Conceptual Overview

Background

With effective light microscopy imaging techniques for fresh specimens, it might be asked:

"Why bother to chemically fix specimens"?

Many kinds of specimens cannot be isolated as individual cells for separate study, and the tissue(s) must be observed intact.

Many softer tissues cannot be cut thin enough for microscopic viewing without some kind of external support in an embedding medium (which requires chemical fixation before the embedding material can be infiltrated into the tissue).

Tissues normally cannot be kept alive for a long time in a state that is suitable for microscopy.

What does chemical fixation accomplish?

It preserves tissues in a life-like (albeit not living) form.

It prepares tissues for the rigors of embedding, staining and sectioning.

What happens when we don't fix tissues?

When living tissue is left in air it dehydrates and shrinks. Left in a fluid (e.g. H₂O), it undergoes osmotic change. But in either case it is subject to attack by molds, bacteria and other microorganisms. Furthermore, a tissue may fall to pieces by "self-digestion" due to autolytic enzymes collectively known as "cathepsins." Cathepsins contain two primary proteinases: carboxypeptidase and aminopeptidase which catalyze the hydrolysis of peptide bonds through either the carboxy or the amino groups.

Thus, there are several requirements of a good chemical fixative agent. It must:

not excessively distort tissue.

infiltrate tissue rapidly for good preservation.

kill or inhibit bacteria and other microorganisms that contaminate and/or degrade specimens.

modify tissues to resist subsequent sample preparation treatments for microscopy.

enable tissue to take up stains.

Some materials that resist fixation (fix very poorly):

Chitin

Cellulose

Starch grains

Silica

Inorganic crystals

Some materials that cannot be retained in their natural sites by any fixative due to solubilization (their loss brings about specimen shrinkage):

Soluble sugars

Low molecular weight carbohydrates

Many lipids

Characteristics of Chemical Fixatives

All fixatives fall into two groups:

COAGULANTS (examples below) bring about specimen shrinkage due to removing the water of hydration of proteins (and, in some cases, lipid conjugates). They may, however, be suitable for light microscopy observations where the magnification used is not high enough to be impaired by the shrinkage.

Ethanol (CH₃ ╖CH₂OH) which is usually used in high concentrations.

Mercuric chloride (HgCl₂) which is a colorless crystal in its normal state, but the standard concentration used in fixation is saturated in water, or about 7%.

Chromium trioxide (CrO₃) which is a brownish-red crystal that is very soluble in water to give an acid solution. It is usually used at 0.5% aqueous concentration.

Acetic acid (CH₃ ╖ COOH) which is a colorless liquid, is used as a fixative in a standard concentration of about 5%.

NON-COAGULANTS usually form (covalent) bridge links with existing protein and/or lipid molecules, and substitute for the water of hydration in proteins. Examples include:

Formaldehyde (CH₂O) which is a colorless gas that is very soluble in water up to a concentration of about 37% (an aqueous solution of that concentration is called formalin), but is used in fixatives at concentrations usually below 5%.

Glutaraldehyde (CHO ╖ CH₂ ╖ CH₂ ╖ CH₂ ╖ CHO) which is a liquid highly soluble in water, but usually used in concentrations at 2-3% (aqueous). It primarily fixes proteins by reacting with the free amino groups of amino acids to form aldamine groups (R-CH=N-R). Due to its being a dialdehyde, it is likely to react at both ends of the molecule and remain as a bridge, or ligand, thus linking adjacent protein molecules together.

Osmium tetroxide (OsO₄) used largely in electron microscopy is seen as pale yellow crystals in the normal state. It is soluble in water to about 7%, but is normally used in fixative solutions at about 1-2% concentration. It reacts with unsaturated lipid chains and aldamine groups. It may also form bridges to stabilize molecules in tissues.

Potassium dichromate (K₂Cr₂O₇) is seen as orange-red crystals that are weakly acid in water where they are soluble to 10%. The standard concentration used in fixatives is about 1.5% (aqueous).

Since most fixatives used for light microscopy individually have limitations, they are often mixed. The object of this mixture is to better confer structural stability to a variety of substrate molecules in the specimen. Traditional embedding methods (paraffin) for light microscopy have often used coagulant fixative agents, e.g. FAA (formalin, acetic acid and alcohol). Most modern embedding procedures for either light or electron microscopy use resin (plastic) embedding agents, and use only non-coagulant fixatives.

Buffers

Buffers are necessary to add to non-coagulant fixatives to adjust for pH, as well as to adjust the osmotic properties of the fixative medium. The pH adjustment is necessary because natural buffer systems in cells are too weak in the environment of fixatives. A big pH shift will radically alter proteins in the cell--both those that are structural as well as those that are enzymatic. By adjusting the pH, autolysis (self-digestion) may be prevented prior to the completion of killing which takes seconds to minutes (and in some cases, hours) to achieve. Furthermore, most chemical fixative reactions proceed fastest at a pH value of about 7.0. Buffers are usually a combination of weak acids and their salts in solution. Their concentration, or molarity, determines the osmotic properties of the fixative solution. The medium needs to be adjusted to a osmolarity about the same as that of the tissue being fixed, so that swelling or shrinkage will not occur.

Dyes and Staining

Color is imparted to specimens, often to specific sites or composition, by dye molecules in order to provide contrast for light microscopy. The chemical reaction which produces a color site is a stain. Dyes for staining cellular components contain two kinds of active chemical groups, chromophoric and auxochromic. Chromophoric groups impart color to the dye, and examples might be carboxyl (-COOH), azo (-N=N-), and nitro (-NO₂) groups which typically contain resonating double bonds or unstable electrons. Auxochromic groups give the dye its ability to attach to the substrate material, and to dissolve and dissociate in water.

Dyes are usually classified as acidic, basic, or amphoteric. An acidic dye is one that has a higher reaction potential with substrate molecules at a low pH and is generally used in staining cytoplasm and other proteins. The net charge on an acidic dye is negative. Examples are: acid fuchsin, Janus Green-B, Orange-G, and methyl blue. A basic dye is generally used for staining the nucleus, and nucleic acids in general. The net charge on the dye ion is positive. Examples are: basic fuchsin, crystal violet, methyl green, and safranin. Amphoteric dyes are ones in which the charge on the dye molecule changes with the pH of the medium.

In some cases, dyes will not bind to substrate molecules without the presence of an intermediate agent that facilitates the reaction. The intermediate agent, usually designated a mordant, is typically composed of a salt of a di- or trivalent metal. The term, mordant, is derived from the Latin term mordere, meaning "to bite," since early dyers thought that it caused color "to bite into" a fabric. The combination of a dye and its mordant is called a lake. Some dyes, such as hematoxylin and carmine require mordants to bind to substrates and, therefore, to impart a stain reaction as a lake. Iron and ammonium alums are common examples of mordants used in staining.

Sudan Black-B is a fat-soluble dye, and is relatively specific for phospholipids such as those in Golgi structures where it remains soluble. Polysaccharide materials, such as starch, cellulose and hemicelluloses in plant cells are often stained with the periodic acid-Schiff (PAS) reaction. The reaction involves the hydrolysis of carbon bonds (C-C) by periodic acid (HIO₄) to form dialdehydes (CHO-CHO). Upon the addition of the Schiff reagent, the specimen takes on a reddish color due to the presence of the aldehydes.

Other dyes that are of particular botanical interest include lacmoid (or cotton blue) as a stain for callose, and phloroglucinol as a stain for lignin.

Subunits:		Feature:
Embedding		Video: Embedding & Sectioning Specimens for Light Microscopy
Microtomy
Selective Stains