This document discusses various fixatives used in histology and cytology techniques. It begins by defining fixation as the process of preserving cells and tissue using physical or chemical methods. Commonly used fixatives include formaldehyde, glutaraldehyde, osmium tetroxide, mercury salts, picric acid, and alcohols. An ideal fixative would be non-toxic, low-cost, and effectively preserve tissue morphology, antigens, and nucleic acids while allowing for long-term storage. Factors like temperature, pH, concentration, and duration impact fixation quality. Proper fixation is important for minimizing artifacts and ensuring high quality staining.

1. FIXATIVES
BY DR.S.MADHUBALA
FINAL YEAR POSTGRADUATE

2. Fixation is the first step of any histological and cytological laboratory technique.
This is a process by which constituents of cells and tissue are fixed in a physical and
partly in a chemical state so that they can withstand subsequent treatment with
various reagents with minimal loss of architecture .
FIXATIVES :a substance that preserves the morphological and chemical
characteristics of cells and tissue and prevents autolytic and putrefactive changes

3. IDEAL FIXATIVE
To date ,a universal or ideal fixative has not identified .
Non toxic ,cost effective ,disposable or recyclable
Prevention of autolysis of the cells .
Inactivation of infectious agents especially prevention of decomposition of tissue by bacteria
Useful for wide variety of tissue types
Preserve small and large specimens and support histochemical ,IHC and ISH
High quality and consistent staining with H&E
Maintaining the volume and shape of the cell as far as possible at the time of processing
Should penetrate and fix rapidly
Shelf life of atleast one year
Compatible with automated tissue processors
Support long term tissue storage and to give excellent microtomy of paraffin blocks.

4. TYPES OF FIXATIVES
PHYSICAL METHODS
Heating, microwaving and freeze drying
CHEMICAL METHODS
Three categories :
1.coagulant fixatives –remove free water from tissue results in
precipitation and coagulation of proteins .
2.cross linking –Adding covalent OH- groups and crosslinking proteins
3.compound fixative :function using mixture of reagents eg. Alcoholic
formalin

6. PHYSICAL METHOD
HEAT FIXATION : Simplest form of fixation
Heat is primarily used to accelerate other forms of fixation and other steps of processing
MICROWAVE FIXATION:A Type of electromagnetic wave - 300MHZ and 300GHZ
The dipolar molecules (water) rapidly oscillate in this electromagnetic field .
Rapid kinetic motion generates uniform heat .
Accelerates the fixation and other steps of processing .
Homogenous increase of temperature within the tissue and every part of tissue is heated .
FACTORS CONTROLLING TEMPERATURE RISE :
1.Dielectric property of the media
2.Thermal properties
3. Radiation level
4.Orientation and shape of the object

7. ADVANTAGE
Rapid processing
No change in volume of tissue
preservation of tissue antigen and good for IHC
Facilitates staining reaction
Reduce time for fixation
DISADVANTAGE
Large amount of toxic gas
Chances of heat injury
APPLICATIONS
Routine surgical pathology laboratory
Electron microscopy after osmium tetraoxide fixation
urgent processing like kidney biopsy

8. • ESSENTIAL PRECAUTION FOR FIXATION IN GENERAL:
Free from excessive blood and mucus
Thin cut in 3-5mm thickness
10 times more than the volume of tissue
Tightly screwed capped bottle

9. CLASSIFICATION

10. NATURE OF FIXATION
1.IMMERSION FIXATION : Commonly used
Liquid fixative such as 10%neutral buffered formalin or cytology smear in 95%ethyl alcohol
2.COATING FIXATION : cytology samples
Fixation of the cells
Impart protecting covering over the smear
No need to carry liquid fixative in jar
PRECAUTION: should be smooth and steady
Distance of 10-12 inches from nozzle and the smear
spray fixative contains alcohol and wax ,wax should be removed before staining
3.VAPOUR FIXATION : Formaldehyde ,glutaraldehyde ,osmium tetraoxide and ethyl alcohol
4.PERFUSION FIXATION :research purpose and whole organ fixation eg.Brain

