Fixation

Cards (173)

  • FIXATION
    First and most critical step in histotechnology
  • primary goal of fixation
    preserve the morphologic and chemical integrity of the cell in as life-like a manner as possible
  • second goal of fixation
    to harden and protect the tissue from the trauma of further handling, so that it is easier to cut and process for microscopy.
  • practical purposes of fixation
    prevent or arrest the degenerative processes which commence as soon as a tissue is deprived of its blood supply.
  • Physical methods of fixation
    heating, microwaving and cryo-preservation
  • Heat fixation
    rarely used on tissue specimens
    its application being confined to smears of microorganisms
  • microwave fixation
    can be regarded as a form of heat fixationis now widely practiced in routine laboratories
  • Cryo-preservation
    usually in the form of freeze drying, has some applications in histochemistry but is not usually applied to diagnostic tissue specimens.
  • Chemical fixation
    usually achieved by immersing the specimen in the fixative solution
  • Additive fixation
    chemical constituent of the fixative is taken in and becomes part of the tissue by forming cross-links
  • Examples of Additive Fixative
    formalin
    mercury
    osmium tetroxide
  • Non-additive fixation
    fixing agent is not incorporated into the tissue, but alters the tissue composition and stabilizes the tissue by removing the bound water attached to H-bonds of certain groups within the protein molecule
  • Example of Non-addditive Fixative
    Alcohol Fixatives
  • Volume
    10-20 times the volume of tissue to be fixed.
  • Hydrogen Ion Concentration
    neutral pH, in the range of 6-8
    Acidity favors formation of formalin-heme pigment that appears as black, polarizable deposits in tissue
  • Temperature
    40°C for regular tissue processing
    electron microscopy and some histochemistry, the ideal temperature is 0-4°C
    Formalin heated to 60°C is sometimes used for the rapid fixation of very urgent biopsy specimens
  • bone marrow continues to undergo mitosis (growth) up to 30 minutes after death when refrigerated
  • Nucleic acids do not react with fixatives to any extent at room temperatures, and chemical reactions including those involved in fixation are more rapid at higher temperatures.
  • Thickness of section
    1 to 2 mm2 for electron microscopy
    2 cm2 wide for light microscopy
  • Brain is usually suspended whole in 10% buffered formalin for 2-3 weeks
  • Formalin and alcohol penetrate the best, and glutaraldehyde the worst. Mercurials and others are somewhere in between.
  • Osmolality
    fixed in a hypertonic solution, the cells may shrink
    fixed in a hypotonic solution, the cells may swell and burst
    Isotonic as well as hypotonic fixatives cause cell swelling and poor fixation (340 mOsm)
    recommend using a normal phosphate buffered saline (PBS) based fixative
    best results are usually obtained using slightly hypertonic solutions (400-450 mOsm)
  • Concentration
    adjusted down to the lowest level possible
    Too high a concentration may adversely affect the tissue and produce artefact similar to that caused by excessive heat
  • Duration of fixation
    Fibrous organs such as uterus or intestinal tract take longer to fix than small or loosely textured tissues such as biopsies or scrapings
  • Prolonged fixation may cause shrinkage and hardening of tissue, and may severely inhibit enzyme activity and immunological reactions, although washing of the tissue in running water considerably restores the activity of some enzymes
  • electron microscopy, it is recommended that diced tissues be fixed for 3 hours and then placed in a holding buffer.
  • Time Interval
    fixed immediately after removal or death to prevent autolysis or putrefaction
  • Simple Fixatives
    made up of only one component substance
  • Compound Fixatives
    made up of two or more fixatives which have been added together to obtain the optimal combined effect of their individual actions upon the cells and tissue constituents.
  • Microanatomical Fixatives
    permit the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of the tissues in question
  • Microanatomical Fixatives
    10% formal saline
    10% neutral buffered formalin
    Heidenhain 's Susa
    Formal sublimate (formal corrosive)
    Zenker 's solution
    Zenker-formal (Kelly 's solution)
    Bouin's solution
    Brasil's solution
  • Cytological Fixatives
    preserve specific parts and particular microscopic elements of the cell itself.
  • Nuclear Fixatives
    preserve the nuclear structures (e.g., chromosomes) in particular. They usually contain glacial acetic acid as their primary component due to its affinity for nuclear chromatin.
    They have a pH of 4.6 or less.
  • Nuclear Fixative
    Flemming's fluid
    Carnoy's fluid
    Bouin's fluid
    Newcomer's fluid
    Heidenhain's Susa
  • Cytoplasmic Fixatives
    preserve cytoplasmic structures in particular.
    They must never contain glacial acetic acid which destroys mitochondria and Golgi bodies of the cytoplasm.
    They have a pH of more than 4.6
  • Cytoplasmic Fixatives
    Flemming's fluid without acetic acid
    Kelly's fluid
    Formalin with "post-chroming"
    Regaud 's fluid (Muller 's fluid)
    Orth 's fluid
  • For RNA
    the precipitant fixatives - ethanol and acetone - give the best quantitative results using frozen tissues as the standard
  • Histochemical Fixatives
    those that preserve the chemical constituents of cells and tissues.
  • Histochemical Fixatives
    Formal Saline 10%
    Absolute Ethyl
    Newcomer's Fluid
    Alcohol Acetone
  • Secondary Fixation
    process of placing an already fixed tissue in a second fixative in order:
    To facilitate and improve the demonstration of particular substances.
    To make special staining techniques possible (with secondary fixative acting as a mordant).
    To ensure further and complete hardening and preservation of tissues.