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Histology simply is the study of normal tissue sectioned as a thin slice, under the microscope. Whereas histopathology is the science concerned with the study of microscopic changes in diseased tissues. The microscopic examination of the changes that occur within the cells in any tissues like tumour cells helps in the diagnosis of disease and disorders. The examination of tissues in histology starts with surgery in order to get biopsy. In histopathology most tissue are fixed before they are examined. Formalin (10% formaldehyde in water) is the most common fixative, to prevent tissues decay. The aim of fixation is to:
After the fixation of tissues, the tissues are then put in different baths of ethanol, followed by toluene or xylene, finally in hot paraffin. During this 12-16 hours process, water will be removed and paraffin will take the place of water. So the soft, wet tissues will turn into a hard block. This allows the sectioning of tissues into very thin sections using microtome. These sections, thinner than the average cell, are layered on a glass slide for staining. The stages of tissue processing involved are: Dehydration; to remove fixative and water from the tissue and replace them with dehydrating fluid. Clearing, i.e. replacing the dehydrating fluid with a fluid that is totally miscible with both the dehydrating fluid and the embedding medium. Impregnation means replacing the clearing agent with the embedding medium. Embedding; the paraffin wax is the most popular embedding medium for histology (Bancroft, et al., 2002)
The aim of all tissue processing is to give sufficient physical support to the tissue to allow sectioning whilst causing minimal damage to the tissue and the knife edge. The total slices of most tissues are almost completely transparent with very little visible detail. In order to produce a good cutting section it depends on the: Knowledge of the equipment used and practical experience (Bancroft, et al., 2002)
In histology laboratories, Automatic Tissue Processor is designed to make the tissue processing faster. The replacing of water with paraffin wax by removing all the water from the tissue sample should be done with optimum speed in order to minimize damage to the tissue which can be caused by dehydration and shrinkage.
To see the tissue under a microscope, the sections are stained with one or more pigments. It is essential to stain different tissue components so that the pathologist can see specific different tissue components that may aid the diagnosis of a particular disease. Also it is important to understand why a dye molecule appears a certain colour and why it becomes attached to a specific site. Haematoxylin and eosin are stains which are most commonly used in histology. Haematoxylin colours nuclei blue, eosin colours the cytoplasm pink. Other compound used to colour tissue sections include salts and numerous artificial dyes originally developed to stain fibres. The science that studies the tissue staining is called histochemistry. In this practical paraffin sections of kidney, lung, skin, liver and bowel were been studied by using Haematoxylin and eosin stain (Stevens, et al., 2002).
The aim of this practical is to stain the given sections smear (kidney, liver, skin, lung and duodenum) by using Haematoxylin and Eosin stain (H&E). Also it is designed to examine and identify the cellular structure of these sections under the microscope.
The H&E stain was carried out on one of each section.
Since the wax must be removed from all sections before staining, they must be TAKEN TO WATER as follows:
HAEMATOXYLIN AND EOSIN
- Place slides in alcohol for 5 minutes
- Place in xylene for 5 minutes
- Mount sections under a cover slip using DPX mountant
This sequence is carried out on ALL stained sections before microscopic examination can be carried out.
Results: nuclei: blue cytoplasm and connective tissues: rResults
The aim of this practical was achieved. The H&E stain procedure was carried out for all the tissue sections and the stain was clear and perfect. The histological structures of each section were identified and the expected histological appearance of the tissues was seen.
The respiratory portion begins with braches of terminal bronchiole known as respiratory known as respiratory bronchioles. This section shows the thin alveolar walls and the capillaries that are involved with gas exchange. The network of respiratory alveoli is clearly visible and the spaces of respiratory alveoli. Also pulmonary artery can be seen, which is filled up with red blood cells.
Alveolar ducts that constitute alveolar sacs and alveoli arise by branching of respiratory bronchioles, is covered by simple squamous epithelial cells, which allows fast and efficient gas exchange between the air space and the capillaries that lie within the alveolar walls.
The two different types of epithelial cells provide a continuous lining to each alveolus. These cells are large, elongated, squamous cells and they are called type I pneumocytes. The diffusion of oxygen happens through the lager type I pneumocytes. The other type is type II pneumocytes which are round in shape. The type II pneumocytes secret a surface-active materials called surfactant, which reduces surface tension and avoid the alveoli collapse during expiration (Stevens, et al., 2002).
At low magnification, it is easy to see that the skin is composed of two layers, the outer epithelial layer is termed the epidermis and the underlying supporting connective tissue layer is called dermis. The epidermis layer is consists of the epithelial tissue cells, which are stained pink, are arranged in a shape known as “stratified squamous”. This kind of arrangement offers the most protection to underlying tissue.
The dermis is attached to an underlying muscle by a loose connective tissue, also called subcutaneous layer. The dermis is the thick layer of connective tissue to which the epidermis is attached. Mostly the dermis layer contains fibroblasts which are responsible for collagen and elastic secretion that give skin its elastic property.
At a low magnification, the liver section gives general view of the liver components. Liver is divided histologically into lobules. The figure (1-3) shows the important parts of liver under 20x objective microscopic power. The lobule is the structural unit of the liver and a lobule is roughly hexagonal in shape, with portal triads around the edges and central vein in the middle. The liver cells are called hepatocytes and they are polygonal shaped cells, their sides can be in contact either with sinusoids or neighbouring hepatocytes. The hepatocytes are arranged into cords, which are separated by vascular sinusoid.
The cords are the solid substance (mostly pink hepatocyte) and these cords are separated by the sinusoids (clear space). In most normal histological preparation of the liver secretions, the blood has been removed from the sinusoids and they appear empty. It can be noted that the portal vein is known by its big whole (Bancroft, et al., 2002).
The nucleus can be seen as dark blue bodies located in the centre of each liver cell.
The cortex is the outermost part of the kidney, containing the glomeruli, the proximal and distal tubules, the cortical collecting ducts, and the peritubular capillaries of the nephrons (the functional units of the kidney). The glomerulus is the most distinctive structure of the kidney. It is clear that the glomerulus is surrounded by proximal convoluted tubules (PCT) and distal convoluted tubules (DST). The PCT is the longest portion of kidney tubular system. It is easy to differentiate the PCT from DCT in that, the DCT stain paler and the nuclei of their cells appear more regularly arranged and have no visible brush border. The glomerulus is composed of groups of capillaries which are surrounded by double layer of thin epithelial cells. One of these layers is attached to outer surface of the capillary and the other one is separated from the capillary by space called Bowman's space (Stevens, et al., 2002).
The Bowman's capsule is the bulbous, distended, closed proximal end of the tubular system and the glomerulus is inside it.
The mucosa of the small intestine is lined by simple columnar epithelium which forms into villi which form into crypts, comparing to the large intestine which is characterised by straight tall crypts and there is no villi. The villi of small intestine are lined by simple tall columnar epithelium with a brush border (Bancroft, et al., 2002).
The proper preparation of the section is very useful in examination and diagnosis of any abnormalities which can occur within the tissue. The H&E stain is considered as the primary diagnostic stain in histology laboratory lab.