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Flashcards in this deck (64)
  • Fixation is the killing, penetration, and hardening of tissues by stabilizing proteins to resist further changes.

    histology fixation
  • The primary goal of fixation is to preserve cells and tissues as close to their living state as possible.

    histology fixation
  • The secondary goals of fixation are to harden tissue for cutting and protect against handling trauma.

    histology fixation
  • The ideal time to perform fixation after tissue removal is 20–30 minutes following interruption of blood supply.

    histology fixation
  • The recommended volume ratio of fixative to tissue for maximum effectiveness is 20 parts fixative to 1 part tissue.

    histology fixation
  • Formalin diffuses into tissue at a rate of approximately 1 mm per hour.

    histology fixation
  • A fixative should have a pH range between 6.0 and 8.0.

    histology fixation
  • The preferred osmolality for fixatives is slightly hypertonic or isotonic; hypertonic causes cell shrinkage, hypotonic causes swelling.

    histology fixation
  • Two additive fixatives are formaldehyde and glutaraldehyde.

    histology fixatives
  • Two non-additive fixatives are acetone and ethanol.

    histology fixatives
  • The recommended fixative for electron microscopy is osmium tetroxide because it preserves lipids and provides excellent ultrastructural detail.

    histology electron_microscopy
  • The fixative traditionally used for general histopathology that is tolerant for mailing specimens is 10% neutral buffered formalin.

    histology fixatives
  • The fixative recommended for enzyme histochemistry is 4% formaldehyde or formol saline.

    histology enzyme_histochemistry
  • 10% formalin is composed of 100 mL of 37–40% formaldehyde diluted to 1 L with distilled water.

    histology formalin
  • Formol corrosive is formed by combining formaldehyde with mercuric chloride.

    histology fixatives
  • The three main methods of paraffin wax infiltration are manual, automatic, and vacuum.

    histology paraffin_infiltration
  • The melting point of routine paraffin wax is 56 °C and it cannot be used for fatty tissues.

    histology paraffin
  • Two substitutes for paraffin wax are Paraplast (melting point 56–57 °C) and Ester wax (melting point 46–48 °C).

    histology paraffin_substitutes
  • The concentration and time for thin celloidin infiltration is 2–4% for 5–7 days.

    histology celloidin
  • The section thicknesses for paraffin, celloidin, frozen, and ultrathin are: Paraffin: 4–6 µm, Celloidin: 10–15 µm, Frozen: 4 µm, Ultrathin (EM): 0.5 µm.

    histology section_thickness
  • The most commonly used microtome for routine paraffin sections is the rotary microtome.

    histology microtome
  • The bevel, clearance, and wedge angles for a microtome knife are: Bevel: 27–32°, Clearance: 0–15°, Wedge: 14–15°.

    histology microtome
  • Honing grinds off nicks with oil stones using 20–30 strokes, while stropping polishes on leather using 40–120 strokes.

    histology knife_maintenance
  • Three methods of deparaffinization are flame, xylene immersion, and 55–60 °C oven incubation.

    histology deparaffinization
  • Three adhesives used for slide attachment are Mayer’s egg albumin, poly-L-lysine, and APES.

    histology adhesives
  • The main reagents and steps in a routine H&E stain are: Xylene (deparaffinize) → descending alcohols (hydrate) → hematoxylin (nuclear stain) → acid alcohol (decolorize) → ammonia water (blue nuclei) → eosin (cytoplasm) → ascending alcohols (dehydrate) → xylene (clear).

    histology h&e_stain
  • In H&E staining, nuclei appear blue–black, cytoplasm appears pale pink, and muscle fibers appear deep pink.

    histology h&e_stain
  • Progressive staining involves applying dye until desired intensity with no decolorization, while regressive staining involves overstaining then removing excess by decolorization.

    histology staining
  • Two metachromatic stains are toluidine blue and methyl violet, which detect glycosaminoglycans.

    histology staining
  • Stains that demonstrate lipids in frozen sections include Sudan Black B, Oil Red O, and Sudan IV.

    histology lipid_staining
  • The Feulgen reaction is a specific histochemical stain for DNA—it hydrolyzes to expose aldehydes, then Schiff’s reagent binds to yield magenta nuclei.

    histochemistry dna staining
  • The gold standard for amyloid detection is Alkaline Congo red which shows apple-green birefringence under polarized light.

    histochemistry amyloid staining
  • To demonstrate myelin with Nissl substance, use Luxol Fast Blue for myelin plus Cresyl Violet counterstain for Nissl.

    histochemistry myelin nissl
  • Two enzyme histochemistry methods are Gomori’s calcium method for alkaline phosphatase and Gomori’s lead method for acid phosphatase.

    histochemistry enzymes methods
  • For rapid fixation of urgent biopsies, the recommended fixative method is Formalin heated at 60 °C.

