Microanatomical,Cytological and Histochemical fixatives

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Some fixatives are made by combining one or more fixative so that the disadvantage of one are reduced by use of another fixative.These types of fixatives are known as compound fixatives.All these compound fixatives have their own advantages and disadvantages and should be used judiciously.Fixatives are divided into three main groups which are Microanatomical fixatives,Cytological fixatives and Histochemical fixatives.

Microanatomical Fixatives

These are fixatives that preserves the anatomy of the tissue fixed.

Cytological fixatives

These are fixatives that preserves the intracellular structures or inclusions of tissues

Histochemical Fixatives

These fixatives are used to preserve the chemical nature of the tissues.Freeze drying technique  is a good example for this purpose

1.Microanatomical Fixatives

Formal saline(Formalin fixation)

It is one of the most widely used fixative in histopathology. In formalin fixative,the tissue can be left for long period without causing excessive hardening or damage. Those tissues which are  fixed for a longer period of time in formal saline occasionally contain a pigment know as formalin pigment.

This pigment may be removed in sections before staining by treating the tissue with picric alcohol or 10% alcoholic solution of sodium hydroxide. Also the formation of formalin pigment can be prevented by buffering or neutralizing the formal saline.

Composition of formal saline

Formaldehyde (40%)100 ml
Sodium Chloride9 grams
Distilled Water900 ml
Formal saline fixation time24 hours at room temperature

Composition of 10% neutral Buffered Formalin

As earlier mention,Buffer formalin fixative has the advantage preventing  the formation of formalin pigment.the following reagents are used for the preparation of 10% formalin solution

Formaldehyde (40%)10 ml
Sodium dihydrogen phosphate 0.4 grams
Disodium hydrogen phosphate (anhydrous)0.65 grams
Distilled water90 ml

Formal calcium

This fixative is useful for demonstration of phospholipids in tissues.It has a near neutral pH and formalin pigment is not formed

Composition of Formal calcium

Formalin 10 ml
Calcium acetate2 grams
Distilled water100ml
Formal calcium fixation time24 hours at room temperature

Heidenhain susa

  • It is an excellent fixative for routine biopsy work.
  • It allows brilliant staining and provide good cytological details of the tissue
  • It also rapidly and evenly penetrates the tissue with minimum shrinkage
  • Disadvantages is that tissue left in it for over a period of 24 hours becomes bleached and excessively hardened
  • Also forms mercury pigments which can be removed by treating it with iodine
Also read  Properties and Principles of Hematoxylin & Eosin staining technique

Composition of Heidenhain susa

Mercuric chloride4.5 grams
Sodium Chloride0.5 grams
Trichloroacetic acid 2.0 grams
Acetic acid4.0 grams
Distilled water 100 ml

Zenker’s fluid

Zenker’s fluid fixative contains potassium-dichromate,mercuric chloride, glacial acetic acid and sodium sulphate.

  • The advantage of using Zenker’s fluid is that,it fixes rapidly with an even penetration

Disadvantage is

  • It is not stable after the addition of acetic acid hence,acetic acid or formalin should be added just before use
  • Also after fixation with Zenker’s fluid,the tissue must be washed in running water in order to remove excess dichromate and mercury pigment must also be removed with Lugol’s iodine

Composition of Zenker’s Fluid

Mercuric chloride5 grams
Potassium dichromate2.5 grams
Sodium sulphate1.0 grams
Distilled water100 ml
Immediately add before use Glacial acetic acid5 ml

Bouin’s fluid(Bouin’s fixative)

Bouin’s solution of bouin’s fixative contains picric acid, 40% formaldehyde acid and glacial acetic acid and is routinely used for the fixation and preservation of testicular and intestinal biopsies because it gives a very good nuclear details.

  • In testes,It is used for Oligospermia and infertility studies
  • It is also a good fixative for glycogen.
  • It has an advantage in that it also rapidly and evenly penetrate the tissues without any shrinkage
  • It us also necessary to remove excess picric acid on the tissue by treating it with alcohol
  • The staining of tissues (Brilliant staining) fixed in Bouin’s fluid is done by trichrome method.

Composition of Bouin’s Fluid

Saturated picric acid (1.2 gm/ 100 ml) 75 ml
Formaldehyde (40%) 25 ml
Glacial acetic acid 5 ml

Zenker’s formal (Helly’s fluid)

Also know as Helly’s fluid,formalin is added instead of acetic acid when in stock.

