Histology COMPOUND FIXATIVES

 COMPOUND FIXATIVES

2.10 COMPOUND FIXATIVES

The compound fixatives are divided into Micro-anatomical and Cytological Fixatives. 

2.10.1Micro-anatomical Fixatives

2.10.1.1 Formol-Saline 10%:

Formal-saline is a miniature physical fixative, yet entirely not a compound one. It is recommended

for fixation of CNS material. It is very safe and is the basis of all museums fixatives

because it is the only fixative that allows the natural colour to be restored to the specimen. 

This is a slow fixative and tissues fixed in it are liable to shrink during dehydration in

alcohol. Rubber gloves must be worn when handling specimen fixed in formal-saline.

Formula

Formalin 40% 100 ml

NaCl 8.5 g

Distilled water 900 ml

2.10.1.2 Neutral Buffered Formalin 10%:

This is recommended for storage of surgical and research specimens. The period of fixation

is 24 hours or longer. 7KLV IL[DWLYHGRHVQ¶WJLYHEURZQSLJPHQWto tissues because formic

acid formation is inhibited. It is also recommended for bone marrow biopsies where cellular

details are of importance. It is safe and fixes the bone marrow biopsy smoothly. However, it

cannot be used in routine busy laboratories because it is costly and difficult to prepare.

Formula

Sodium dihydrogen phosphate 3.5g NaH2PO4

Disodium hydrogen phosphate 6.5g Na2HPO4

Formalin 40% 100ml 

Distilled water 900mllkjb bjp, ?

2.10.1.3 Alcoholic Formaldehyde 10%:

The advantage of using this fixative is that the dehydration process in also initiated, so

tissues can directly be taken to absolute alcohol rather than taking them to lower

concentration of alcohol. The formula involves the mixing of two fixatives.

Formula

Formalin 40% 100ml

95% Alcohol 900ml

Calcium Acetate 0.05 g (for neutrality)

2.10.1.4 Formol Calcium:

This fixative is recommended for lipid fixation.

Formula

Formalin 40% 100ml

CaCl2 10% 100ml

Distilled water 900ml

2.10.1.5 Formol Sublimate:

This is recommended for routine post-mortem material. 7KHF\WRORJLFDOGHWDLOVDQG5%&¶V

are well preserved. No hardening or shrinkage is cause by this fixative.

Formula

Saturated HgCl2 900ml

Formalin 40% 100ml

2.10.1.6 %RXLQ¶V Fluid:

It is a compound fixative of picric acid. It is poorly penetrating fixative.

Formula

Saturated picric acid 75ml

Formalin 40% 25ml

Glacial acetic acid 05ml

Advantages:

i. This fixative fixes nuclei properly due to presence of glacial acetic acid.

ii. %RXLQ¶VIOXLGimparts a yellow colouration to tissues which is helpful in handling of

tiny fragmentary biopsies, e.g. rectal biopsy or needle biopsy from kidney. 

16

iii. It is also recommended for glycogen demonstration.

iv. It is also recommended for delicate tissue biopsies, e.g. embryos or testicular biopsy

2.10.1.7 *HQGUH¶V)OXLG:

This fixative is widely used for glycogen preservation because of the combined effect of

picric acid and alcohol. 

Formula

Glacial Acetic Acid 5ml

Picric acid in 95% alcohol 80ml

Formalin solution 40% 15ml

2.10.1.8 =HQNHU¶V6ROXWLRQ:

This is recommended for the fixation of small pieces of liver and spleen.

Formula

Mercuric Chloride 5g

Potassium dichromate 2.5g

Sodium Sulphate 1.0g (optional)

Distilled water 100ml

Add 5ml of icy acidic corrosive not long before use.

Merits:

i. It is an excellent fixative for nuclei and of connective tissue fibers.

ii. It is recommended for tissues which are to be stained by one of the trichrome

techniques.

Demerits:

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ii. Prolonged immersion causes brittleness.

iii. It is not recommended for frozen sections.

2.10.1.9 Zenker-Formol:

This fixative is recommended for pituitary tissue and bone marrow. It gives excellent

nuclear fixation and cytoplasmic organelles are well preserved.

