Stains and microscope

• Tissue preparation


  1. Fresh tissue

  2. Whole mount

  3. Smear

  4. Stained sections
• Histologic staining


  • Chemical basis for staining
  • Acid-base (hematoxylin and eosin, H & E)
  • Trichrome
  • Silver stains
  • Lipid stains
  • Periodic acid-Schiff's and few others
• Tissue processing for microscopic studies


  Demonstrate appropriate light microscopic techniques
• Tissue Preparation and Staining is an important procedure, and when done accurately the results obtained can guide the treatment course, management, and prognosis
• There are various processes that a tissue sample/specimen has to undergo before it can be analyzed via microscopy
• The histological stains chosen for a given specimen depend on the investigational inquiry or study method ordered/required
• Tissue Preparation


  1. Fixation

  2. Dehydration

  3. Clearing

  4. Infiltration

  5. Embedding

  6. Trimming
• Fixation


  • Prevent tissue degradation
  • Preserve components of the tissue
  • Harden the tissue
• Fixative


  Transforms the contents of the cell from a semifluid state to a semisolid state and prepares the cell contents for visualization with stains, dyes, or metallic salts
• Methods of Fixation


  1. Immersion fixation

  2. Perfusion fixation
• Perfusion fixation


  Fixative is preceded by a wash of physiological saline containing heparin and a substance that will paralyze the vessel walls, then perfused through the intact vascular system of the organism
• Immersion Fixation


  • Involves the immersion of the tissue sample in a fixative
  • Penetrates rapidly
  • Allows the fixative to penetrate all sides of the tissue sample
• Freezing


  • Rapid diagnosis is required
  • Fresh tissue sample is retrieved from the patient
  • Immersion in liquid carbon dioxide or in a substance (such as isopentane)
  • Cooled extremely rapidly by dry ice
  • Prevents maximum tissue disruption
• Dehydration


  • Goal is to remove the water from the tissue
  • Alcohol can be used to remove water from the tissue
• Types of Fixatives


  • Formalin
  • Picric acid
  • Aldehydes
  • Alcohols
  • Mercuric chloride
  • Acetic acid
• Clearing


  Process by which alcohol is removed in organic solvents in which both alcohol and paraffin are miscible
• Clearing Method


  1. Tissue is passed from 100% alcohol through changes of the clearing reagent

  2. Progressively removes the alcohol from the tissue and replaces it with the clearing reagent
• Infiltration


  Process which the tissue is then placed in melted paraffin until it becomes completely infiltrated with this paraffin
• Infiltration Method


  1. Tissue sample is taken from the last step in the clearing reagent and placed in melted embedding medium (wax; such as paraffin, paraplast or bioloid)

  2. Sample is then progressively passed through several changes of the embedding material
• Embedding


  Process by which paraffin-infiltrated tissue is placed in a small mold with melted paraffin and allowed to harden
• Embedding Method


  1. Tissue is removed from the last infiltration step and immediately placed in melted medium in a vacuum oven

  2. Tissue sample is oriented in an embedding mold

  3. Mold is then filled with melted medium and allowed to cool so the medium hardens
• Trimming/Sectioning


  • Involves mounting the sample on a microtome and sectioning (slicing) it into sections
  • Preferred thickness is 4-5 micrometers so that it can be placed on a microscope slide for examination
• Trimming/Sectioning Method


  1. Sections are cut using extremely sharp metal or glass knives

  2. Sections are removed from the edge of the knife either as individual sections or as ribbons of sections

  3. Sample is placed in water to float (usually warmed) or in other fluids depending on the specific technique
• Light Microscopy


  • For paraffin embedded tissue: Sections range in thickness from about 5 to 12 mm
  • For plastic embedded tissue: Sections range in thickness from about 0.5 to 2.0 mm
  • Special techniques may require sections up to 40 to 70 mm in thickness
• Electron Microscopy


  Sections range in thickness from about 80 to 110 nm
• After the sections are cut, and before they are mounted, ensure the glass slides are clean and treat the slides so that the sections will not come off during the staining process
• Staining


  • To highlight
  • Preference
  • Visualize
  • Differences
• Various tissue samples have commonalities, for example; epithelium, connective tissue, muscle tissue, and nervous tissue may appear similar however, they look very distinct structurally after staining
• In certain cases, multiple stains can be performed on multiple slides (micrographs) to gather the full range of structural information on the sample
• Acidic dyes


  • Carry negative charges and are, therefore, known as anionic dyes
  • Attracted to positive charges within the tissue
  • Example: Eosin Y
• Basic dyes


  • Carry positive charges and are, therefore, known as cationic dyes
  • Attracted to negative charges within the tissue
  • Examples: Hematoxylin and toluidine blue
• Hematoxylin and Eosin (H&E)


  • Most commonly used combination stain
  • Hematoxylin stains proteins a blue color, while Eosin stains proteins a pink color
  • Defines intracellular organelles and proteins
• Hematoxylin


  • Most commonly used nuclear stain
  • Complexes with nucleic acids such as DNA and RNA in the nucleus and cytoplasm
  • Structures that bind hematoxylin are termed "basophilic"
• Eosin


  • Acidic dye that stains basic structures "eosinophilic" or "acidophilic"
  • Stains membranes and most proteins
  • Stains collagen red/orange, actin pink, and elastin glassy red
• Trichrome Stains


  Mixture of acidic dyes used to demonstrate connective tissue (collagen stains blue), other cellular constituents (nuclei and cytoplasm generally stain red), and blood cells (erythrocytes stain yellow)
• Gram Stain


  • First-line lab test in bacterial identification
  • Bacteria with thick peptidoglycan layers retain crystal violet dye (gram positive)
  • Bacteria with thin peptidoglycan layers turn red or pink (gram negative) with counterstain
• Giemsa Stain


  Used to identify Chlamydia, Rickettsia, Trypanosomes, Borrelia, Helicobacter pylori, Plasmodium
• Periodic Acid-Schiff Stain


  • Stains glycogen, mucopolysaccharides
  • Used to diagnose Whipple disease
• Silver Stain


  Used to identify Helicobacter pylori, Legionella, Bartonella henselae, and fungi
• Fluorescent Antibody Stain


  • Used to identify many bacteria, viruses, Pneumocystis jirovecii, Giardia, and Cryptosporidium
  • Example is FTA-ABS for syphilis