  • It is an excellent microanatomical fixative used especially for kidney,bone marrow,blood containing organs and  spleen tissues fixation
  • It is necessary to remove excess dichromate and mercuric acid pigment by washing of the tissue in running water

Composition of Zenker’s Formal (Helly’s fluid)

Mercuric chloride5 grams
Potassium dichromate2.5 grams
Sodium sulphate1.0 grams
Distilled water 100 ml
Then add formalin immediately before use5 ml of formalin

B5 Stock solution

It is widely used for the fixation of lymph node biopsies in order to improve the cytological details and to enhance immunoreactivity with anti-immunoglobulin antiserum used in phenotyping of B cells neoplasm

Also read  Fixation and properties of an ideal fixatives in Histopathology

Composition of B5 Stock solution

Mercuric chloride12 grams
Sodium acetate2.5 grams
Distilled water200 ml

In order to prepared B5 Working solution add 20 ml of B5 stock solution with 2 ml formalin

Gender’s Fluid

A better fixative for glycogen

Composition of Gender’s Fluid

Saturated picric acid in 95 % alcohol80 ml
Formalin15 ml
Glacial acetic acid5 ml

2.Cytological fixatives

It is divided into Nuclear fixatives and Cytoplasmic fixatives

A) Nuclear fixatives

These fixatives gives a good nuclear fixation and includes

Carnoy’s Fluid

  • It rapidly penetrates and gives an excellent nuclear fixation
  • It is a good fixatives for carbohydrates
  • Also glycogen and nissl substance are preserved
  • It causes considerable shrinkages of tissue
  • This fixative dissolves most of the cytoplasmic elements and for small pieces of tissues of thickness 2-3 mm,only 15 minutes is need for xiation

Composition of Carnoy’s Fluid

Absolute alcohol75 ml
Glacial acetic acid10 ml
Chloroform30 ml
Fixative time1-2 hours

Clarke’s Fluid

  • It is a rapid and good nuclear fixative which preserves the cytoplasmic elements
  • It is also an excellent for smear or coverslip preparation particularly of cell culture or chromosomal analysis

Composition of Clarke’s Fluid

Absolute alcohol75 ml
Glacial acetic acid25 ml

B.Cytoplasmic Fixatives

Champy’s Fluid

  • It preserves the mitochondrial fat and lipids but can’t be kept hence freshly prepared before used.
  • It has a poor and uneven penetration
  • Also tissue must be washed overnight after fixation with champy’s fluid

Composition of Champy’s Fluid

3g/dl of potassium dichromate7 ml
1% chromic acid7 ml
2 g/dl osmium tetroxide4 ml

Formal saline and Formal Calcium

The fixation in formal saline the by post chromatization gives a good cytoplasmic fixation

3.Histochemical fixatives

Cryostat is one of  the mostly used histochemical fixation method Here,sections are rapidly frozen or freeze dried.

Such sections are usually unfixed but if delay is inevitable,then vapour fixatives can be used.

Vapour Fixatives

  • Formaldehyde

The vapour is obtained by heating paraformaldehyde solution (heat fixing) or paraformaldehyde fixation  at a temperature between 50° C to 80 °C.Block of tissues to be fixe requires 3-5 hours whereas section requires half and hour to one hour

  • Acetaldehyde

Heated at 80 °C for 1-4 hours to obtain vapour

  • Glutaraldehyde which causes glutaraldehyde crosslinking
Also read  Papanicolaou stains : Principle ,Procedure and Interpretation

50% aqueous solution heated at 80 °C for 2 minutes to 4 hours

  • Acrolein/chromy chloride

Used at temperature of 37 ° C for 1-2 hours

Secondary tissue fixation methods

It is sometimes necessary to submit the tissue for a second fixation like mercuric chloride for a period of 4 hours.This provide a firmer texture to the tissues and gives  brilliance to the staining

Post chromation of tissues

This involves the treatment of tissues with 3% potassium dichromate following a normal fixation.This process  or technique is used as a mordant to the tissues and is carried out either before processing when the tissue is left for 6-8 days in potassium dichromate solution or after processing when the sections are directly immersed in potassium dichromate solution for 12-24 hours.

Follow to Post chromation,washing of the tissues in running water is essential

Washing out of Tissues

After the use of certain tissues,it is urgent and essential that these tissues should be thoroughly washed in running water in order to entirely remove the fixative.

The washing process should be carried out ideally for 24 hours.Mostly those tissues treated with potassium dichromate,picric acid and osmium tetroxide needs to be washed thoroughly with clean water followed by dehydration with alcohol.

Summary of Tissues and fixatives of choice

TissuesFixatives if choice Time required for Fixation
RoutineFormalin10 -12 hours
Gastrointestinal TractBuffered formaldehyde4-6 hours
Testicular biopsyBouin’s fluid fixative4-6 hours
Liver BiopsyBuffered formaldehyde4-12 hours
Bone marrow biopsyBouin’s fluid in running2 hours followed by thorough washing in running water overnight
Spleen and blood filled cavitiesZenker’s fluid1-6 hours
Lymph nodeB512-18 hours
Mitochondria,nissl substances and phosphatidesCarnoy’s fluid1-2 hours
Chromosome/Cell cultureClarke’s fluid1-2 hours

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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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