College of Education Biology Dept. Microscopic Slides Preparation for Histopathology Examination By Saman M. Mohammad-Amin PhD. Biotechnology 16.3.2017 Seminar Hall, 10.00 Am

Microscopic Slide Preparation Examining cells, fragments of tissues or organs, after their vital processes have been stopped by fixation. Allows the examination of the structure and main characteristics of cells.

Microscopic Slide Preparation, Purposes and Usage Purpose: preserving cellular structures and the relations between them indefinitely Used in : Clinics Morphopathology Forensics Research purposes Educational purposes

Steps for Microscopic Slides Preparation Recoltation Fixation Dehydration Clearing Infiltration 6. Embedding 7. Sectioning 8. Staining 9. Mounting 10. Labeling

Recoltation may obtained by : Obtaining the sample to be examined from humans, living animals or corps. Recoltation may obtained by : Biopsy Necropsy (Autopsy)

Fixation A physical or chemical process that stops as soon as possible vital cellular processes, maintaining with minimal alteration the shape, volume, spatial and molecular relation between elements. Formaldehyde, Glutaraldehyde, Acetic acid, Metyl alcohol and Osmium tetraoxide used as the fixative , each with it’s advantages and disadvantages.

Dehydrating (fluid) agent: Dehydration To remove fixative and water from the tissue and replace them with dehydrating fluid. Dehydrating (fluid) agent: Ethanol Methanol Isopropanol Acetone

Clearing Clearing is the transition step between dehydration and infiltration with the embedding medium. Clearing fluid: Toluene and xylene Benzene Chloroform Cedar wood oil Clove oil

Infiltration media (materials): Is the saturation of tissue cavities and cells by a supporting substance which is generally, the medium in which they are finally embedded. Infiltration media (materials): Paraffin wax , Gelatin , Celloidin and Plastics

Embedding Is the process by which tissues are surrounded by a medium such as agar, gelatin, or wax which when solidified will provide sufficient external support during sectioning.

Sectioning Is a real art and needs much skills and practices. It is important to have a properly fixed and embedded block. This is done with a Microtome. The important thing for proper sectioning is a very sharp knife.

Staining Intends to increase the contrast between cell or tissue components by modifying refraction indexes of the morphologic substrate, using dyes, it done as follows: A. Deparaffinization B. Hydration C. Staining: The most common stain applied for histological study is Hemotoxylin and Eosin. D. Dehydration E . Clearing

There are two types of mounting media : Histological sections which need to be examined for any length of time or to be stored, must be mounted under a cover-slip. There are two types of mounting media : 1. Aqueous media: Used for material which is unstained such, (glycerin). 2. Resinous media :For routine staining such, (Canada balsam).

Labeling It will be noted: Celullar type Recoltation source The fixative The staining Date

Normal tissues appearances Prostate Kidney

Breast Histopathology Normal breast tissue Cancerous breast tissue

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