Fixation Faculty of Applied Medical Sciences Presented by: Histopathology Teaching Assistant Walaa Mal

Content Aldehydes Oxidizing agents Cross linking fixatives Introduction Definition Purpose of fixation Features of fixation Characteristics of fixatives Fixation process Types of fixation Types of fixatives Cross linking fixatives Aldehydes Oxidizing agents Precipitating fixatives Other fixatives References

Introduction Tissue block is taken by biopsy, surgical excision or postmortem. Numerous techniques can be used to prepare tissue for microscopical examination depend on: Structures examined Nature of examined tissues Urgency of investigation Fresh or preserved specimens Fresh tissue can be examined as: Smear: e.g. screening of cancer cervix Frozen section using cryostat as urgent conditions e.g. during surgery Method: The fresh tissue is rapidly frozen using liquid nitrogen or carbon dioxide. Sectioning is carried out in the refrigerated cabinet of a Cryostat which is an apparatus designed to keep the microtome knife and tissue at a sub-zero temperature throughout the sectioning.

Definition of Fixation In the fields of histology, pathology, and cell biology, fixation is a chemical process by which biological tissues are preserved from decay, either through autolysis or putrefaction. Fixation terminates any ongoing biochemical reactions, and may also increases the mechanical strength or stability of the treated tissues.

Purpose of fixation The purpose of fixation is to preserve a sample of biological material (tissue or cells) as close to its natural state as possible in the process of preparing tissue for examination. To achieve this several conditions must usually be met. The aims of fixation are: Prevent postmortem (PM) degeneration Prevent autolysis. It is effective against hydrolytic enzymes Stop the bacterial effect Harden the tissues, as fixation causes coagulation of proteins Fixation has a mordanting effect, facilitating subsequent staining of tissues.

Features of fixatives First, a fixative usually acts to disable intrinsic biomolecules – particularly proteolytic enzymes – which would otherwise digest or damage the sample. Second, a fixative will typically protect a sample from extrinsic damage. Fixatives are toxic to most common microorganisms (bacteria in particular) which might exist in a tissue sample or which might otherwise colonise the fixed tissue. Finally, fixatives often alter the cells or tissues on a molecular level to increase their mechanical strength or stability. This increased strength and rigidity can help preserve the morphology (shape and structure) of the sample as it is processed for further analysis such as Nuclear Morphometry System. Note: Even the most careful fixation does alter the sample and introduce artifacts that can interfere with interpretation of cellular ultrastructure. A prominent example is the bacterial "mesosome", which was thought to be an organelle in gram-positive bacteria in the 1970s, but was later shown by new techniques developed for electron microscopy to be simply an artifact of chemical fixation.

Characteristics of a good fixative It must kill the cell quickly without shrinkage or swelling It must penetrate the tissue rapidly It must inhibit bacterial decay and autolysis Harden the tissue and render it insensitive to subsequent treatment as staining It should allow tissue to be stored for long time It should be simple to prepare and economical is use.

Fixation process Fixation is usually the first stage in a multistep process to prepare a sample of biological material for microscopy or other analysis. Therefore, the choice of fixative and fixation protocol may depend on the additional processing steps and final analyses that are planned. For example, immunohistochemistry utilises antibodies which bind to a specific protein target. Prolonged fixation can chemically mask these targets and prevent antibody binding. In these cases, a 'quick fix' method using cold formalin for around 24 hours is typically used.

Types of fixation There are generally three types of fixation process: Heat fixation: After a smear has been allowed to dry at room temperature, the slide is gripped by tongs or a clothespin and passed through the flame of a Bunsen burner several times to heat-kill and adhere the organism to the slide. Heat-fixation method can be successfully used for preparing Gram-negative and Gram-positive bacteria samples for studies Perfusion: Fixation via bloodflow. The fixative is injected into the heart with the injection volume matching cardiac output. The fixative spreads through the entire body, and the tissue doesn't die until it is fixed. This has the advantage of preserving perfect morphology, but the disadvantages that the subject dies and the cost is high (because of the volume of fixative needed for larger organisms) Immersion: The sample of tissue is immersed in fixative of volume at a minimum of 2/3rds greater than the volume of the tissue to be fixed. The fixative must diffuse through the tissue in order to fix, so tissue size and density, as well as the type of fixative must be taken into account. Using a larger sample means it will take longer for the fixative to reach the deeper tissue.

Types of fixatives Crosslinking fixatives Precipitating fixatives Aldehydes Oxidising agents Precipitating fixatives Ethanol Methanol Acetone Other fixatives Picric acid Mercuric chloride

Crosslinking fixatives “Aldehyde” Crosslinking fixatives act by creating covalent chemical bonds between proteins in tissue. This anchors soluble proteins to the cytoskeleton, and lends additional rigidity to the tissue. By far the most commonly used fixative in histology is the crosslinking fixative formaldehyde (also named formalin). Formaldehyde is thought to interact primarily with the residues of the basic amino acid lysine. Another popular aldehyde for fixation is glutaraldehyde. It is believed to operate by a similar mechanism to formaldehyde. As a somewhat larger molecule, glutaraldehyde may not penetrate thicker tissue specimens as effectively as formaldehyde. On the other hand, glutaraldehyde may offer a more rigid or tightly linked fixed Some fixation protocols call for a combination of formaldehyde and glutaraldehyde, so that their respective strengths complement one another. These crosslinking fixatives – especially formaldehyde – tend to preserve the secondary structure of proteins and may protect significant amounts of tertiary structure as well.

Crosslinking fixatives “Oxidising agents” The oxidising fixatives can react with various side chains of proteins and other biomolecules, allowing the formation of crosslinks which stabilise tissue structure. Osmium tetroxide is often used as a secondary fixative when samples are prepared for electron microscopy. It is not used for light microscopy as it penetrates thick sections of tissue very poorly. Potassium dichromate, chromic acid, and potassium permanganate all find use in certain specific histological preparations.

Precipitating fixatives “Denaturing fixatives” Precipitating (or denaturing) fixatives act by reducing the solubility of protein molecules and (often) by disrupting the hydrophobic interactions which give many proteins their tertiary structure. The precipitation and aggregation of proteins is a very different process from the crosslinking which occurs with the aldehyde fixatives. The most common precipitating fixatives are ethanol and methanol. Acetone is also used. Acetic acid is a denaturant that is sometimes used in combination with the other precipitating fixatives. The alcohols, by themselves, are known to cause shrinkage of tissue during fixation while acetic acid alone is associated with tissue swelling; combining the two may result in better preservation of tissue morphology.

Other fixatives Other fixative agents include Picric acid Mercuric chloride.

References Ryter A (1988). "Contribution of new cryomethods to a better knowledge of bacterial anatomy". Ann. Inst. Pasteur Microbiol. 139 (1): 33–44. PMID 3289587. Friedrich, CL; D Moyles, TJ Beveridge, REW Hancock (2000). "Antibacterial Action of Structurally Diverse Cationic Peptides on Gram-Positive Bacteria". Antiomicrobial Agents and Chemotherapy 44 (8): 2086–2092.

Bunsen burner

Covalent chemical bonds

Transmembrane receptor