Microtomy-Staining

• Microtomy


  The process by which processed tissue, most commonly a paraffin embedded tissue, is trimmed and cut into uniformly thin slices or "sections" to facilitate studies under the microscope

  
• Microtomy


  • Requires precision and hand-eye coordination combined with a delicate touch by the experienced histotechnologist/cian
  • Hand and eye coordination is to avoid accidents while performing the processes
• Microtome


  The basic instrument that is capable of cutting a section at a predetermined thickness by sliding the block into a cutting tool, usually a steel knife, glass or diamond blade, which is fixed and attached to the machine

  
• 3 essential parts of a microtome

  • Block holder where the tissue is held in position
  • Knife Carrier and Knife for actual cutting of tissue sections
  • Pawl, Ratchet Feed Wheel and Adjustment Screws to line up the tissue block in proper position with the knife, adjusting the proper thickness of the tissue for successive sections

  
• Principle of microtome

  A spring-balanced teeth or pawl is brought into contact with, and turns a ratchet feed wheel connected to a micrometer screw, which is in turn rotated, moving the tissue block at a predetermined distance towards the knife for cutting sections at uniform thickness
• Kinds of microtomes

  • Rocking microtome
  • Rotary microtome
  • Sliding microtome
  • Freezing microtome
  • Cryostat or cold microtome
  • Ultrathin microtome
• Rocking (Cambridge) microtome


  The simplest among the different types of microtomes, consisting of a heavy base and two arms the lower arm resting on pivots and a supporting column, and attached to the micrometer screw, at the base of which is found the ratchet wheel with feed mechanism
• Rocking (Cambridge) microtome

  • A section is cut as the tissue passes to the knife edge in a slightly curved plane, in 10-12 u thickness
  • Available in two sizes, has been used to cut small and large blocks of paraffin tissues
  • Not recommended for serial section since tissue are cut into slightly curved plains
  • Not currently favored by some laboratories because of the restriction of size of tissue block that can be cut and difficulty in orienting the block
• Rotary (Minot) microtome


  Invented by Minot in 1885-86 to cut paraffin embedded tissues, and is currently the most common type used for both routine and research laboratories, especially for sectioning paraffin-embedded tissues
• Rotary (Minot) microtome

  • Electrically driven rotary microtomes are available and can be ideally used to produce ribbons for serial sections
  • The cutting section is in perfectly flat plane, allowing excellent serial sections to be cut
  • More stable than rocking microtome
  • More complex in design and structure, so more expensive
• Sliding microtome


  Developed by Adams in 1789, there are two types: Base-Sledge Microtome and Standard Sliding Microtome
• Base-Sledge microtome


  • Consists of two movable pillars holding the adjustable knife clamps, allowing the knife to be set at an angle for cutting celloidin sections
  • Favored in laboratories where very hard tissue or large blocks are usually sectioned
• Standard Sliding microtome


  • The block remains stationary while the knife is moved backward and forward during the process of sectioning
  • For cutting celloidin embedded tissue blocks
  • Inherently more dangerous because of the movable knife, which makes it difficult to attach the knife card
  • Recommended for cutting of extremely hard or rough tissue block
• The sliding microtome is the most dangerous type of microtome due to movable exposed knife
• Freezing microtome


  Invented by Queckett in 1848, used to cut undehydrated thin to semi-thin sections of fresh, frozen tissues, especially in instances when rapid diagnosis is required, when histological demonstration of fat is needed, when certain neurological structures are to be studied, and when sensitive tissue constituents to be studied are damaged or destroyed by heat
• Cryostat or cold microtome


  A refrigerated apparatus used for freezing the tissue into the block holder to the correct degree of hardness that allows for easier and faster sectioning, consisting of a microtome, usually a rotary microtome, kept inside a cold chamber which has been maintained at a temperature between -5° to -30°C
• Cryostat or cold microtome


  • Provides a means of preparing thin sections of fresh frozen tissues especially for fluorescent antibody staining techniques or histochemical enzyme studies
  • Most commonly used for rapid preparation of urgent tissue biopsies for intraoperative diagnosis
• Ultrathin microtome


  An instrument equipped with a glass or gem grade diamond knife used to cut very thin sections (typically 60 to 100 nanometer) of tissue embedded in epoxy resin, which are then stained with an aqueous solution of an appropriate heavy metal salt and examined with a transmission electron microscope (TEM)

  
• Types of microtome knives

  • Plane-concave
  • Biconcave
  • Plane-wedge
• Microtome knives


  • The cutting edge has an angle of 27-32° (Bevel Angle)
  • Glass knives are used for trimming and semi-thin sectioning of tissue block
  • Diamond knives are used to cut any type of resin block, are brittle and expensive but very durable
• Honing


  Removal of gross nicks on the knife edge (also called as the coarse honing) to remove blemishes, grinding the cutting edge of the knife on a stone (carborundum)
• Types of hones

  • Belgium yellow
  • Arkansas
  • Fine carborundum
• Stropping


  The process whereby the "burr" formed during honing is removed and cutting edge of the knife is polished
• Types of tissue sections

  • Paraffin Sections
  • Celloidin Sections
  • Frozen Sections
• Factors to consider in sectioning

  • Cutting depends upon the type of the tissue
  • The size of the block
  • The model or the type of the microtome
• Sections usually 4-6µ thickness for routine histologic procedure
• The knife is usually tilted at 0-15° angulation on a microtome
• Staining


  The process of applying dyes on the sections
• Aims of staining

  • Make the cell structure visible
  • Show variation in structure
  • Indicate the chemical nature and tissue entities
• Differential action of dyes

  The depth of coloration is affected by chemical affinity, density, and permeability + diffusion of dye
• Aims of Staining

  • Make the cell structure visible
  • Show variation in structure
  • Determine if tissues are normal or abnormal
  • Indicate the chemical nature and tissue entities

  
• Differential action of dyes

  The depth of coloration is affected by: Chemical affinity, Density, Permeability + diffusion of dye
• Chemical affinity


  Certain parts of cells and tissues that are acidic in nature like nucleus have greater affinity than basic dyes. The basic constituent like the cytoplasm takes more of the acid stain.
• Density and permeability


  Facilitates the contrasting stain used like the hematoxylin stains the nuclear details and the eosin that brings out the cytoplasmic details of the cells and the tissue architecture
• Tissue staining can be classified into three major groups

  • Histological Staining
  • Histochemical Staining (Histochemistry)
  • Immunohistochemical staining
• Histological Staining


  A process whereby the tissue constituents are demonstrated in sections by direct interaction with a dye or staining solution producing coloration of the active tissue component. Used to demonstrate the general relationship of tissues and cells with differentiation of nucleus and cytoplasm.
• Histological Staining Examples

  • Micro-anatomic stains
  • Bacterial stains
  • Specific tissue stains (e.g. muscles, connective tissue and neurologic stains)
• Histochemical Staining (Histochemistry)


  A process whereby various constituents of tissues are studied through chemical reactions that will permit microscopic localization of a specific tissue substance.
• Histochemical Staining Examples

  • Perl's prussian blue for hemoglobin
  • Periodic Acid Schiff for carbohydrates
• Immunohistochemical staining


  A combination of immunologic and histochemical techniques that allow phenotypic markers to be detected and demonstrated under the microscope, using a wide range of polyclonal or monoclonal, fluorescent labeled or enzyme-labeled antibodies.