Tissue preparing and processing- Lecture 2

Practical pathology- lecture 2:

Paraffin embedded technique:
This technique involving the following steps:
1-fixation, this step should be done as soon as possible after taking the specimen:
The purpose of fixation is the following:
• To prevent tissue autolysis and putrefaction.
• To preserve tissue details.
• To harden the tissue.
Types of fixatives:
1- formalin.
2- Ethyl alcohol.
3- Zenker fluid.
4- Bouins solution.
The most common fixative used in the histopathological lab. is the formalin, which is (formaldehyde gas soluble in water), the concentration of formalin is 40%, it diluted ten times and used in fixation of the specimens in concentration of 10%. Practically we use one formalin container for two hours.
2- dehydration:
The paraffin used in this technique is immiscible with water, so the water in the tissue should be removed, so this removal of water called the dehydration and done by immersion of increasing strengths of ethyl alcohol, from 50% or 70%, till 100% alcohol. Practically we use five containers of increasing strengths of ethyl alcohol, two hours for each.
3-Clearing:
We use the xylene after dehydration of the tissue and this step is to substitute for ethanol by an organic solvent. We use two container of xylene, two hours for each.
4-impregnation: after clearing of the tissue, impregnated with paraffin wax heated to 56-60 °C , We use two container of paraffin jars, two hours for each.
5- blocking or casting of the specimens by embedding them in liquid (heated) paraffin using the L- Shaped models.
Cutting the paraffin blocks:
The specimens sections by using a machine called microtome.
Staining techniques:
Hematoxylin – eosin stain:
It’s the most commonly used stain, the nuclei stained blue in color, while the cytoplasm stained pink.
There are long list of special stains that are specific for certain substances and of diagnostic importance.
Staining procedure:
1- xylene 2 min. (twice)
2- absolute alcohol two min.
3- decreasing ethanol concentration (rehydration), one min. for each concentration.
4- Wash in tap water.
5- Hematoxylin for 5-10 min.
6- Running water.
7- Eosin 3-5 min.
8- Absolute alcohol two min.
9- Xylene two min (twice).
10 – Canada balsam or DBX.
11- Cover slide.