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Histology Techniques - Tissue Fixation and Fixatives

  • What is fixation
  • Fixation Artefacts
  • Common Fixative Types
  • Fixatives
  • More Fixatives

Tissue fixation is used for several reasons, including prevention of putrefaction from bacteria, autolysis from enzyme degradation and loss of soluble substances. Most tissue fixation also helps enhance staining in later techniques like immunohistochemistry, if required and also routine haematoxylin and eosin staining.

Fixation preserves 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 usually must be met.

First, a fixative usually acts to prevent autolysis. This is self-destruction of the cell by the body own enzymes, which are released by lysosomes rupturing after death.  They cause more damage to soft tissue like brain than to harder tissue like collagen. Fixatives do this by disabling proteolytic enzymes and so prevent any damage occuring to the tissue from their activity.

Second, a fixative typically protects a sample from putrefaction. This is the destruction of the cell by bacteria which are normally commencals, but after death they spread quickly causing decomposition. Fixatives are toxic to most common microorganisms like bacteria, that might exist in a tissue sample or which might otherwise colonise the fixed tissue. In addition, many fixatives chemically alter the fixed material to make it either toxic or indigestable for opportunistic microorganisms, so helping to prevent putrefacation.

Finally, fixatives often alter the cells or tissues on a molecular level to increase their mechanical strength and stability. This increased strength and rigidity helps to preserve the morphology of the sample, which is important later on to acheive accurate diagnosis for patients.

 

Despite the care taken sometimes even the most careful fixation will effect the tissue and introduce unwanted artifical artifacts that can interfere with interpretation of cellular structure.

One very well known historical example of this phenomenon wass the bacterial mesosome which at first was believed to be organelle in gram-positive bacteria. However this was later shown by new techniques developed for electron microscope to be an chemical fixation artifact.

Standardization of fixation and other tissue processing procedures must be careful to account for artifacts by understanding what procedures introduce which kinds of artifacts. Researchers must know what types of artifacts to expect with each tissue type and processing technique to accurately interpret sections with artifacts, or idealy select techniques that will minimize artifacts production in the areas being analysed.

Here is a list of the main types of histology fixatives, as well as some of their advantages and disadvantages

Fixatives Pros and Cons
FixativesEffectPositive FeaturesNegative FeaturesType
Neutral Buffered Formalin (10% NBF)Forms cross links between protein groups, pH dependant.Tolerant fixative as substances can be left in indefinitely without harm. Cross links can be broken easily using water, making protein groups available for other reactions. Tissue remains soft which is good for dissection and processing. Red blood cells and cytological detail well preservedSubstance can be Toxic, Carcinogenic, Sensitizing agent hazardous, tissue should be 1cm maxCompound Fixative, aldehyde
GluteraldehydeForms cross links between protein groups, pH dependant. Main difference has 2 aldehyde groups and quicker reaction.Very good tissue morphology for later compared to formalin fixationPoor Tissue Penetration, difficult to remove from tissueAldehyde
Acetic AcidPowerful precipitator of nucleoprotein.Good DNA preservationCauses tissue to swell., Poor cytoplasmic staining and mitochondria stainingDenaturing Agent
Ethanol Dissolves fats, denatures proteins and nucleic acids.Preserve chemical reactivity of many cellular materials. Useful in combination fixative eg acetic alcoholTissue shrinkage, may become brittle in timeDenaturing agent

Here is a list of further types of less common fixatives that can be used, as well as some of their advantages and disadvantages

Fixatives Pros and Cons
FixativesEffectPositive FeaturesNegative FeaturesType
Pottasium DichromatepH, High pH acts as non-coagulant , acidic pH as coagulant. Strong fixative effect on certain lipids, useful for myelinated nerve fibre study.Health hazard in the laboratory, Personal protective equipment required around it. Also dissolves nucleic acidsOxidising agent
Osmium TetroxideAdditive fixative turns protein into a gel. Adds on double bonds too.Good for fixing lipids as it adds on a double bondHealth hazard can cause irritation of eye, nose and throat, also very expensive making extensive use impracticalOxidising Agent
Picric AcidNormally used in combination with other fixatives. Denatures proteins.Excellent glycogen preservation, enhances acidic stainingDamages basic dye stainingDenaturing Agent
Bouin SolutionContains picric acid, formaldehyde and glacial acetic acidGood preservation for tissue eg seminal vesicles, good for glycogen, good for small biopsies due to yellow dyePoorer for cytological detail, poor tissue penetration, small tissue onlyMicroanatomical fixative

Here is a list of the least common types of fixatives that can be used, as well as some of their advantages and disadvantages

Fixatives Pros and Cons
FixativesEffectPositive FeaturesNegative FeaturesType
Zenker SolutionContains mercuric chloride, potassium dichromateExcellent nuclear staining and connective tissue, especially for trichrome stainsPoor penetration of tissue, must be 05cm or less, tissue becomes brittle after 24 hours exposure. Tissue after fixation must be washed in water for several hours not usable for frozen sections. Solution does not keep well after acetic acid added.Microanatomical fixative
Carnoy FluidAlcohol, chloroform and acetic acidAllow rapid penetration of tissue by staining, especially staining with acidic dyes. Good for chromosomes fixing, lymph nodes and small biopsies. Rapid fixation after only ½ hour – 3hours, preserves glycogen, after fixation can transfer direct to alcoholShrinkage of tissue excessive, red blood cells destroyed and only useable on small tissueCytological fixatives - Nuclear
Mercuric ChlorideFixes tissue by attaching to proteins and forms crosslinksAllow rapid penetration of tissue by staining, especially staining with acidic dyesExcessive exposure damages tissue and makes microtomy difficultDenaturing Agent
Flemming FluidContains chromic acid, aqueous osmium tetroxide and glacial acetic acid. Also version without acetic acidGood for preservation of nuclear structures, especially chromosomes and permanently preserves lipids. Only small volumes required 1:10 ratio tissue to fluid.Poor penetration so only useful on small tissue samples, solution deteriates rapidly, requires 24 hours washing in water before dehydrationCytological fixatives - Nuclear