11. 5.FREEZE DRYING
Tissue is cut into thin sections and rapidly frozen at very low temperature .Ice within the tissue is
removed by vacuum chamber in higher temperature .
STEPS: 1.Thin cut tissue sections rapidly frozen at -160 ⁰C by immersing it into liquid coolant(Liquid
nitrogen, propane, isopentane )-QUENCHING
2.Ice removed by placing tissue in vacuum chamber at -30 to -50 ⁰C
3. water is removed by sublimation
4.Tissue is gradually warmed at 4 ⁰C and impregnated with embedding medium
ADVANTAGE : For enzyme study ,no change of proteins, no shrinkage of tissue, preservation of
glycogen

12. COMMONLY USED FIXATIVES IN LAB
FORMALDEHYDE: Pure formaldehyde vapour dissolved in water as formaldehyde 37-40%
concentration (FORMALIN). 10% of this formalin is used to Make NBF
RATE OF PENETRATION : 1mm/hr
VOLUME OF FORMALIN :10 times the volume of tissue
completely immersed in formalin
should not be direct contact with container .
formalin soaked gauze in between the tissue
Formalin is buffered with phosphates to pH 7.2-7.4
REMOVAL OF FORMALIN : washing for 24hour in water will remove 50% of formalin from tissue
PRECAUTION: irritant to eye ,skin ,toxic for inhalation .CARCINOGENIC ELEMENT.

13. ADVANTAGE
Penetration rate is high
Cell morphology well preserved
Cheap,stable,easy to make
Effective fixation for routine laboratory
staining of tissue .
DISADVANTAGE
Slow fixation
Reversible and can be removed by
washing
Fails to preserve acid
mucopolysaccharides
Highly vascular tissue –dark brown
granules
Skin exposure –dermatitis ,inhalation
bronchitis
Not good for staining fat and enzymes

14. PREPARATION OF DIFFERENT FORMALIN SOLUTION
1. 10% NEUTRAL BUFFERED FORMALIN
Formaldehyde 40%:100ml
Distilled water :900ml
Sodium phosphate ,monophasic, monohydrate :4g
sodium phosphate,dibasic,anhydrous :6.5g
2.Carson’s modified milling’s phosphate buffered formalin –Better ultrastructural
preservation
3.Formal (10% formalin),calcium acetate – fixative for lipid
4. Formal (10% formalin),saline
5. Formal (10% formalin),zinc,unbufferd-IHC
6.Formal ,buffered zinc

15. GLUTARALDEHYDE
Uses: Electron microscopy
Mechanism: Extensive crosslinking of proteins
ADVANTAGE : DISADVANTAGE:
1.Better fixation of ultrastructure 1.Poor penetration and tissue should be<0.5mm thick
2.Less cell shrinkage 2.less stable compound
3.Preservation of protein is better 3.no lipid fixation
4.Good cross linking with collagen 4.polymerizes above pH 7.5
5.Less irritant 5.Costly
Commercially as 25%or 50%solutions in 10ml

16. OSMIUM TETRAOXIDE
Uses :Electron microscopy
It react with unsaturated bonds in the lipid molecules and fixes them .
ADVANTAGE : DISADVANTAGE
1.Very good fixative for lipid 1.Doesn’t fixes proteins and carbohydrate
2.Preserves cytoplasmic organelles 2.Clumping of DNA
3.Does not make the tissue hard 3.Poor penetration
4.Stain lipids in frozen section 4.Tissue swelling
5.Harmful vapour
6.Expensive
Commercially available as 0.1 -1gm.Aqueous solution of 4%OsO4 is made .
1%OsO4 in buffer solution of pH 7.2 is used

17. DEHYDRANT FIXATIVES
METHYL ALCOHOL AND ETHYL ALCOHOL
• Dehydrant fixatives
• Used in cytology smears
The tissue or smear containing water should not be put directly in the higher
concentration of alcohol – distortion of the cells due to rapid rush of fluid from the
cell
• Graded alcohol
• LAB USE: 95%Ethyl alcohol
• PREPARATION :Absolute alcohol 950ml
water :50ml
TIME OF FIXATION :15-30MINS

