Fixation (preparation method)

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In biology and medicine , especially pathology , fixation refers to the preservation of structures of histological or anatomical objects for the purpose of further investigations. The fixatives used depend on the further use of the preparation produced in this way.

Methods

method Example of scope Details of the procedure
Formaldehyde , paraformaldehyde Conventional histology and immunohistology, as well as anatomy Soaking in buffered formaldehyde solution 40 g / L. Formaldehyde acts as a denaturant , e.g. B. with formalin or Bouin solution .
Cryopreservation Quick cut Freezing at around −30 ° C, for example with dry ice
Alcohols (e.g. methanol or ethanol ), acetone or diethyl ether Cytodiagnostics Displacement of the intracellular water and simultaneous denaturation by soaking. Usually an aqueous solution of the displacer is used, the concentration of the solution is gradually increased and 100% fixative is used at the end.
Crosslinking agents such as glutaraldehyde Transmission electron microscopy , tanning Osmium tetroxide , uranyl acetate or tungstophosphoric acid are mostly used as contrast media .
heat microbiology Heating the preparation in a flame (e.g. that of a Bunsen burner ) or using microwaves
color-retaining fixing solutions Macroscopic preparation For example Jores I solution, Kaiserling I solution, Romhànyische solution and other solutions.
Salts of heavy metals Histology, tanning Formation of poorly soluble protein salts, e.g. B. in chrome tanning or in fixation with copper salts according to Hollande or with mercury salts according to Zenker

During fixation in microscopy , the examination material is fixed on the glass of the slide . For this purpose, heating can be used, whereby organic substances of the object, such as proteins and polymer carbohydrates ( slime ), are changed in such a way that they stick the object to the glass surface ( heat fixation ). The action of certain chemicals, such as formaldehyde , can change polymeric components of the object (for example denaturation ) in such a way that they adhere the object to the glass surface. When microorganisms are smeared on a glass slide, they are usually largely killed by the fixation. Only after fixation, if necessary, are colored to emphasize the structures of the objects, because the objects can then no longer be rinsed off so easily and because their structures are then no longer so easily changed by the coloring process. Fixation is always accompanied by denaturation, which reduces autolysis . Before a microsection , a biological tissue is embedded after fixation with paraffin . this creates an FFPE fabric .

When whole animals or their parts are fixed by infusion or injection into blood vessels , perfusion is also used, e.g. B. in the Thiel fixation . For fixation according to Walter Thiel , a fixation solution with boric acid , ethylene glycol , ammonium and potassium nitrate, chlorocresol , sodium sulfite and formaldehyde is used. The subsequent storage takes place in a more dilute solution ( barrel solution ).

In immunology , the process of undoing the effects of a fixation is called antigen unmasking . The cross-linking and modification of biomolecules with formaldehyde can be reversed by heating or by adding bases .

Fixation in the anatomy

In the anatomy both whole body, as well as individual can bodies are fixed. In macroscopic anatomy in particular, this is possible with body donors. Such preparations are then used in preparation courses for medical teaching. The first description of the fixing properties of methanal in relation to macroscopic anatomy (human medicine) took place in 1894 by F. Blum. He observed a transition of the tissue from a "firm, soft aggregate state to a much more resistant, harder modification". He first noticed it through contact with his own skin and subsequent hardening of the epidermis . He then put an "anthrax mouse" in formaldehyde for one night and then found that it felt like an alcohol preparation . Blum further described that a 10-fold dilution (starting value: 40% solution) hardened faster than ethanol, and this even with larger tissue. Macroscopically, the tissue was better preserved than after treatment with ethanol, while no significant shrinkage could be seen. In the brain , the white and gray matter could even be clearly differentiated. After dehydration and embedding in celloïdin microscopy, susceptibility to histological staining was still detectable even after days. Blum cooperated with Weigert, who carried out the actions on the brain and spinal cord , while Blum himself carried out the processing of the other organs. Meister, Lucius and Brüning zu Höchst a. M. (today Sanofi SA ) had drawn attention to the product "Formol" (product name) and gave it to Blum for review. The father of the practicing physician F. Blum, senior teacher J. Blum, used formaldehyde for the first time in 1893 to preserve zoological and botanical preparations. Today, formaldehyde is the most widely used fixative and preservative. Today's methods are often still based on the formalin method, but other methods of restraining a corpse are also available. In particular, movable and often color-retaining fixations are moving more and more into the focus of medical education and training due to their extremely realistic value. The best-known solutions include so-called alcohol and salt solutions and the Thiel solution.

