Portion 1: Steps of histological slide preparation & H&E staining rules.

Histology

The study of structure, development, and function of cells, tissues, and organs

General histology

Deals with four main tissues of the body

Four main basic tissues of the body

Epithelial tissue, supporting and connective tissue including blood, muscle tissue, and nervous tissue

Special histology

Studies organ systems of the body dealing with the microscopic structure of organs composed of different tissues

Microscopic anatomy

Alternative name for special histology

Step 1 of histological slide preparation

Cutting of small tissue samples

Fixation

The second step of histological slide preparation designed to terminate cell metabolism and prevent enzymatic degradation by autolysis

Fixative purpose in slide preparation

Kill pathogenic microorganisms such as bacteria, funguses, or viruses

Simple fixative

10 percent neutral formalin solution

Complex fixatives

Fixatives which contain several ingredients

Dehydration step of slide preparation

Tissue is passed through a series of alcohol to remove water

Ascending concentration sequence for dehydration

Passed through 50 degree, 70 degree, 96 degree, to absolute alcohol

Reason for ascending concentration in dehydration

Performed to avoid deformation of the tissue

Step 4 of histological slide preparation

Paraffin embedding

Automated embedding machine example

Tissue-Tek

Step 5 of histological slide preparation

Tissue sectioning with a microtome

Microtome section thickness for light microscopy

Usually 3 to 7 micrometers thick

Step 6 of histological slide preparation

Staining of the sections

Deparaffinization requirement

In case of paraffin embedded tissues, paraffin must first be removed from the sections using xylene followed by ethanol

Most commonly used stain in histology

Hematoxylin-eosin

Step 7 of histological slide preparation

Mounting

Mounting medium options

Neutral resin like Canadian balsam or synthetic mounting medium like Eukitt

Coverslip placement

A thin coverslip is placed over the section after applying the mounting medium

Hematoxylin-eosin staining characteristic for nuclei

Blue

Hematoxylin-eosin staining characteristic for cytoplasm

Varying shades of red

Hematoxylin-eosin staining characteristic for collagen

Very pale pink

Hematoxylin-eosin staining characteristic for cartilage and calcium deposits

Dark blue

Hematoxylin-eosin staining characteristic for red blood cells

Bright red