18. ACETONE
Dehydrant
USED : Enzyme study and immunocytochemistry and in detection of rabies.
Fixation should be short (1hour) at 4⁰C on small specimens
DISADVANTAGE :Poor fixative for morphological preparation due to cell shrinkage
CLARKE’S CARNOY’S METHACARN’S
Absolute ethanol 60ml Acetic acid 10ml Acetic acid 10ml
Glacial acetic acid 20ml Absolute alcohol 60ml 100%methanol
Chloroform 30ml chloroform 30ml

19. PICRIC ACID FIXATIVES
BOUIN’S FIXATIVE
Reacts with aminoacid and forms picrate .USES: Testicular biopsy and fixation of embryos
ADVANTAGE DISADVANTAGE:
connective tissue stains and glycogen Hydrolysis of nuclei acid
Rapid penetration Produces yellow stain
REMOVAL OF YELLOW STAIN: 1. wash in 70%ethanol 2.Dipping in lithium carbonate in 70%alcohol
PREPARATION: Picric acid solution (1%distilled water ):1500ml
Formaldehyde 40%:500ml
Glacial acetic acid :100ml
HOLLANDE’S SOLUTION –GI biopsies and endocrine tissue
GENDER’S FLUID- Glycogen

20. MERCURY SALT CONTAINING FIXATIVES
Non coagulant cross linking fixatives
ADVANTAGE: Excellent staining of nuclei and connective tissue(trichrome stain)
Quick fixation
DISADVANTAGE: Rate of penetration of fixative reduces after few millimetres
Intolerant fixatives
radio-opaque
corrodes metal
Brown mercury pigments

21. ZENKER’S FLUID
Good fixative for nuclear chromatin and collagen
Bloody (congested) specimens
PREPARATION :Mercuric chloride 50g
potassium dichromate 25g
Distilled water 950ml
Sodium sulphate 2.5g
Glacial acetic acid 50g(lastly added)
HELLY’S FLUID :Good cytoplasmic fixative, for bone marrow and intercalated discs
PREPARATION: SOLUTION A SOLUTION B
Distilled water :250ml 35% formaldehyde
Potassium dichromate 6.3g
Mercuric chloride 12.5g
Sodium sulphate 2.5g Just before use mix 95ml of solution A and 5ml of Solution B

22. B5 FIXATIVES
Used for bone marrow ,lymph node ,spleen and other hematopoietic tissues.
Stock solution A Stock solution B
Mercuric chloride 12g 35%formaldehyde
Sodium acetate 2.5g
Distilled water 200ml
Before use is 20ml of solution A with 2ml of stock solution B
CARNOY LEBRUN SOLUTION SCHAUDINN’S SOLUTION OHLMACHER’S SOLUTION
Absolute alcohol 15ml Distilled water 50ml Absolute alcohol 32ml
Chloroform 15ml mercuric chloride 3.5g chloroform 6ml
Glacial acetic acid 15ml Absolute alcohol 25ml Glacial acetic acid 2ml
Mercuric chloride 8g Mercuric chloride 8g

23. SPECIAL FIXATIVES
Dichromate fixative : Neuroendocrine tissue
MILLER’S REGAUD’S ORTH’S SOLUTION
Potassium dichromate 2.5g potassium dichromate 3g potassium dichromate 2.5g
Sodium sulfate 1g Distilled water 80ml sodium sulfate 1g
Distilled water 100ml 20ml formaldehyde Distilled water 100ml
10ml formaldehyde
Chromaffin reaction ?
FIXATIVES FOR DNA,RNA,PROTEIN ANALYSIS:
HEPES-glutamic acid buffer mediated organic solvent protection effect (HOPE)
Z 7

24. SECONDARY FIXATION
It is the process tissue is fixed by the sequential application of two fixatives.
POSTMORDANTING
The first fixative is primary fixative and second fixative is secondary fixative .
PURPOSE: When primary fixative is not totally effective the tissue can withstand second harsher
fixatives .
Advantage: Tissue can be cut more easily
Better quality of staining
Eg. trichrome staining ,chromaffin reaction