literature

  • Maria Mulisch, Ulrich Welsch (ed.): Romei's microscopic technique. 18th edition. Spektrum Akademischer Verlag, Heidelberg 2010, ISBN 978-3-8274-1676-6 . ( limited preview in Google Book search)
  • Gudrun Lang: Histotechnology. Springer-Verlag, 2013, ISBN 978-3-7091-1190-1 . Chapter 5: Fixation.
  • Siegfried Schwerin: Anatomical dry, wet and bone preparations. Springer-Verlag, 1952, ISBN 978-3-662-30466-2 .
  • Walter F. Steinmann: Macroscopic preparation methods in medicine. Thieme-Verlag, 1982, ISBN 3-13-623901-6 .

Individual evidence

  1. ^ A b Mareike Dartsch: The ventral vesicourethral suspension apparatus as part of the male continence system. Dissertation . Medical Faculty of the University of Leipzig, 2012, p. 35.
  2. ^ H. Fraenkel-Conrat, BA Brandon, HS Olcott: The reaction of formaldehyde with proteins; participation of indole groups; gramicidin. In: The Journal of biological chemistry. Volume 168, Number 1, April 1947, ISSN  0021-9258 , pp. 99-118. PMID 20291066 .
  3. ↑ H. Fraenkel-Conrat, HS Olcott: The reaction of formaldehyde with proteins; cross-linking between amino and primary amide or guanidyl groups. In: Journal of the American Chemical Society. Volume 70, Number 8, August 1948, ISSN  0002-7863 , pp. 2673-2684. PMID 18876976 .
  4. ↑ H. Fraenkel-Conrat, HS Olcott: Reaction of formaldehyde with proteins; cross-linking of amino groups with phenol, imidazole, or indole groups. In: The Journal of biological chemistry. Volume 174, Number 3, July 1948, ISSN  0021-9258 , pp. 827-843. PMID 18871242 .
  5. ↑ F. Blum: Note on the use of formaldehyde (formol) as a hardening and preservative. In: Anat. 9, 1894, pp. 229-231.
  6. ↑ F. Blum: The formaldehyde as a hardening agent. In: Zschr. F. Micro. and microsc. Tech. 10, 1896, pp. 314-315.
  7. ↑ J. Blum: Formol as a preservation liquid. In: Zool. Number 10, 1893, pp. 450-452.
  8. ↑ Reinhard Hildebrand: Formalin. In: Werner E. Gerabek , Bernhard D. Haage, Gundolf Keil , Wolfgang Wegner (eds.): Enzyklopädie Medizingeschichte. Volume 1: AG. Walter de Gruyter, Berlin / New York 2007, ISBN 978-3-11-019703-7 , p. 410.
  9. ↑ S. Hayashi et al .: Saturated salt solution method: A useful cadaver embalming for surgical skills training. In: Medicine Baltimore. Vol. 93, No. 27, 2014, pp. 196-197. ISSN  0025-7974
  10. ↑ R. Coleman, I. Kogan: An improved low-formaldehyde embalming fluid to preserve cadavers for anatomy teaching. In: J. Anat. 192, 1998, pp. 443-446.
  11. ↑ J. Weigner: Nitrite curing salt-ethanol-polyethylene glycol 400 solution for the fixation and preservation of organs and animal bodies for teaching and research. In: The taxidermist. Vol. 57, 2011, pp. 34-53, ISSN  0032-6542
  12. ^ W. Thiel: The preservation of whole corpses in natural colors. In: Ann. Anat. 174, 1992, pp. 185-195.