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Published online: 2019-08-29
THIEME
Review Article 51
Fixation and Fixatives: Roles and Functions—A Short
Review
1 1 2 1 1
Himanshu Singh Kundendu Arya Bishen Deepti Garg Hemani Sukhija Dheeraj Sharma
Urvashi Tomar1
1Department of Oral and Maxillofacial Pathology, Index Institute of Address for correspondence Kundendu Arya Bishen, MDS, PhD,
Dental Sciences, Malwanchal University, Indore, Madhya Pradesh, India Department of Oral and Maxillofacial Pathology, Index Institute
2Department of Oral and Maxillofacial Pathology, Bhojia Dental of Dental Sciences, Malwanchal University,Indore 452016,
College, Baddi, Himachal Pradesh, India Madhya Pradesh, India (e-mail: kundenduarya@gmail.com).
Dent J Adv Stud 2019;7:51–55
Abstract Fixation is considered as physiochemical process where cells or tissues are fixed chem-
Keywords ically. Fixatives perform various functions such as prevention of autolysis and tissue
► fixatives putrefaction. Various fixative agents include formaldehyde, glutaraldehyde, osmium
► fixation tetroxide, glyoxal, picric acid, and so on. A detailed search on PubMed, Google scholar,
► formaldehyde and Scopus database showed very few articles on “fixation” and “fixative.” Keeping this
► glutaraldehyde fact in mind, a comprehensive review on fixation and fixatives was prepared. The main
► glyoxal aim of this review is to make pathologists and laboratory technicians familiar with the
► osmium tetroxide basic aspects and different types of fixatives.
► picric acid
Introduction as putrefaction (bacterial attack) of tissues. Autolysis seems
Fixation is known as a physiochemical process in which cells to be a frequent issue in enzyme-rich tissues, and rigorously
or tissues are fixed chemically. As a result, the tissue or cell autolyzed tissue does not get stained properly for microscop-
can combat the successive treatment by different reagents ic examination. On the other hand, bacterial invasion can be
with negligible disfigurement of morphology.1 blocked by following the strict antiseptic methods.
An ideal fixation involves complicated progression of Another important function is conserving the association
chemical episodes. An ideal fixative is presumed to transmit in between cells and extracellular substances. Fixatives sta-
mechanical toughness to tissue so that it resists destruction bilize the cell component by making them insoluble, there-
due to further processing steps. It prevents the autolysis, by reducing the alteration by subsequent treatment and
putrefaction of tissue as well as tissue component degrada- also preventing osmotic damage of tissue, which may cause
1,2 shrinkage or swelling, thus preserving the cellular and tissue
tion. Fixation should be able to preserve the cellular struc- structure in life-like state.
ture and tissue architecture in life-like manner.2 Fixation also performs various other functions such as
For the purpose of tissue processing in the histopatholo- making tissue firm, so that gross cutting becomes much easi-
gy, fixation of tissue is considered as necessary and essential er. Also, fixatives help make the tissue more easily permeable
step. Fixation amends the physio-chemical state of tissues so for subsequent reagents and play a role in emphasizing the
that it remodels the reactiveness of cellular components for dissimilarity in refractive index and thus help in increased
3
stains. Fixatives can be classified in different ways, as shown 1,2,4
in ►Tables 1 –3. visibility of different elements of tissue.
Functions of Fixative Factors Affecting Fixation and Fixatives
Length of Fixation
Fixatives perform various functions. The primary function The ideal time of fixation is experimentally determined for
of fixatives is to prevent autolysis (enzymes attack) as well different types of tissue. If time period for fixation is longer,
received DOI https://doi.org/ ©2019 Bhojia Dental College and
April 6, 2019 10.1055/s-0039-1693098 Hospital affiliated to Himachal
accepted ISSN 2321-1482. Pradesh University
May 20, 2019
published online
August 29, 2019
52 Fixation and Fixatives Singh et al.
Table 1 Classification of fixatives based on chemical Size
composition Tissue thickness is one of the important factors for fixation.
Fixatives Examples If the sample size is large, it is unfavorable for the fixative
1. Physical agents Heat, microwaves to penetrate and reach to the deeper part of the tissue,
4
which would result in autolysis of epithelium. Ideally 4- to
2. Aldehydes Formaldehyde, acrolein, 6-mm-thick specimen is best suited for complete penetration
glutaraldehyde by fixatives.
