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Introduction to Histology INTRO
INTRO

Why do we study histology?

1- To understand the structures and the function of the components of the cell and

tissues (morphology is a rough indicator of function)

- Morphology: the structure and the components of the cell and tissues

2- To Gain insight into detailed morphological Features.

3- To compare normal structure VS abnormal (pathology)

Methods of the cell study

❖ Microscopic studying:

- to study the structure of the cell and tissues ,we use the microscope. -

There are different types of microscopes as you know, we will focus on

light microscope and electron microscope.

➢ Light microscope:
1- Conventional (used in lab)

2- Confocal

3- Phase contrast

➢ Electron microscope:
1- Scanning

2- transmission

- We choose the type of Microscope depending on the size of the object or the structure which you want to study

Microscopy
➢ Simply, preparing slides and see them under the microscope. -

The specimen is preserved as close as possible to Original form

in the Living state.

- Stains are used to show contrast colors.

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➢ The most two important features of microscope are: 1-

Resolution: The smallest distance between two structures

at which they can be seen as two separate objects.

- In Light Microscopy: 0.2 µm Vs Electron: 3 nm.

2- Magnification: Light 1000-1500. Vs Electron; 120,000.

➢ In general, a given type of radiation cannot be used to probe structural details much smaller than its own wavelength.
.

➢ This is a fundamental limitation of all microscopes.

➢ The ultimate limit to the Resolution of a light microscope is therefore set by the Wavelength of Visible Light, which

ranges from about 0.4 µ m (for violet) to 0.7 µ m (for deep red).

➢ In practical terms, bacteria and mitochondria, which are about 500 nm (0.5 µ m) wide, are generally the smallest

objects whose shape can be clearly discerned in the light microscope.

How the microscope works?

➢ The light pass from the lamp to be condensed by the condenser then it will pass

through the specimen then to the objective lens (first magnification occur here)

then to the ocular lens (the second magnification occur) then to your beautiful

eyes.

➢ So, notice that there are two magnification processes and the total is the multiplication of them together.

Light VS Electron microscopy

Light Microscopy Electron Microscopy
Beam Light Electron Beam
Magnification 1000-1500 times 120000 times
Magnification 0.2µm 1nm
section Thickness 1-90 µm 2-10 nm
Fixatives 4% Formaldehyde 4% Glutaraldehyde
Embedding Parafin Resins
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Steps in Tissue Preparation for Microscopic Study

1- Collection of Tissue:

Soon after death, Why? To prevent the degradation of the tissue.

2- Cutting:

Thickness should not exceed .5cm (Light) & 1-2-mm (Electron). Why? Because fixative may not penetrate whole tissue.

3- Fixation:

Chemicals that interact with amine groups OR Cross-link with Proteins. Why? From its name, it will fix the tissue as in
living state as possible.

Note: No fixatives for Enzymatic Studies & During Surgical Procedures. We use Freeze Fracture (Immerse tissue in Liquid
Nitrogen -70-800(, Why?

- Because fixation will kill the specimen so we will not use it while studying living things like when we study

enzymatic activity, and in case of surgery we don’t have enough time to make this preparation and we cut the route

by using liquid nitrogen.

➢ To sum up, keep in mind these points:

- Fixation is used to keep the specimen as living state as possible.

- Fixation kills the specimen.

- Fixation and the rest of steps need long time that we don’t have during surgery.

4- Dehydration:

- Removal of water by immersing tissue block in gradual concentration of Alcohol (70%-100%).

- Why we use it? To prevent it from mixing with the paraffin in the next step.

- Why it gradual (70%-100%)? Because we don’t need to harm the tissue by suddenly dehydration of it.

5- Clearing:

- Removal of Alcohol with Xylene. To prevent Alcohol from mixing with the Paraffin & to give the specimen transparent
look.

- So, we use xylene to remove alcohol which removes the water.

6- Embedding:

Paraffin/Resin. Maintain internal architect of the tissue and provide the block with certain solidity to facilitate its
sectioning
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7- Sectioning:

Use of Microtom

8- Staining:

To give colors to what we see in order to understand it.

Acidophils/Basophils stains OR Mixed types as Hematoxylin (base, blue color) eosin (acid, pink color).

Notes:

➢ Basophilic structure takes blue such nucleus, and Eosinophilic structure takes pink such cytoplasm.

➢ Basophilic means love basic so basophilic structure don’t mean the structure itself is basic, no it is an acidic it to bind

can so structure

➢ How to memorize this forever?

Remember inside the nucleus there is a nucleic acid, and from its name it’s negative in charge and will bind to basic

stains which is positive in charge, and notice the (b) in the beginning of each basophilic and blue.

9- Mounting:

We put the specimen on Glass Slides and then covering it by chip of glass.

➢ This picture shows a section of Renal Tubules Stained with Contrast Colors.

Nuclei appear Pink Color and The Cytoplasm Sky-Blue Color. The

Extracellular Areas are Stained Purple.

Stains natural or chemical dyes

❖ classified as basophilic or eosinophilic.
1- H&E Stain:

- hematoxylin (basic dye): stains acidic (basophilic) structures.

- eosin (acidic dye): stains basic structures (cytoplasmic proteins) pink color.

2- periodic acid–schiff reaction (PAS):

- selectively stains carbohydrates (mucin of goblet cells) as deep red color called magenta.

- Also basement membranes, brush border cells of GIT, kidney, glycogen & cartilages are PAS positive.
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3- masson’s trichrome: used to stain support tissue, gives three colors:

- nuclei blue

- collagen green

- cytoplasm muscle, RBS, keratin bright red

4- van gieson (CT stain):

- Collagen red

- nuclei blue

- cytoplasm & RBCs yellow

5- azan (CT stain): Special epithelium stain.

- nuclei bright red

- BM & mucin blue

- RBCs orange

6- giemsa stain: standard blood cells smear & bone marrow.

- nuclei blue

- cytoplasm pale blue

- RBCs pink

7- sliver and gold: used specially for nervous tissue studies.

8- nissl stain: used to demonstrate rER of neurons.