Importance Of Fixation And Decalcification

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Fixation and Decalcification are the common technique for technicians to preparations of patient or other samples. Both process are important for further diagnostics and clinical test. After processing fixation, the sample will become favor to support the high quality and consistent staining with H&E and for the long-term storage of paraffin blocks. Decalcification is specific for obtaining satisfactory paraffin sections of bone. The dense collagen of cortical bone become obviously tough via removing inorganic calcium from the organic collagen matrix, calcified cartilage and surrounding tissues.

Principle of Fixation
Fixation is the by the constituents of cells and tissue are fixed in a physical and chemical state
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It is disable intrinsic biomolecules, particularly proteolytic enzymes—which otherwise digests or damages the sample.
Fixatives are toxic to most common microorganisms (bacteria in particular) that might cause extrinsic damage to tissue sample. Then at the molecular level they increase the mechanical strength or stability of cells which help to preserve the morphology (shape and structure) of the sample. And also alteration of refractive indices of cell components so that unstained components are easily visualized.

Principle of decalcification
Insoluble salt converted to soluble salt by the action of decalcifying agent so that tissue become soft. Then, chelating agent binds to calcium ions present in the bone and decalcification is carried out. Thus, removing the calcium salt from bone, teeth and other calcified tissues and making them amenable, it can make bone flexible and easy for pathological investigation. This is necessary in order to obtain soft section of bone using the microtome.

Precaution & improvement:

Wear gloves while cutting the sample by the
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The formalin will volatile and may inhealed by nose and skin.
The pH value should be maintain in pH 6 - 8. If not, it may change tissue structure and detrimental for preservation.

All sample are used a hour to fixation, the result show as below:

Processing with 10% formol saline
After the fixation with 10% formol saline, the layer was detected of the pork liver internal side. There are red color layer and pink color layer. Red color meaning the formol saline not fully penetrate in the liver therefore it look like before the fixation.Also the pink side and pink surface is affected by formol saline but causing the weak RBC lysis power of formol saline, so it still look pink.

the hardness is between the liver processing with acetic acid and processing with 3%potassium dichromate.

biggest area of bloody surface remained between three types of reagent.

Processing with acetic acid
After the fixation with acetic acid,there are grey surface and dark red internal side. That show that the acetic acid penetrated in the pork liver but the RBC in the surface are lysed

It is the hardest between three types of

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