25. FIXATIVES OF CHOICE

26. FIXATIVES FOR INDIVIDUAL TISSUES
TISSUE FIXATIVE
EYE 10%NBF
BRAIN Perfusion technique 10%NBF
BREAST 10%NBF
LUNGS 10%NBF
LYMPHOID TISSUE 10%NBF ,B5 or Zinc
TESTIS 10%NBF or Bouins’s fixative
MUSCLE BIOPSY 10%NBF
RENAL BIOPSY 10%NBF-Light microscopy
Glutaraldehyde –EM
Isopentane and liquid nitrogen -IF
GIT 10%NBF

27. TISSUE CHANGES DURING FIXATION
1.VOLUME CHANGES
Osmium tetraoxide –cell swelling
Formaldehyde –shrinkage of the volume by 33%
Glutaraldehyde –tissue shrinkage
2.HARDENING OF TISSUE
3.INTERFERENCE OF STAINING
Formaldehyde-Inactivates 80% of ribonuclease enzyme
Osmium tetraoxide –inhibits H&E staining
4.CHANGES OF OPTICAL DENSITY BY FIXATION

28. FACTORS AFFECTING FIXATION
1.pH
Better in Neutral pH
BUFFER : Phosphate, bicarbonate, tris and acetate.
2.TEMPERATURE:
Higher temperature(60-65⁰C)-fixation time reduced
In very high temperature the antigen in tissue may be destroyed
For enzyme histochemistry –lower temperature 0-4⁰ C
3.DURATION OF FIXATION
D=k√ T D-depth of penetration ,T-Time duration ,k-coefficient of fixative eg..0.79 for 10%NBF
4.OSMOLALITY: Mildly hypertonic fixative( 400-450mOsm) for best morphological Study
Electrolyte or sucrose can be added to maintain osmolarity
Hypertonic fixative –shrinkage of cell
Hypotonic fixative –swelling of cell

29. 5.CONCENTRATION
Optimal concentration of formaldehyde is 10%
Very low concentration –prolongs the time of fixation
Higher concentration –hardening ,shrinkage and artefacts.
White precipitates
Ethanol - <70% do not remove free water from tissue
6.AGITATION :
Increases the rate of penetration .Optimal agitation is needed.
Slow agitation –no effect of fixation
Rapid agitation – deterimental effect on delicate tissue

30. FIXATION ARTEFACT
1.FORMALIN PIGMENT : Unbuffered formalin
Formic acid +hemoglobin
acid formaldehyde haematein
Brown black granular birefringe pigment
REMOVAL : PICRIC ACID METHOD(BARRETT):
1.Section is immersed in xylene followed by alcohol to bring in water
2.immerse in 1.8% picric acid in absolute ethyl alcohol for 5mins to 2hours
3.wash for 10mins
4. 90%alcohol for 2mins
5.re-stained
KARDASEWITSCH’S METHOD LILLIE’S VEROCAY’S SCHRIDDE’S
70%Ethyl alcohol 100ml acetone 50ml NaOH 75%alcohol 200ml
28%ammonium water H2O2 50ml KOH 25% Liquor ammonia1ml

32. 2.MERCURY PIGMENT: Dark brown irregular deposits
Removal: Iodine converts it into mercuric chloride which is removed by sodium thiosulphate
3.FUZZY STAINING: Nuclear and cytoplasmic details are obscured looks fuzzy or hazy
Cause: improper fixation
4.PROLONGED FIXATION :
Shrinkage of tissue followed by separation
may have holes or empty spaces
5.DICHROMATE DEPOSIT :chromium salt with alcohol forms Yellow brown precipitate
REMOVAL: 1% HCL in 70% alcohol for 30 mins

33. REFERENCES
1.Bancroft’s theory and practice of histological techniques,8th edition
2. Cellular Pathology Technique - Culling - 4th edition
3.Handbook of Medical & laboratory technique.