histopath

• Formaldehyde:

  • Most widely used fixative
  • Produced from the oxidation of methyl alcohol
  • 37% - 40% weight in volume soluble in water
  • Commercially available as 35% - 40% gas by weight
  • 40% pure stock solution is unsatisfactory as it overhardens the outer layer of tissue and affects staining adversely
  • Commonly used as a 4% solution, giving 10% formalin for tissue fixation
  • Buffered to pH 7 with phosphate buffer
  • Fixation time: 24 hours for tissue <5mm thick, complete in 6 - 12 hours at room temperature
• • Advantages: cheap, readily available, easy to prepare, stable especially if stored in buffered solution, compatible with many stains, does not overharden tissue, penetrates well, preserves fat and mucin, preserves glycogen, preserves but does not precipitate proteins, allows tissue enzymes to be studied, does not make tissue brittle, recommended for nervous tissue preservation, allows restoration of natural tissue color, allows frozen tissue to be prepared easily, does not require washing out unless stored for long periods of time, tolerant fixative, used for mailing specimens
• • Disadvantages: fumes irritating, sinusitis, allergic rhinitis, excessive lacrimation, irritating to skin, allergic dermatitis, may produce considerable shrinkage, soft fixative that does not harden some cytoplasmic structures for paraffin embedding
  • Precautions: proper ventilation to prevent inhalation of toxic fumes, rubber gloves to avoid dermatitis, change fixative every 3 months to prevent bleaching, immerse tissue in 70% alcohol to restore natural tissue color
• 10% Formol-Saline:

  • Simple microanatomical fixative
  • Formaldehyde (40% by weight volume) diluted to 10% with NaCl
  • Used for CN tissues and general post-mortem histochemical examination
  • Fixation time: 24 hours at 35°C (95°F) or 48 hours at 40 - 45°C (65 - 77°C)
• • Advantages: even penetration and fixation, preserves microanatomic and cytologic details with minimal shrinkage and distortion, large specimens may be fixed for a long time, preserves enzymes and nucleoproteins, demonstrates fats and mucin, does not harden tissue, facilitates dissection, ideal for most staining techniques and silver impregnation, restores natural tissue color upon immersion in 70% alcohol
  • Disadvantages: slow fixative, tissues tend to shrink during alcohol dehydration
  • Precautions: change fixative every three months
• 10% Neutral Buffered Formalin or Phosphate Buffered Formalin:

  • Preservation of surgical, post-mortem, and research tissues
  • Formulation: Sodium dihydrogen phosphate (anhydrous) 3.5 gm, Disodium hydrogen phosphate (anhydrous) 6.5 gm, Formaldehyde 40% 100 mL, Distilled water 900 mL
  • Fixation time: 4 - 24 hours
  • Advantages: similar to formol-saline, prevents precipitation of acid formalin pigments on post-mortem tissue, best fixative for iron pigments and elastic fibers, requires no post-treatment, inert towards lipids especially neutral fats and phospholipids
• • Disadvantages: longer to prepare, time-consuming, reduced PAS positivity of mucin, gradual loss of basophilic staining, reduced reactivity of myelin to Weigert’s iron hematoxylin
• Formal-Corrosive (Formal Sublimate):

  • Routine post-mortem fixative
  • Formulation: Sat.Aq.Mercuric Chloride 90mL, Formaldehyde 40% 10mL
  • Fixation time: 3 - 24 hours
  • Advantages: penetrates small tissues rapidly, minimum shrinkage and hardening, excellent for many staining procedures like silver reticulum methods, brightens cytoplasmic and metachromatic stains better than formalin, well-preserved cytological structures and blood cells, fixes lipids especially neutral fats and phospholipids, no need for washing out
• • Disadvantages: slow penetration, should not be more than 1 cm thick, forms mercuric chloride deposits, does not freeze tissue sections, inhibits determination of extent of tissue decalcification
• Alcoholic Formalin (Gendre’s Fixative):

  • Post-fixation with phenol-formalin (6 hrs) enhances immunoperoxidase studies
  • Formulation: 95% Ethyl alcohol saturated with picric acid 80mL, Strong formaldehyde solution 15 mL, Glacial HoAc 5mL
  • Advantages: faster fixation, rapid diagnosis, preserves glycogen, good for microincineration technique, fixes sputum and coagulates mucus
  • Disadvantages: produces gross hardening, partial lysis of RBC, poor preservation of iron-containing pigments, poorer morphological picture compared to glutaraldehyde
• Glutaraldehyde:

  • Two formaldehyde residues, 3 carbon chains
  • Used for routine light microscopic work
  • Fixation time: 2.5% for 2 - 4 hours, 4% for 6 - 8 hours up to 24 hours, less than 4 mm thick
  • Advantages over formalin: more stable effect on tissues, firmer structure especially to CNS tissue, preserves plasma proteins better, less tissue shrinkage, better cellular structure, enzyme histochemistry and electron microscopy
  • Disadvantages: expensive, less stable, slow penetration, makes tissue brittle, reduces PAS positivity of reactive mucin
• • Precautions: immerse tissue in concentrated glacial acetic acid and aniline oil to reduce PAS positivity of reactive mucin, keep the vial refrigerated during the fixation process, swirl the vial to ensure the specimen is in contact with fresh solutions all the time
• Mercuric Chloride 5 gm
• Penetrates and hardens tissue rapidly and well
• Fine detail nuclear components
• Precipitates all protein
• Greater affinity to acid dyes, preferred in cytoplasmic staining
• Excellent trichrome staining
• Routine fixative of choice for the preservation of cell detail in tissue photography
• Brilliant metachromatic staining of cells, renal tissues, fibrin, connective tissues, and muscles
• Marked shrinkage, add acid to counteract
• Rapid hardening of outer layer, incomplete fixation at the center, thin sections only
• Beyond first 2-3 mm, penetration is slow, do not use more than 5mm thick
• 1. 2 days fixation results in dull and brittle tissue
• Prevents adequate fixing of fatty tissues, difficult cutting frozen tissues
• Lysis of RBC, removes iron from hemosiderin
• Inert to fats and lipids
• Forms black granular deposits
• Reduces demonstrable glycogen
• Add Saturated Iodine Solution in 96% alcohol for dehydration
• Freshly prepared always
• Do not use metallic caps and bottles
• Remove mercury deposits from deparaffinised section with iodine in ethanol and sodium thiosulfate
• Ultrastructural preservation is poor
• Satisfactory in trichrome and immunoperoxidase techniques
• Deteriorates rapidly upon addition of Glacial acetic acid
• Corrosive to metals
• Zenker’s fluid
• Mercuric chloride stock solution + glacial acetic acid
• Acetic acid prevents turbidity and formation of a dark precipitate