HP Lec Midterms

• Funktion der Fixation


  • Stabilisierung
  • Erhaltung
• Erhaltung


  Erhaltung der Gewebestruktur
• Fixative


  • Alkohol
  • Formalin
  • Neutral gepuffertes Formalin
• Hauptgruppen von Fixativen


  • Quervernetzende Fixative
  • Formaldehyd und Formalin
• Bei großen, weichen Präparaten wie dem menschlichen Gehirn dauert es 2-4 Wochen in neutral gepuffertem Formalin, bis sie fest genug sind, um geschnitten zu werden
• Fixative


  • 10% Formal-Saline
  • 10% neutral gepuffertes Formalin
  • Zinkformalin (ungepuffert)
  • Formol-Korrosiv (Formol-Sublimat)
  • Paraformaldehyd
  • Karnovsky-Fixativ (4% Paraformaldehyd-1% Glutaraldehyd in 0,1 M Phosphatpuffer)
  • Glutaraldehyd
• Formol-Zenker


  A fixative composed of formaldehyde and dichromate components
• Formol-Zenker is unstable due to the reaction between formaldehyde and dichromate components, producing formic acid and chromic ions, causing the solution to turn greenish within a few hours
• Advantages of Formol-Zenker


  • It penetrates and hardens tissues rapidly and well
  • Nuclear components are shown in fine detail
  • It precipitates all proteins
  • It has a greater affinity to acid dyes and is preferred in lieu of formaldehyde for cytoplasmic staining
  • Trichrome staining is excellent
  • It is the routine fixative of choice for preservation of cell detail in tissue photography
  • It permits brilliant metachromatic staining of cells
  • It is recommended for renal tissue, fibrin, connective tissue and muscle
• Disadvantages of Formol-Zenker


  • It causes marked shrinkage of cells (this may be counteracted by addition of acid)
  • It rapidly hardens the outer layer of the tissue with incomplete fixation of the center; therefore, thin sections should be made
  • Penetration beyond the first 2-3 millimeters is slow; hence, not more than 5 mm. thickness of tissues should be used
  • If left in fixative for more than 1-2 days, the tissue becomes unduly hard and brittle
  • It prevents adequate freezing of fatty tissues and makes cutting of frozen tissues difficult
  • It causes considerable lysis of red blood cells and removes much iron from hemosiderin
  • It is inert to fats and lipids
  • It leads to the formation of black granular deposits in the tissues
  • It reduces the amount of demonstrable glycogen
  • Compound solutions containing mercuric chloride deteriorate rapidly upon addition of glacial acetic acid to formalin
  • It is extremely corrosive to metals
• Precautions for Formol-Zenker


  • Sections must be cleared of mercury deposits before immunostaining. Black deposits may be removed by adding saturated iodine solution in 95% alcohol, the iodine being decolorized with absolute alcohol in the subsequent stages of dehydration
  • Compound solutions must always be freshly prepared
  • The use of metallic forceps and of metal caps to cover the bottles containing the fixative should be avoided
  • Contact of mercuric fixatives with personal jewelries should be avoided
  • Mercury-containing solutions (Zenker's or B-5) should always be discarded into proper containers. Mercury, if poured down a drain, will form amalgams with the metal that build up and cannot be removed
• Zenker's Solution


  A fixative composed of mercuric chloride, potassium dichromate, distilled water, and glacial acetic acid
• Advantages of Zenker's Solution

  • It produces a fairly rapid and even fixation of tissues
  • Stock solutions keep well without disintegration
  • It is recommended for trichrome staining
  • It permits brilliant staining of nuclear and connective tissue fibers
  • It is recommended for congested specimens (such as lung, heart and blood vessels) and gives good results with PTAH and trichrome staining
  • It is compatible with most stains
  • It may act as a mordant to make certain special staining reactions possible
  • It is a stable fixative that can be stored for many years
• Disadvantages of Zenker's Solution