Formula

Mercuric Chloride 5.0g

Potassium dichromate 2.5g

Sodium Sulphate 1.0g (Optional)

Distilled water 100ml

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2.10.2 Cytological Fixatives

The cytological fixatives are divided into two groups as follows:

Nuclear Fixatives Cytoplasmic Fixatives

i. FlemPLQJ¶V Fluid L )OHPPLQJ¶V Fluid without Acetic

Acid

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iii. Formalin with Post Chroming

2.10.2.1 )OHPPLQJ¶V)OXLd:

This fixative is recommended for the preservation of nuclear structure, e.g. chromosomes. It

is the only fixative which can permanently preserve fats. This fixative is relatively costly

but small volumes are required for fixation. The fixative is poorly penetrating so only be

used for small biopsies. It must be prepared immediately before use because it deteriorates

rapidly. 

Formula

Chromic acid 1% 15ml

OsO4 Aqueous 04ml

Glacial Acetic Acid 01ml

2.10.2.2 &DUQR\¶V)OXLG

This fixative is best for lymph glands and urgent biopsies but excessive shrinkage is caused

E\WKHVROXWLRQDQG5%&¶VDUHDOVRKDHPolyzed.

Formula

Absolute alcohol 60ml

Chloroform 30ml

Glacial acetic acid 10ml

2.10.2.3 )OHPPLQJ¶VFluid without Acetic Acid:

It LVUHFRPPHQGHGIRUPLWRFKRQGULD7KHIRUPXODLVVLPLODUWR)OHPPLQJ¶VIOXLGEXWDFHWLF

acid is not added and the merits and demerits are also similar.

2.10.24 +HOO\¶V)OXLG

This is same like Zenker-Formol.

2.11 Removal of Pigments

When stained sections are examined microscopically, a deposit or pigment is frequently

observed. This may be either artificial or natural in origin. 

a. The artificial pigments are of two main types as follows.

x Mercuric chloride precipitate

x Formaldehyde precipitate

b. The natural pigments are also of two main types:

x Exogenous pigments, which consist of foreign matter absorbed by the body during

life. The most commonly encountered is carbon which occurs as a jet-black pigment

in sections of lung and bronchial glands. Other example is tattooing ink.

x Endogenous pigments, which are produced within the organism, e.g. haemosiderin

which can be demonstrated by Prussian blue reaction.

2.11.1 Mercuric Chloride Precipitate:

The removal of HgCl2 pigment is done as follows:

Solution 1: Solution 2:

Potassium iodide 02g Sodium thiosulphate 05g

Iodine 01g Distilled water 100ml

Distilled water 100ml

Procedure:

i. Bring sections to water and immerse in solution 1 for 10 minutes to remove HgCl2

deposit

2HgCl + I2 Æ HgCl2 + HgI2

ii. Rinse in water and place in 5% sodium hyposulfite for 1-2 minutes to remove iodine.

2Na2SO2O3 + I2 Æ 2NaI + Na2S4O6

iii. Wash sections in running tap water for 2-5 minutes to remove the sodium

thiosulphate crystals, and then stain.

2.11.2 Formalin Precipitate:

This removal of formaldehyde precipitate is achieved by:

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Formula

KOH, 1% aqueous solution 01ml

Ethyl alcohol, 80% 100ml

Procedure:

i. Bring sections to 80% alcohol.

ii. Treat with alcoholic hydroxide solution for 10 minutes.

iii. Wash with 2 changes of water.

iv. Transfer to 80% alcohol for 5 minutes.

v. Wash in water and continue with staining. 

%DUUHWW¶s Alcoholic Picric Acid Method:

i. Deparaffinize with Xylene and wash in absolute alcohol.

ii. Immerse in saturated alcohol picric acid (85%) for 30 minutes.

iii. Wash in absolute alcohol to remove the picric acid.

iv. Bring section to water and continue staining in the normal way.

Box-2

What is Secondary Fixation?

Following fixation with formalin, it is sometimes advantageous to refix the tissue for a further 4

hours in a second fixative. The fixatives usually selected for this purpose are picric acid or osmium

tetraoxide or mercuric chloride. Formal-VXEOLPDWH IRUPDOLQ  PHUFXULF FKORULGH DQG =HQNHU¶V

formol can also be sued for secondary fixation. It has following advantages:

a. This secondary fixation has the advantage of imparting a firm texture to the tissue.

b. In many cases, secondary fixation improves the subsequent staining results.

What is Post Chroming?

In order to facilitate certain staining procedures, fixed tissues are immersed in 3% K2Cr2O7 for

several hours prior to staining. This procedure is termed post chromatization or post-chroming and

is used mainly with tissues fixed in formalin. This procedure should be followed by washing out

because K2Cr2O7 is a oxidizing agent.