    fixation biopsy histochemistry
  • The fixative used for neurochemical substances like acetylcholine is Microwave fixation or 0–4 °C glutaraldehyde for EM/histochemistry.

    fixation neurochemistry
  • The silver impregnation stain used for reticulin fibers is Gomori’s silver impregnation (or Gordon & Sweet’s method).

    staining reticulin histochemistry
  • The stain used for myeloid peroxidase activity is the Peroxidase reaction which turns myeloid granules green to dark blue.

    staining myeloid peroxidase
  • The best stain for detecting Helicobacter pylori is the Warthin-Starry silver stain (or Gimenez method).

    staining helicobacter histochemistry
  • The metallic impregnation method used for astrocytes is Cajal’s gold sublimation.

    staining astrocytes histochemistry
  • Three special fixatives and their uses are: Bouin’s for embryos/pituitary/endometrium, Carnoy’s for chromosome prep, and Newcomer’s for histochemical preservation of glycogen.

    fixation histochemistry special
  • The purpose of gelatin infiltration is to stabilize tissue without dehydration/clearing during frozen sections and enzyme studies.

    infiltration gelatin histochemistry
  • The embedding medium that is water-soluble for enzyme histochemistry is Carbowax (polyethylene glycol).

    embedding enzyme histochemistry
  • The typical steps and alcohol concentrations for tissue dehydration are: 70% → 80% → 90% → 100% ethanol, usually 1–2 changes at each grade.

    dehydration alcohol histochemistry
  • Three common clearing agents are: Xylene (fast, but toxic), toluene (good penetration, slower), and chloroform (gentle, carcinogenic).

    clearing agents histochemistry
  • The recommended volume ratio and time for paraffin infiltration in an Autotechnicon is 4 changes of wax over 1 h with constant agitation.

    paraffin infiltration histochemistry
  • Vacuum infiltration is used to remove air faster, ideal for urgent biopsies and lungs/brain specimens.

    infiltration vacuum histochemistry
  • The process that fills tissue cavities and spaces before embedding is infiltration/impregnation with paraffin, celloidin, gelatin, or resin.

    embedding infiltration histochemistry
  • The steps for gelatin embedding include: 10% gelatin + 1% phenol for 24 h → 20% gelatin + 1% phenol for 12 h → fresh 20% gelatin + 1% phenol cool until solid → 10% formalin for 12–24 h.

    embedding gelatin histochemistry
  • Double embedding involves infiltrating first with celloidin then embedding in paraffin for fragile tissues.

    embedding double histochemistry
  • Three types of embedding molds and their uses are: Leuckhart’s brass for routine blocks, compound unit for multiple specimens, and peel-away plastic for no trimming.

    embedding molds histochemistry
  • To cool a paraffin block to solidify, refrigerate at –5 °C or immerse the mold in cold water.

    paraffin cooling histochemistry
  • The speed of frozen sectioning in a cryostat is determined by temperature (–5 to –30 °C), freezing agent, and section thickness.

    frozen sectioning cryostat
  • Four freezing agents are: Liquid nitrogen (ultra-cold, expensive), isopentane–LN2 (rapid, flammable), dry ice (–78 °C, slices thicker), and CO₂ gas (fast, uneven freeze).

    freezing agents cryostat
  • The principle of decalcification is to remove mineral salts to soften tissue; agents include acids (HCl) for speed and chelators (EDTA) for preservation.

    decalcification agents histochemistry
  • Formalin pigments can be removed using Kardasewitsch: 70% ethanol + 28% ammonia; Lillie’s: H₂O₂ + ammonia; or Picric acid.

    formalin pigments removal
  • A mordant is a substance that bridges dye to tissue (e.g., alum); an accentuator intensifies selectivity/intensity (e.g., phenol).

    mordant accentuator staining
  • The difference between direct and indirect staining is that direct staining involves the dye binding tissue directly, while indirect staining requires a mordant for binding.

    staining direct indirect
  • The PAS reaction mechanism involves periodic acid oxidizing carbohydrates to aldehydes → Schiff’s reagent binds aldehydes producing a magenta color.

    pas reaction staining
  • The PAS reaction mechanism involves periodic acid oxidizing carbohydrates to aldehydes.

    biochemistry pas reaction
  • In the PAS reaction, Schiff’s reagent binds to aldehydes producing a magenta color.

    biochemistry pas color
  • The PAS reaction is used to identify carbohydrates by detecting aldehydes.

    biochemistry pas identification
  • The first step in the PAS reaction is the oxidation of carbohydrates to aldehydes by periodic acid.

    biochemistry pas mechanism
  • The final product of the PAS reaction is a magenta color due to the binding of Schiff’s reagent to aldehydes.

    biochemistry pas color