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6 histotechniques
 

6 histotechniques

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  • In most text books available, Histotechniques are explained from the perspective of pathology. In anatomy, it the the same process but there is alteration in some steps. For example tissue procurement is seldom discussed because this step is crucial only to anatomists who is preparing a slide for teaching learning purpose unlike a pathologist who gets tissue from patient for tissue diagnosis. Similarly grossing is usually described in detail which is not very important to an anatomist.Decalcification for bone – addedFrozen section: fixation omitted
  • Though Histotechniques are done in various steps, they should not be viewed in isolation. Each step usually complement the other . For example fixation with osmium tetroxide will also stain the myelin. Fixation not only preserves the tissue but also fortifies it against harmful effects of chemicals used in further processing.Hardening of tissue by fixation aids in section cutting.
  • The first thing that is needed for Histotechniques is the tissue itself. In pathology tissues are available from patients which are to be processed and examined to provide tissue diagnosis. In anatomy tissue are processed at majority of times for preparation of slides for teaching learning process.Hence in anatomy we need source of ‘normal’ tissues. Easiest source of tissue is our set up is PM bodies. Certain organs need to be obtained from smaller animals because they are larger in human and sections of whole organs cannot be accommodated in glass slide. Commonly used animals are rabbit, dog, squirrel, gunniepigs etc. for luminated organs like large blood vessels, parts of GIT, brain and Genitourinary tract. For certain organs we need specific animals e.g. pig for liver. If few specific organs are needed tissue can be acquired from butchers instead of sacrificing the the whole animals.
  • The Committee for the Purpose of the Control & Supervision of Experimental Animals (CPCSEA). CPCSEA mentions that, animals should not be used for repetition of experiments or experiments whose results are already well established. Use of animals which are very old, diseased and dying , and ones dead due to some other experimentation is not clearly mentioned.
  • Unlike in pathology again processing of tissue is planned in Anatomy. If tissue needs to be transported, the best medium for transportation is fixative itself. Washing for removing excess blood, mucasetc should always be done with isotonic saline. These precautions are basically to prevent swelling or shrinkage of tissue and prevent crushing of tissue. Labeling and marking is necessary for correct orientation of tissue in block and subsequently in slide.Collapse of luminated structure e.g. can be prevented by stuffing them with paraffin wax, for its proper demonstration.
  • 1,3)Maintained in as lifelike as possible but certain changes are necessary. Coagulation of tissue is necessary but is a major artifact since living tissue are in fluid or semi-fluid state.Properties of a good fixative: cheap, easily available, non irritant, non- toxic, non-inflammable, easy to store, no change on storage etc.Ideally , fixed tissue should tell more about life rather than looking more ‘life like’
  • 5) Fixation aids in optical differentiation of cells and tissue constituents by altering their refractive indices, in various degrees. This is valuable since refractive index of some elements of cell is so close to that of surrounding structures making them invisible in living state when examined under ordinary microscope.5) Fixatives may have both facilitating and inhibiting action on dyes. Carmalum, a nuclear stain stains less strongly when fixed with formalin but stains strongly when mercuric chloride is used for fixation.5) Fixatives can act as mordant- direct link b/w tissue and stain, e.g. potassium dichromate if mixed with formalin aids in demonstration of myelin sheath with hematoxylin.
  • Aldehydes : react with basic amino acid residues of proteins, form cross links b/w protein molecules leading to polymerization and increased molecular weight. Max cross linking is formed by Glutaraldehyde followed by formaldehyde and hydroxyadipaldehydeOsO4; form cross links with proteins with rapid increse of viscosity followed by decrese in viscosity – secondary liquefactionHgCl2 ; commonly used as secondary fixatives, reacts with amino acid residues espthiol, imidazole, phospate and hydroxyl group. Special affinity for histidine. Ultrastrucural preservation poor.
  • What will the audience be able to do after this training is complete?Briefly describe each objective and how the audience will benefit from this presentation.
  • Apart from acidity of fixative, tissue is also acidic due to accumulation of CO2 following anoxia.
  • Chemical reaction will reduce effectiveness of both buffer and fixative.
  • Chemical reaction will reduce effectiveness of both buffer and fixative.
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  • For ideal penetration of tissue, coagulum formation makes tissue impermeable to fixatives.Time is inversely proportional to size of tissue.
  • Fixative is a pure chemical substance e.g. 100% formaldehyde or 40% formalin, fixing fluid is that is actually used for fixation , made by dilution of or mixing fixative with some other component. E.g. buffered normal 10% formalin.General ; suitable for most tissues, specific ; for a particular tissue or tissue component eg TestisCompound : When 2 different fixatives are used one after another e.g. Chromaffin reaction : aldehyde + potassium dichromate; potassium dichromate oxidese formaldehyde to formic acid ->Cr +++, enters into catacholamines. Mercuric chloride-formalin & Helly’s fluidPreserves orientation of tissue components Vs preservation of cell componentsSingle VS multiple componentsLight microscopy Vs Histochemistry, electron microscopy etc.Chemical : Aldehyde, oxidising agents, protein denaturating agents, other cross linking agents, Physical – heat, microwaveUnknown mechanism – HgCl2, picric acidNewer : Carbodiimides like demethylsuberimidate and p-benzoquinone etc.
  • Add slides to each topic section as necessary, including slides with tables, graphs, and images. See next section for sampletable, graph, image, and video layouts.
  • Add slides to each topic section as necessary, including slides with tables, graphs, and images. See next section for sampletable, graph, image, and video layouts.
  • Formic acid -> formalin pigmentsFormic acid : add borx to diluted formalin -> red color with phenolphthaline,Allow diluted formalin to stand on layer of calcium carbonate, addition of caco3 on conc formalin can cause explosion.Add 2% calcium acetate -> preservation of phospolipidsParaformaldehyde -> cause turbidity, filter with filter paper.
  • Good protein fixativeNo effect on carbs. – glycogen held by proteinsLipids preserved – not fixedFavors staining of acidic structures with basic dyes – nucleiDiminishes effects of acid dyes on basic structures
  • Turbidity can be removed by filtering with filter paper
  • Glacial -> solidifies at about 17 degrees.
  • Add a case study or class simulation to encourage discussion and apply lessons.
  • Discuss outcomes of the case study or class simulation.Cover best practices.
  • Discuss outcomes of the case study or class simulation.Cover best practices.
  • Discuss outcomes of the case study or class simulation.Cover best practices.
  • Discuss outcomes of the case study or class simulation.Cover best practices.
  • Discuss outcomes of the case study or class simulation.Cover best practices.

6 histotechniques 6 histotechniques Presentation Transcript