  • Penetration is poor
  • It is not stable after addition of acetic acid
  • Prolonged fixation (for more than 24 hours) will make tissues brittle and hard
  • It causes lysis of red blood cells and removes iron from hemosiderin
  • It does not permit cutting of frozen sections
  • It has the tendency to form mercuric pigment deposits or precipitates
  • Tissue must be washed in running water for several hours (or overnight) before processing. Insufficient washing may inhibit or interfere with good cellular staining
• Precautions for Zenker's Solution

  • Do not let tissues stay in solution for more than 24 hours
  • Solutions must always be freshly prepared
  • Tissues should be cut thin (2-3 mm.) and hollow organs should be opened to promote complete penetration and fixation
  • Tissues must be washed out thoroughly in running water to permit good staining
  • It produces mercury pigment which should be removed from sections prior to staining and can produce chrome pigment if tissue is not washed in water prior to processing. After water washing, tissue should be stored in 70% ethanol
  • Mercuric deposits may be removed by immersing tissues in alcoholic iodine solution prior to staining, through a process known as de-zenkerization
• Zenker's Solution is an excellent fixative for bone marrow, extramedullary hematopoiesis and intercalated discs of cardiac muscle
• Zenker's Solution produces mercury pigment which should be removed from sections prior to staining and it can produce chrome pigment if tissue is not washed in water prior to processing
• After water washing, fixed tissue should be stored in 70% ethanol. Because of the low pH of this fixative, formalin pigment may also occur
• Never use metal forceps to handle tissue fixed with Zenker's Solution
• Helly's Solution


  A fixative composed of mercuric chloride, potassium dichromate, distilled water, and 40% formaldehyde
• Advantages of Helly's Solution

  • It is an excellent microanatomic fixative for pituitary gland, bone marrow and blood containing organs such as spleen and liver
  • It penetrates and fixes tissues well
  • Nuclear fixation and staining with Helly's solution is better than with Zenker's
  • It preserves cytoplasmic granules well
• The disadvantages of Helly's solution are similar to Zenker's except that brown pigments are produced if tissues (especially blood containing organs) are allowed to stay in the fixative for more than 24 hours due to RBC lysis. This may be removed by immersing the tissue in saturated alcoholic picric acid or sodium hydroxide
• Lillie's B-5 Fixative


  A fixative composed of 4% aqueous formaldehyde, 0.22M mercuric chloride, and 0.22M acetic acid. Acetic acid induces the coagulation of nuclear chromatin, while mercuric chloride ensures rapid structural stabilization and enhances staining with various dyes
• Tissues fixed with mixtures containing mercuric chloride develop a brown crystalline precipitate, likely mercurous chloride (Hg2Cl2), referred to as mercury pigment
• Due to its mercury content, B-5 is subject to toxic waste disposal regulations, applying not only to the fixative solution but also to any subsequent solutions contaminated with mercury
• Practical Applications of Lillie's B-5 Fixative

  • Despite its mercuric chloride content and consequent problems with disposal, this solution is popular for fixation of hematopoietic, bone marrow biopsies and lymphoid tissue
  • Rapid fixation can be achieved in 1 1/2 - 2 hours
  • It produces excellent nuclear detail, provides good results with many special stains, and is recommended for immunohisto-chemical staining
  • Sections will require the removal of mercury pigment prior to staining
  • Tissue should not be stored in this fixative but placed in 70% ethanol instead
  • Over-fixation hardens the tissue and makes cutting difficult
  • The two working solutions are kept separate, since the mixture is unstable. Mix just prior to use
  • Some B-5 solutions will form precipitate on standing, but this is of no consequence
  • As with all mercuric chloride fixatives, the mercury pigment can be removed by de-zenkerization
• Heidenhain's Susa Solution


  A fixative recommended mainly for tumor biopsies especially of the skin; it is an excellent cytologic fixative
• Advantages of Heidenhain's Susa Solution

