Uploaded by Fatima Rizvi

01 Introduction

Tuesday, January 9, 2018
9:35 PM
- Levels of Organization of the Body:
○ Atoms -> Molecule -> Organelles -> Cells -> Tissues -> Tissues -> Organs -> Organ Systems -> Multicellular
▪ Body structures and functions result from chemical changes that occur within cells or fluids.
- Concept of Histology
○ Histology is the study of the structure and function of cells and their organization into the hierarchy of the
○ Histology is most commonly studied by examining cells and tissues through the process of sectioning and
staining (makes them visible) and using microscopic examination using a LM or EM.
○ Understanding histology allows us to better understand physiology and be able to use various diagnostic
methods to provide improved pathology.
▪ Normal tissue found within the samples aids in differentiating the diseased portion and helps with
- Microscopy
○ Using a microscope involves the process of understanding how different types of microscopes work.
▪ Visualization of details within the cells and tissues requires tissue preparation which is dependent on
the type of microscope.
□ Different structures require different microscopes to be used.
- Tissue preparation for light microscopy (LM)
1. Collection
2. Fixation
3. Dehydration
4. Clearing
5. Infiltration & Embedding
6. Sectioning & Mounting
7. Staining
- For an electron microscope (EM), there are similar steps with the exceptions that:
○ Sample sizes must be smaller.
○ Special fixatives like epoxy resins are used instead of paraffin wax.
- LM with staining produces colored images because:
○ Cell structures allow the stain to be picked up in different ways, and thus, can have different components
be different colors.
○ A mixture of wavelengths of light are used to illuminate each part of the specimen.
- LM Tissue Preparation
1. Collection
▪ Collect a sample from the body through biopsy or necropsy. The sample is cut down into 1 cm cube
so chemicals can easily penetrate the small volume.
2. Fixation
▪ Preserves the cell and tissue structure by cross-linking proteins and inactivating degradative
□ A common fixant is formalin in an aqueous solution of formaldehyde at 37% dilution.
 Formalin reacts with the amine group of proteins that inactivate degradative enzymes.
 Formalin washes away ions and small molecules from the samples.
 These, in turn, prevent autolysis and post-mortem degeneration, bacterial growth, and
help preserve the structure of the tissue and readies it for dehydration.
1 Introduction to Histology Page 1
help preserve the structure of the tissue and readies it for dehydration.
 Formalin does not preserve lipid structures.
□ An alternative to chemical fixation for the samples is to freeze them (normally done with
compressed CO2 or liquid nitrogen).
 This process differs in that:
◊ It preserves lipids.
◊ Frozen tissue is sectioned in a cryostat, mounted in a glass slide, and then stained.
◊ It can provide immediate diagnostic information without the time taking process of
 Freezing might take barely 10 minutes to conduct whereas fixation can take
from 12 to 60 hours.
3. Dehydration
▪ Removes tissue water.
▪ The tissue sample is transferred through a series of increasingly concentrated ethyl alcohol solutions,
ending in absolute alcohol
4. Clearing
▪ Remove the alcohol.
▪ Clearing increases the refractive index of the tissue and make it easier to visualize.
▪ Clearing is also done to prepare the sample for embedding media (such as paraffin wax) to infiltrate
the tissue.
▪ To clear the sample, the tissue has to be treated with a solvent that mixes with (miscible with) both
alcohol and the embedding media that is to be used (most likely paraffin wax).
□ Most samples use toluene for clearing or other similar organic solvents such as xylene,
chloroform, or benzene.
□ During this process, the alcohol used for dehydration is removed by the organic solvents.
 This is important since ethyl alcohol is immiscible with wax.
5. Infiltration & Embedding
▪ Infiltration
□ Process where the tissue is placed in melted paraffin until it becomes infiltrated.
□ The clearing solvent used evaporates in the oven temperature.
▪ Embedding
□ The paraffin-infiltrated tissue is placed into a small mold with the melted paraffin and allowed
to solidify into a small cube at room temp.
6. Sectioning (slicing) & Mounting
▪ Sectioning
□ Very thin slices are cut from the paraffin-embedded tissue using a microtome.
□ Each section is cut ~5 - 10 um in thickness normally. They can even be cut down to 2 um or less.
 1 um = 1 x 10-6 m.
▪ Mounting
□ The section is mounted onto a glass slide.
□ The paraffin wax is removed using an organic solvent and tissue is rehydrated with decreasing
concentration of alcohol (reverse of dehydration).
□ The section is usually stained at this stage and a coverslip is applied to stop movement.
7. Staining
▪ Provides visual contrast and may help identify specific tissue components.
□ Various dyes stain tissue components more or less selectively. They form salt linkages with
1 Introduction to Histology Page 2
□ Various dyes stain tissue components more or less selectively. They form salt linkages with
oppositely charged tissues.
○ The risk with any of these steps is that there is a chance of introducing a foreign artifact into the sample.
- Hematoxylin & Eosin (H&E)
○ H&E are the most commonly used stains for light microscopy.
▪ They behave like basic or acidic compounds.
▪ Hematoxylin:
□ Behaves like a basic (cationic) dye.
 Caries a net + charge on its colored portion.
 Basic dyes are described by the general formula: dye+ Cl-.
□ It stains acidic compounds of the cells and tissues called basophilic regions (base-loving).
 Basophilic molecules include:
◊ In the nucleus:
 DNA in Heterochromatin (condensed heterochromatin is dark blue color)
 Nucleolus
– RNA in the nucleolus
◊ Ribosomes in the RER
◊ Glycosaminoglycans in the EC matrix
 Long chains of polysaccharides made by hundreds of disaccharide repeating
□ Hematoxylin produces a blue/purple color.
□ In pancreatic acinar cells, only hematoxylin is used. (Acinar cells means cells that look like a
bunch of grapes)
□ With hematoxylin, parts of the cell that include the nucleus and areas containing cytoplasmic
RNA are heavily stained.
□ Other basic dyes include: Methylene blue, Toluidine blue, and Thionine.
 These frequently react with polyanionic tissue components and impart to it, a color
different from that of the dye itself. This instance is called metachromasia.
◊ Examples of metachromasia:
 Mast cell granules
– Type of cell in the connective tissue.
– There are several different types of mast cell granules within the
cytoplasm throughout connective tissue. Some contain histamine,
heparin, sulfates, and etc.
 Cartilage
– Contains lots of proteoglycans and glycosaminoglycans.
 Mucin
 Amyloids
▪ Eosin
□ Acts as an acidic (anionic) dye.
□ Acid dyes carry a net - charge on the colored portion & generally given the formula: dye- Na+.
□ Acidic dyes stain basic components with a net + charge and are called eosinophilic regions or
1 Introduction to Histology Page 3
□ Acidic dyes stain basic components with a net + charge and are called eosinophilic regions or
acidophilic regions.
 Acidophilic molecules are usually:
◊ Proteins (cytoplasmic or EC)
◊ Cellular structures containing basic components similar such as some proteins with
basic amino acids.
 Mitochondrion
 Most secretory vesicles
 Cytoplasmic filaments
 EC structures such as collagen and elastic fibers
□ Eosin stains a pink/red/orange color.
□ In pancreatic acinar cells, only eosin is used.
□ Eosin has an overall staining effect.
 The nuclei are less conspicuous than with hematoxylin.
□ Other acidic dyes include Orange G., Acid fuchsin, and aniline blue.
1 Introduction to Histology Page 4