In the name of Allah the most gracious the most merciful

Histology Methods of Study

The purpose of this course is to enable the students to understand miss phosphatic anatomy of cells and their products, how the cells form tissues and how the tissues are incorporated into larger organs.

Must understand the gross anatomy of structures.

The professor said she will rely on the questions in the text book for the theory tests (first and final). The final will include 20% of the first material.

The second test will be based on what is done in the lab; it will be about a week before the final. The style of the questions will represent a micrograph of a section as seen under the microscope so that the image would be focused. For example the question will be divided into two sections for the first section you will need to identify and the following section will be questions related to the previous section.

Now what does histology mean?

The study of the body tissues and how they are arranged to constitute organs. Of course the tissues are made up of cells and extra cellular matrix. Due to the small size of the cells and extra cellular matrix components, histology is dependent on the use of the microscopes why? To give us a magnified image.

There are specific steps we need to do to prepare the tissue to be seen under the microscope:

1. Fixation2. Embedding and sectioning3. Staining

The importance of fixation of tissue for a microscope is to preserve the structure and molecular composition of the tissue by avoiding tissue digestion by proteolytic enzymes, decrease in size, by bacteria which is called apoptosis.

By the way in histology, the suffix (–ology) means the study of something. Autolysis in the cell means, the breakdown of substance within the cell itself.

Fixation can be done by either of the 2 methods:

1. Physical (for example by changing the temperature of the tissues by freezing them

2. Chemical( using fixatives; and one of the best fixatives for routine light microscopy is 37% formaldehyde)

Now in embedding, the tissue needs to be embedded in a solid medium in order to facilitate sectioning. Examples of embedding materials or media are

1. Paraffin for light microscopy2. Resin for light and electron microscopy

Now the step before the last, sectioning, which is done by using the microtome. Microtome is a device that uses steal or glass blades. The blades are used to section/slice the ready tissue embedded in the medium into 1-10 micrometer(a measuring unit for length) {1micrometer= 10-3 millimeter=10-6 meter} the 1-10 micrometer is for the light microscope. However, for the electron microscope you need much thinner sections using nanometer {1nanometer=10-3micrometer=10-

9meter} which is from 50-80 nanometer.

The last step for tissue preparation for microscopes is staining. Staining is used to enable to study the tissue microscopically since most tissues are colorless. So this staining makes the tissue components conspicuous and distinctive. Staining is done by using dyes. Dyes can behave acidic or basic. They have the tendency to perform electrostatic linkages with ionisable radicals of the tissues.

Hematoxylin is an example of a basic dye. It stains basophilic structures to blue or purple. The suffix (-philic )means like/love. So this means that this dye likes bases so it binds to it. So basophilic dyes stains basophilic tissue components. These components are also anionic (meaning they have a negative net charge). Hematoxylin stains DNA and RNA. DNA and RNA are found in the nucleus.

Eosin is an example of acidic dyes stain acidophilic tissue components, like acids. These components are cationic (having a positive net charge). Eosin stains the cytoplasm giving it a pink color.

Referring to slide #7,the 2 images belong to the same tissue(columnar epithelium lining of the small intestine which later on we will learn about it and about the GIT. No need now to know exactly what it is) but different parts of the tissue. They are stained with deferent dyes. The image on the left is stained with hematoxylin and eosin. We have hematoxylin stained nucleus which appears blue. The pinkish part is stained by eosin, the cytoplasm of the cell. The image on the right, the oval structures are called goblet cells (mucin secreting cells) areas rich of oligosaccharides (oligo- some) as well as the surface of the cell but it is not well stained not well appeared, including polysaccharides. It is stained with periodic acidic Schiff. As you can see the goblet cells, labeled G, is intensely stained even at the surface of the cell. Here you can only see the goblet cell and the cell surface. However, a counter stain was used in order to provide additional information. The counter stain used here was hematoxylin only that’s why you can see the hematoxylin stained nuclei. A counter stain: is used in a different procedure than the original one to provide additional information.

There are 2 classes of microscopy:

1. Electron microscopy (based on the interaction between electrons and tissue)a)transmissionb) scanning

2. Light microscopy (based on the interaction of light and tissue. There are many kinds but we should focus on the bright-field microscopy which we will be using throughout the course.

The bright field microscopy has two components

1.optical

2. Mechanical

The optical constitutes of three systems of lenses

1. Condenser: collects and focuses light to produce column of light for the object to be illuminated and observed.

2. Objective: enlarge /magnify the illuminated object and project it towards the eye piece

3. Ocular/eyepiece: further magnifies the image and projects it on to the viewer’s retina or on a radiographic film

Ocular an objective lens produces magnification. Therefore, the total magnification is obtained by the multiplication of magnification of objective lens x ocular lens

The objective lens causes the resolving power which is the smallest distance between 2 particles at which they can be seen as separate objects. The maximal resolving power is 0.2 micrometer which means this means if there is an object that is smaller or thinner than 0.2 micrometer it cannot be distinguished. They will be seen as one object.

The resolving power determines the quality of the image.The objective lenses with higher magnification will have higher resolving power.

Must identify the parts of the microscope on slide#10.

Best of luck to all!Amineh Al-Farraj