  • It penetrates and fixes tissues rapidly and evenly
  • It produces minimum shrinkage and hardening of tissues due to the counter-balance of the swelling effects of acids and the shrinkage effect of mercury
  • It permits most staining procedures to be done, including silver impregnation, producing brilliant results with sharp nuclear and cytoplasmic details
  • It permits easier sectioning of large blocks of fibrous connective tissues
  • Susa-fixed tissues may be transferred directly to 95% alcohol or absolute alcohol, thereby reducing processing time
• Disadvantages of Heidenhain's Susa Solution

  • Prolonged fixation of thick materials may produce considerable shrinkage, hardening and bleaching; hence, tissues should not be more than 1 cm. thick
  • RBC preservation is poor
  • Some cytoplasmic granules are dissolved
  • Mercuric chloride deposits tend to form on tissues; these may be removed by immersion of tissues in alcoholic iodine solution
  • Weigert's method of staining elastic fibers is not possible in Susa fixed tissues
• After using Heidenhain's Susa fixative, the tissue should be transferred directly to a high-grade alcohol, e.g. 96% or absolute alcohol, to avoid undue swelling of tissues caused by treatment with low-grade alcohol or water
• Osmium tetroxide (Osmic Acid; OsO4)


  A fixative that fixes conjugated fats and lipids permanently, preserves cytoplasmic structures well, and adequately fixes materials for ultrathin sectioning in electron microscopy
• Advantages of Osmium tetroxide


  • It fixes conjugated fats and lipids permanently
  • It preserves cytoplasmic structures well, e.g. Golgi bodies and mitochondria
  • It fixes myelin and peripheral nerves well
  • It produces brilliant nuclear staining with safranin
  • It adequately fixes materials for ultrathin sectioning in electron microscopy
  • It precipitates and gels proteins
  • It shows uniformly granular nuclei with clear cytoplasmic background
  • Some tissues (e.g. adrenal glands) are better fixed in vapor form of osmium tetroxide
  • Osmium tetroxide completely permeabilizes cell membranes
  • It penetrates tissue blocks in a gradient
• Disadvantages of Osmium tetroxide

  • It is very expensive
  • It is a poor penetrating agent, suitable only for small pieces of tissues (2-3 mm.thick)
  • It is readily reduced by contact with organic matter and exposure to sunlight, forming a black precipitate which settles at the bottom of the container
  • Prolonged exposure to acid vapor can irritate the eye, producing conjunctivitis, or cause the deposition of black osmic oxide in the cornea, producing blindness
  • It inhibits hematoxylin and makes counterstaining difficult
  • It is extremely volatile
• Precautions for using Osmium tetroxide

  • Eyes and skin may be protected by, working in a fume hood or wearing protective plastic masks or gloves while using osmium tetroxide
  • It should be kept in a dark-colored, chemically clean bottle to prevent evaporation and reduction by sunlight or organic matter
  • It should be kept in a cool place or refrigerated to prevent deterioration
  • Addition of saturated aqueous mercuric chloride solution (0.5 to 1 ml/ 100 ml of stock solution) will prevent its reduction with formation of black deposit
  • Block osmic oxide crystals may be dissolved in cold water
  • To prevent contact of tissues with black precipitate formed in the bottom of the jar, the tissues may be wrapped in cotton gauze and suspended in the fluid by means of a thread
  • Osmic acid-fixed tissues must be washed in running water for at least 24 hours to prevent formation of artefacts
• Flemming's Solution


  A fixative composed of aqueous chromic acid, 1% aqueous osmium tetroxide, and 2% glacial acetic acid
• Advantages of Flemming's Solution

  • It is an excellent fixative
• Precautions when using osmium tetroxide

  • Wear protective plastic masks or gloves
  • Keep in a dark-colored, chemically clean bottle to prevent evaporation and reduction by sunlight or organic matter
  • Keep in a cool place or refrigerated to prevent deterioration
• Preventing reduction of osmium tetroxide

  Addition of saturated aqueous mercuric chloride solution (0.5 to 1 ml/ 100 ml of stock solution)
• Preventing contact of tissues with black precipitate


  Wrap tissues in cotton gauze and suspend in the fluid by means of a thread
• Washing osmium tetroxide-fixed tissues

  Wash in running water for at least 24 hours to prevent formation of artefacts