3. Coagulants Methyl alcohol, ethyl alcohol,
acetic acid Osmolarity
4. Oxidizing agents Osmium tetroxide If osmolarity of tissue as well as fixative is same, it will pre-
7
5. Miscellaneous Picric acid, mercuric chloride vent swelling or shrinkage of the tissue.
Table 2 Classification of fixatives based on number of struc- Various Fixating Agents Used in
tures fixed Histopathology
Fixatives Examples Formaldehyde or Formalin
1. Simple fixatives e.g., Formaldehyde, picric acid, Formaldehyde was discovered in 1859 by Butlerov. In 1889
osmium tetroxide Ttrillat was the first who manufactured formaldehyde com-
2. Compound fixatives e.g., Bouin’s fluid, formol saline, mercially as industrial reagent. In 1892, Ferdinand Blum recog-
Zenker’s fluid 8,9
nized that formalin could give benefit when used as a fixative.
The most routinely used solution for fixation of tis-
Table 3 Classification of fixatives based on type of structures sue—10% formalin solution v/v—is nothing but an aqueous
fixed suspension of formaldehyde In 10% neutral buffered form,
Fixatives Examples formaldehyde is found to be the most commonly used fix-
ative in pathology. Reaction between the formaldehyde
1. Histochemical Formaldehyde, glutaraldehyde, vapor and macromolecules of tissue seems to be complex. Form-
fixatives fixatives aldehyde reacts with nucleic acids as well as proteins, and
2. Microanatom- Bouin’s fluid, 10% formalin, Zenker’s it penetrates between nucleic acids and proteins and forms
ical fixatives fluid, formol calcium, Heidenhain’s stabilized shell of nucleic acid-protein complex.10-13 As com-
susa, Helly’s fluid, Rossman’s fluid, pared with other fixatives, formaldehyde causes lesser tissue
3. Cytologic Champy’s fluid, glacial acetic acid, shrinkage, with exceptions being acetone and ethanol. Form-
fixatives alcohol, formol saline, Carnoy’s fluid, aldehyde seems to harden tissue more when compared with
Clarke’s fluid, Newcomer’s fluid, other fixatives. The lipids are conserved, but carbohydrates
Flemming’s fluid
4
are not fixed by formaldehyde.
it results in over–cross-linking, and samples become brittle. Formalin comprises 37 to 40% formaldehyde and 60 to
If time period for fixation is short, sufficient amount of pen- 63% water by weight. After continuous storage for long peri-
4 ods, accumulations of white deposits are observed in the
etration in tissues and cross-linking will not occur. For oral solution. These are the precipitates of paraformaldehyde. By
soft tissue, overnight fixation is sufficient. storing formalin at low temperature, these white deposits
Temperature can be avoided. Also, 10% methanol may be added into the
Temperature of fixative during fixation may affect the tis- formalin to minimize the polymerization reaction that pro-
sue architecture. Rate of fixation is increased with increase duces paraformaldehyde precipitate. It also contains a slight
in temperature, but increased temperature will also increase amount of formate ions. These are obtained from Cannizza-
autolysis rate. If temperature is low or decreased, the dif- ro reaction. In this reaction, two molecules of formaldehyde
fusion rate also decreases, which results in extended pen- react together. One molecule condenses to form methanol
14
5 and second molecule gets oxidized to form formic acid. The
etration time. For electron microscopic studies, 0°to 4°C is solution is acidic in reaction because of formic acid, but acid-
appraise as ideal temperature. ic nature of solution can be counterbalanced with incorpora-
Concentration tion of magnesium carbonate in little proportion.15
Fixative agents need prolonged time for fixation if concen- Glutaraldehyde
tration is low. If concentrations of fixing agent are high, it Glutaraldehyde was found in 1963 by Sabatini et al as par-
results in damaging of cellular structures as well as obliterat- ticular fixative for ultrastructural researches. Glutaraldehyde
6
ed enzyme activities. Different fixatives have different ideal comprises two aldehyde groups that are divided by three
concentration that is determined experimentally; for exam- methylene bridges. Although penetration rate of glutaralde-
ple, ideal fixative for oral soft tissue is formalin used in 10% hyde is found to be slower when compared with formaldehyde,
concentrated solution.
