardening action on specimens.

Ø  The materials fixed in Rawlin’s FAA can be stored for many years.

Ø  Fixation time :18 hours

Ø  A lower percentage of alcohol may be used for fixing delicate materials.

Ø  For woody materials, decrease the acetic acid and increase formalin content.

Ø  After fixation, wash in alcohol and store in alcohol.

(III). Chromo-Acetic-Formalin Mixture (CRAF)

Ø  Most common CRAF is Navaschin’s Formula.

Ø  Navaschin’s CRAF formula contains two solutions, Solution A and Solution B


            Solution A:      Chromic acid 1%         :           15 ml

                                          Glacial Acetic acid      :           10 ml

                                          Distilled water            :           90 ml

            Solution B:     Formalin                      :           40 ml

                                         Distilled water            :           60 ml

Ø  Mix equal quantities of Solution A and Solution B just before use.

Ø  Fixation time 12 hours.

Ø  Washing is not required.

Ø  Navaschin’s original formula has been modified by many workers.

Ø  They are named as CRAF-I, CRAF-II and so on.

(I). CRAF-I Preparation

Chromic acid 1%       :           20 ml

Acetic acid 1%           :           75 ml

Formalin                    :           5ml

(II). CRAF-II Preparation

Chromic acid 1%       :           20 ml

Acetic acid 10%         :           10 ml

Formalin                    :           5 ml

Distilled water          :           65 ml

Ø  Both CRAF-I and CRAF-II should be prepared just before use

Ø  After the addition of formalin, a colour change takes places (in 2-3 hours).

Ø  Subsequently the colour changes to olive green or deep green.

Ø  Once the green colour is developed, the solution becomes a fixative.

Ø  Fixation time: 12 hours

Ø  Washing in water is not required.

The Technique of Fixing

Ø  Materials should be fixed as soon as possible after the collection.

Ø  If possible, the material should be fixed at the collection point itself.

Ø  Should know the properties of fixing reagent.

Ø  Select proper fixative according to the requirement.

Ø  Larger specimens should be cut into small pieces.

Ø  Place the specimen in a bottle with a cap.

Ø  Add the required quantity of fixative.

Ø  Specimens should sink in the fluid (use aspirator if necessary).

Ø  Required incubation time should be provided.

Ø  Wash after fixing, use proper preservatives.

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