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    Fixation

    Tissue
    Processing Dehydration

    Clearing

    Impregnation

    Embedding and blocking

    Section cutting (Microtomy)

    Routine staining (H & E)


    Fixation – Denaturation & precipitation of protein
    i. Prevents putrefaction and autolysis
    ii. Hardens the tissue which helps in section cutting.
    iii. Makes cell insensitive to hypertonic or hypotonic solutions
    Ideal fixative
    • It should be cheap and easily available
    • It should be stable and safe to handle
    • It should be rapid in action
    • It should cause minimal loss of tissue.
    • It should give even penetration.
    • It should retain normal colour of the tissue.
    • It should not impart its own colour to the tissue.
    Formalin
    • This is the most commonly used fixative in routine practice

    • Formalin is commercially available as 40 % formaldehyde & is considered as

    100% formalin.

    • For routine fixation, 10% formalin is used which is prepared by dissolving 10

    ml of commercially available formalin in 90 ml of water.


    Formalin
    Duration of fixation depends upon the size and thickness of the tissue, type of

    tissue and its density

    It takes 6-8 hours for fixation of a thin piece of tissue 4 mm thick at room

    temperature

    The amount of fixative should be 15 to 20 times the volume of the specimen.


    Other fixatives
    • Gluteraldehyde – Electron microscopy
    • Bouins Fluid (Picric acid)- Renal & testicular biopsy,
    demo of glycogen
    • Carnoy’s Fixative (Alcohol) – for cytologic smears &
    endometrial curratage
    • Osmium Tetraoxide- CNS & Electron microscopy, lipid
    Dehydration
    • This is a process in which water from the tissues and cells is
    removed so that this space so created is subsequently taken
    up by wax
    • Dehydration is carried out by passing the tissues through a
    series of ascending grades of alcohol: 70%, 80%, 95% and
    absolute alcohol
    • alternatives such as methyl alcohol, isopropyl alcohol or
    acetone can be used
    Clearing
    • This is the process in which alcohol from the tissues and
    cells is removed (dealcoholisation) and is replaced by a fluid
    in which wax is soluble
    • It also makes the tissue transparent
    • Xylene is the most commonly used clearing agent
    • Alternatives- Toluene,
    • benzene (which is carcinogenic),
    • chloroform (which is poisonous)
    • cedar wood oil (which is expensive and very viscous)
    Impregnation
    • This is the process in which empty spaces in the tissues and
    cells after removal of clearing agent are taken up by molten
    paraffin wax
    • This hardens the tissue which helps in section cutting
    • Impregnation is done in molten paraffin wax which has the
    melting point ranging from 54-62°C.
    Embedding and Blocking
    • Embedding of tissue is done in molten wax
    • Wax blocks can be conventionally prepared using metallic L
    (Leuckhart’s) moulds; nowadays plastic moulds of different
    colours for blocking are also available
    • Same plastic moulds with detachable plastic covers can be
    used for the twin purpose of tissue capsule as well as
    moulds for blocking
    Embedding and Blocking
    Vacutainers

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