Dental Journal of Advance Studies Vol. 07 No. 2/2019
Fixation and Fixatives Singh et al. 53
glutaraldehyde is more effective cross-linker for proteins than Glyoxal
16-18
formaldehyde and it also inhibits enzyme activity. Glyoxal was first advocated in 1943. They are also known
When polymerization of aqueous solution of glutaralde- as ethanedial or oxalaldehyde. Glyoxal is considered as
hyde occurs, it forms oligomeric and cyclic compounds, and alternative fixative to formalin because it is a dialdehyde
also forms glutaric acid by oxidation. For stability, it requires in nature. It is a bifunctional aldehyde. Its individual alde-
19,20
pH of 5 and storage at 4°C. At room temperature, glutaral- hyde groups are potentially reactive, and also cross-links
21
dehydes are not able to cross-link the nucleic acids. can be established. Glyoxal fixed tissues may demonstrate
Glutaraldehyde preserves the ultrastructure of the tissue, precise cellular details, lysed erythrocytes, and disinte-
thereby it is used in electron microscopy studies, but owing 26-28
grated microcalcifications.
to poor penetration and overhardening properties, it is not According to Harke and Hoffler, glyoxal does not evapo-
used as tissue fixatives for light microscopy. On exposure to rate from the solution, and as per Henry law constant, glyox-
oxygen, glutaraldehyde becomes unstable and breaks down al is virtually nonvolatile with consideration to the aqueous
with decrease in pH. Glutaraldehyde can act as sensitizer, phase. For microwave fixation, glyoxal is the chief component
and its exposure may result in respiratory tract, skin, and used in the fixatives. It does not produce vapors at room tem-
4
digestive tract irritation. perature, so it is considered as less dangerous in use than
29
formaldehyde. By molecular weight, glyoxal is the third
Osmium Tetroxide smallest aldehyde after formaldehyde and acetaldehyde. It
Osmium tetroxide is type of fixative that is water soluble and contains two carbon atoms. Glyoxal is commercially manu-
also soluble in nonpolar solvents. Osmium tetroxide seems to factured as aqueous solution that contains hydrates such as
react with proteins side chains that cause cross-linking. The trimers, dimmers, and ring strucutres.30
reactive groups of osmium tetroxide include various groups
such as disulfide, carboxyl, hydroxyl, sulfydryl, amide, and so Picric Acid
22
on. During fixation by osmium tetroxide, either due to slow Picric acid is an example of a coagulant fixative. It forms
rate of reaction or due to restricted penetration of osmium picrates with basic protein groups, which causes coagulation.
tetroxide into tissue, large amounts of carbohydrates as well For the purpose of demonstration of DNA or RNA, picric acid
as proteins are eradicated. fixatives are not used as picric acid and can hydrolyze nucleic
For electron microscopic studies, osmium tetroxide is acids. Also, picric acid is seen to disintegrate calcium depos-
used as secondary fixative, and it also performs well as stain its in samples. Although picric acid is not able to fix most car-
and imparts contrast when observed under electron micro- bohydrates and lipids, picric acid is the most advised fixative
scope. Osmium tetroxide is also helpful for staining of lip- to preserve glycogen. Brighter staining is seen by picric acid
ids in frozen sections. It is observed that fixation by osmium fixatives.4,18,31
tetroxide causes swelling in tissue, which can be decreased Picric acid is an acidic solution. Therefore, sometimes it
21
by adding sodium chloride or calcium chloride to fixatives. gets washed out by alcohol. To avoid this, lithium carbonate
It is traditionally sold as crystalline solid that is sealed in is added, which acts as a neutralizer. Luna reported that if
glass ampule. It is seen that osmium tetroxide crystals con- picric acid is present in the tissue or not completely removed,
vert from solid state to vapor state. Continued exposure to distortion or obliteration of cellular structures will occur as
osmium tetroxide vapors can cause deposition into cornea, 4,32
outcome.
which eventually leads to blindness.23,24
Ethanol and Methanol
Mercuric Chloride For ethanol and methanol, fixation initiates at 50 to 60%
Mercuric chloride can also be used as a tissue fixative for histo- concentration and greater than 80% concentration, respec-
pathology. It chiefly reacts with cysteine and also reacts with tively. They are known to be coagulants that cause protein
amines, amides, sulfydryl groups, and ammonium salts, and denaturation. They cause interruption in hydrogen and
results in tissue hardness. It acts as a strong protein coagulant. hydrophobic bonding by substituting water in tissue envi-
With acid dyes, it shows strong staining affinity. It also ronment, which results in change in tertiary structure.
