Bouin solution, or Bouin's solution, is a compound
fixative used in
histology
Histology,
also known as microscopic anatomy or microanatomy, is the branch of biology which studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures vis ...
. It was invented by French biologist
Pol Bouin and is composed of
picric acid
Picric acid is an organic compound with the formula (O2N)3C6H2OH. Its IUPAC name is 2,4,6-trinitrophenol (TNP). The name "picric" comes from el, πικρός (''pikros''), meaning "bitter", due to its bitter taste. It is one of the most acidic ...
,
acetic acid
Acetic acid , systematically named ethanoic acid , is an acidic, colourless liquid and organic compound with the chemical formula (also written as , , or ). Vinegar is at least 4% acetic acid by volume, making acetic acid the main component ...
and
formaldehyde
Formaldehyde ( , ) (systematic name methanal) is a naturally occurring organic compound with the formula and structure . The pure compound is a pungent, colourless gas that polymerises spontaneously into paraformaldehyde (refer to section F ...
in an aqueous solution. Bouin's fluid is especially useful for fixation of
gastrointestinal tract
The gastrointestinal tract (GI tract, digestive tract, alimentary canal) is the tract or passageway of the digestive system that leads from the mouth to the anus. The GI tract contains all the major organ (biology), organs of the digestive syste ...
biopsies because this fixative allows crisper and better
nuclear
Nuclear may refer to:
Physics
Relating to the nucleus of the atom:
* Nuclear engineering
*Nuclear physics
*Nuclear power
*Nuclear reactor
*Nuclear weapon
*Nuclear medicine
*Radiation therapy
*Nuclear warfare
Mathematics
*Nuclear space
*Nuclear ...
staining
Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Stains and dyes are frequently used in histology (microscopic study of biological tissues), in cytology (microscopic study of cells), and in the ...
than 10% neutral-buffered formalin. It is not a good fixative when tissue
ultrastructure
Ultrastructure (or ultra-structure) is the architecture of cells and biomaterials that is visible at higher magnifications than found on a standard optical light microscope. This traditionally meant the resolution and magnification range of a co ...
must be preserved for
electron microscopy
An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a hi ...
. However, it is a good fixative when tissue structure with a soft and delicate texture must be preserved. The acetic acid in this fixative
lyses red blood cell
Red blood cells (RBCs), also referred to as red cells, red blood corpuscles (in humans or other animals not having nucleus in red blood cells), haematids, erythroid cells or erythrocytes (from Greek ''erythros'' for "red" and ''kytos'' for "holl ...
s and dissolves small iron and calcium deposits in tissue. A variant in which the acetic acid is replaced with
formic acid
Formic acid (), systematically named methanoic acid, is the simplest carboxylic acid, and has the chemical formula HCOOH and structure . It is an important intermediate in chemical synthesis and occurs naturally, most notably in some ants. Es ...
can be used for both fixation of tissue and decalcification. The effects of the three chemicals in Bouin solution balance each other. Formalin causes cytoplasm to become basophilic but this effect is balanced by the effect of the picric acid. This results in excellent nuclear and cytoplasmic
H&E stain
Hematoxylin and eosin stain ( or haematoxylin and eosin stain or hematoxylin-eosin stain; often abbreviated as H&E stain or HE stain) is one of the principal tissue stains used in histology. It is the most widely used stain in medical diagnos ...
ing. The tissue hardening effect of formalin is balanced by the soft tissue fixation of picric and acetic acids. The tissue swelling effect of acetic acid is balanced by the tissue shrinking effect of picric acid.
Hydrated sections of formaldehyde-fixed tissue are usually pre-treated with Bouin solution to obtain correct results in the
trichrome stain
Trichrome staining is a histological staining method that uses two or more acid dyes in conjunction with a polyacid. Staining differentiates tissues by tinting them in contrasting colours. It increases the contrast of microscopic features in c ...
s for contrasting colours in collagenous and cytoplasmic (muscle) fibres. The trichrome methods were devised for tissues fixed in acidic mixtures containing mercuric chloride, which are now used only on a small scale.) Only the picric acid component of Bouin's solution is needed to bring about this correction of trichrome staining.
When using Bouin solution, several potential problems can arise. Due to the formalin in the solution, formalin pigment may be present when viewing tissue sections under the microscope. Wet tissue should be fixed in Bouin solution for less than 24 hours. Excess picric acid should be washed out of tissue using several alcohol and water solutions or staining quality may deteriorate over time. Wet tissue fixed in Bouin solution should be stored in an alcohol and water solution rather than Bouin solution. Since Bouin solution contains formaldehyde, picric acid and acetic acid, appropriate safety precautions for these substances should be taken and regulations followed. In particular, noting that picric acid can be explosive, sensitive to friction and shock when dry and in contact with some metals can form unstable metal picrates.
Under the name "Bouin's fluid" this fixative is also widely used for marine invertebrates. It is prepared as follows: picric acid, saturated aqueous solution – 75 ml; formalin, 40% aqueous solution – 25 ml; acetic acid, glacial – 5 ml.
Variations
Gendre solution
Gendre solution is an alcoholic version of Bouin solution. An alcoholic solution saturated with picric acid is used instead of an aqueous solution saturated with picric acid when making this solution. This solution is useful when
glycogen
Glycogen is a multibranched polysaccharide of glucose that serves as a form of energy storage in animals, fungi, and bacteria. The polysaccharide structure represents the main storage form of glucose in the body.
Glycogen functions as one o ...
and other
carbohydrate
In organic chemistry, a carbohydrate () is a biomolecule consisting of carbon (C), hydrogen (H) and oxygen (O) atoms, usually with a hydrogen–oxygen atom ratio of 2:1 (as in water) and thus with the empirical formula (where ''m'' may or ma ...
s must be preserved in tissue. It is prepared by a mixing saturated solution of picric acid in 95% ethanol (80ml) with formalin (37-40% formaldehyde) (15 ml) and glacial acetic acid (5ml). Gendre's fixative contains more picric acid than Bouin's, because of greater solubility (6.23%w/v) in ethanol than in water (1.23%w/v).
Hollande solution
Hollande solution is a version of Bouin solution that contains
copper acetate
Copper is a chemical element with the symbol Cu (from la, cuprum) and atomic number 29. It is a soft, malleable, and ductile metal with very high thermal and electrical conductivity. A freshly exposed surface of pure copper has a pinkish-or ...
. The copper acetate stabilizes red blood cell membranes and the
granules of
eosinophil
Eosinophils, sometimes called eosinophiles or, less commonly, acidophils, are a variety of white blood cells (WBCs) and one of the immune system components responsible for combating multicellular parasites and certain infections in vertebrates. A ...
s and
endocrine
The endocrine system is a messenger system comprising feedback loops of the hormones released by internal glands of an organism directly into the circulatory system, regulating distant target organs. In vertebrates, the hypothalamus is the neu ...
cells so that there is less lysis of these cell components than occurs in regular Bouin solution. Hollandes fixative is made by adding 6.25g cupric acetate, 10g picric acid (wet powder), 25ml formalin (37-40% formaldehyde) and 2.5 ml glacial (100%) acetic acid to 250ml water.
[Gray, P. 1954. The Microtomist's Formulary and Guide. Reprinted 1975. Huntington, NY: Kreiger, p.220.]
References
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Pathology
Histology