reacts with phosphate remnants of nucleic acids and ade- Ethanol causes mispresentation of cytoplasmic as well as
quately fixes the nucleoproteins. Therefore, because of these nuclear details, but sometimes it can be used for preservation
reasons, it is observed that mercuric fixatives are the major of glycogen. Methanol is more commonly used for fixation of
component of some fixatives such as Helly’s fixative and b-5 18,33,34
exfoliative cytology smears and blood films.
fixatives.4,18
Nowadays, mercurial fixatives are not routinely used Acetone
except for fixation of hematopoietic tissues. Mercury-based Acetone is another fixative agent used in histopathology. It
fixatives show some characteristics features. They are toxic acts as an efficacious lipid solvent that results in tissue brit-
in nature and should not be allowed to come in contact with tleness. Apart from tissue fixation, they are primarily used
metals. These fixatives have slow penetration capacity, so the as an agent for dehydration in tissue processing. Because of
thickness of the specimens being fixed by mercuric fixatives extremely volatile as well as flammable nature, they are not
25 18,33
should be thin. recommended for use in automatic tissue processor.
Dental Journal of Advance Studies Vol. 07 No. 2/2019
54 Fixation and Fixatives Singh et al.
Acetic Acid Genipin is available as a crystalline white powder that is
Acetic acid is considered as a noncoagulative fixative agent. It soluble in acetone, methanol, and ethanol. Genipin demon-
43,44
acts by causing nuclear proteins coagulation. Incidentally, it strates its cross-linking properties at pH 7.4–8.5. It is a
stabilizes and assists to prevent nucleic acids loss. Acetic acid, known cross-linking agent and has proved its potential in
when combined with ethanol, is used as an effective cytolog- various biomedical application such as dentistry, articular
ical fixative that helps in conservation of nucleic acids, but if cartilage tissue engineering applications, nerve regeneration,
45-47
it is used singly, it results in swelling of cells. Time required and so on.
for fixation by acetic acid is less as penetration of acetic acid
35
is faster into tissues. Conclusion
Potassium Dichromate Fixation is considered as key step in histopathology pro-
Potassium dichromate is also a noncoagulant fixative, but if cedure. Each and every fixative has its own advantage and
used in combination with acid solution, it acts as a coagulant disadvantage. Various different fixatives perform various
fixative. It is seldom used alone for fixation because chro- functions, and various factors such as size, temperature, and
mate ions will link with few lipids and makes them insoluble. osmolarity have direct effect on fixation procedure.
Chromium seems to react with hydroxyl as well as car- Note
boxyl groups. By increasing the amount of reactive basic Prior to this publication, this study was not presented at
groups, the affinity of tissues for eosin staining will boost up. any conference or convention.
It conserves mitochondria but dissolves DNA. It is suggested
that tissues that are fixed with chromate fixatives have to be Funding
washed completely in water before processing of tissues any None.
further. This step is important as it avoids establishment of Conflict of Interest
4,18
chromate suboxide that is insoluble. None declared.
Bouin’s Fixative
Bouin’s fixative is known as noncoagulant picrate fixative References
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Bouin’s fixative is considered as good fixative for conserving Techniques. 5th ed. Philadelphia, PA: Churchill Livingstone
delicate as well as soft tissue structures. The major portion Elsevier; 2002;63–108
of Bouin’s fixative contains picric acid with little quantity of 2 Culling CFA, A Handbook of Histopathological and Histochem-
acetic acid as well as formaldehyde. In the samples that have ical Techniques. 4th ed. London: Butterworth Publication;
to be undertaken in situ hybridization, Bouin’s solution cannot 1985:27–77
36-38 3 Hayat MA, Stains and Cytochemical Methods. New York, NY;
be used because it decreases the severity of hybridization. London, UK: Plenum Press;1993
Acrolein 4 Freida L, Carson CHC. Histotechnology: A Self Instructional
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2,18,33 Oxford University Press;2018
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43,44
collagen in tissues.
Dental Journal of Advance Studies Vol. 07 No. 2/2019
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