# Histology/Slide Preparation

The purpose of dynamic histology is to examine tissue structures at the microscopic level in order to understand their physiological and anatomical functions. For example, if a patient is undergoing a biopsy, the doctor cannot just look at the lump of extracted tissue and decide whether it is cancerous (malignant) or benign (non-malignant). The tissue must be fixed, sliced into very thin sections to be properly mounted, stained, and finally examined via light or electron microscopy. Tissues are generally sectioned in slices 5 to 10 micrometers thick, although thinner slices are required for examination with a transmission or scanning electron microscope. A typical cell is about 10 micrometers thick, so this size of section will usually allow a layer that is only one cell thick.
As one can imagine, it would not be easy to take a piece of fresh tissue and cut it this thin. Imagine trying to slice a chicken breast - even with a very fine razor blade, the tissue is too soft to allow for even cutting.
The most common process for deriving sections is by embedding the tissue in paraffin wax and sectioning it on a microtome, a mechanical apparatus onto which a sharp blade is mounted to section the hard tissue. Although similar processes are employed to prepare tissue sections for both electron and light microscopy, different chemical products are used.

## Slide Preparation
### Fixing

The first step of processing the tissue is to "fix" it. The fixation step for light microscopy is usually done by immersing the tissue in formalin for several hours to several days. The fixation step keeps the tissue from decomposing or being ruined by bacteria and enzymatic activity. Alternatively, the subject could be perfused with the fixative chemicals, in which case the fixation process would be carried out more effectively, namely for subsequent electron microscopy analysis.
There is also another type of fixation called cryofixation, which relies on the use of liquid nitrogen, at a temperature of about -196 degrees Celsius. As the name implies, the tissue section is frozen in order to prevent tissue breakdown by enzymatic activity. Brain tissue, for example, will start decomposing via proteolysis about thirty minutes following death due to the release of proteases from lysosomes.

### Dehydrating

The ultimate goal is to get the tissue immersed in wax, and to have all the water in the tissue displaced by wax. The water in the tissue needs to be removed first. The dehydration step is done with successively stronger concentrations of ethyl alcohol.

### Clearing

Alcohol will not mix with wax, therefore a clearing step replaces the alcohol in the tissue with a clearing agent, typically xylene, which will mix with the wax.

### Penetration

The tissue is then put into a warm paraffin wax bath for several hours. The paraffin can now enter the tissue and each individual cell. The entire piece of tissue is filled with the warm wax.

### Embedding

The tissue is finally put into a block of wax. The block of wax gives the tissue support and shape for the next step - cutting.

## Concepts Quiz

Refer to Wikipedia histology articles to answer the following questions:

### Fixation

* What are the main purposes of fixation?
* What is the difference between autolysis and putrefaction?
* List the properties of an ideal fixative:
* What percentage of formalin is routinely used in histology?
* What percentage of formaldehyde is used to make it?
* Primary fixatives contain __________ __________, while compound fixatives contain ____________.
* What two fixatives are used for electron microscopy (EM)?
* Fixative used should be _____ times the volume of tissue to be fixed.
* The rate of fixation is affected by what four variables?
* How do fixatives prevent autolysis and putrefaction?
* How to fixatives harden tissue?
* One way to slow down autolysis is with temperature. Circle the following true statements.
** Cold (2-8°C) slows down / speeds up autolysis
** Warm (37°C) slows down / speeds up autolysis
** Hot (57°C) slows down / speeds up autolysis
* Autolysis affects complex organs such as the ___________ and the __________ more rapidly than other tissues.
* Explain the phenomenon of coagulation of tissue proteins.
* Explain cross-linking of tissue proteins.
* State the advantages, the disadvantages, and the main uses of the following fixatives:
** Formalin
** Acetic Acid
** Picric Acid
** Alcohol (ethanol)
** Gluteraldehyde
** Mercuric Chloride
** Potassium Dichromate
** Osmium Tetroxide
** Zenker’s Fluid
** Helly’s Fluid
** Bouin's Fluid
** Carnoy's Fluid

### Artifacts
#### Formalin Pigment

* What does it look like? [It looks like birefringent in polarized light]
* What is the reason for it? [Due to oxidation of formalin to para-formaldehyde at cold environment; and oxidation of formalin to formic acid at room temperature leading to formation of formalin precipitates called"formalin pigments" which are artifact pigments]
* How do you prevent it? [Can be prevented by two mechanisms, removing formic acid produced in formalin, and removing of hydrogen ions from formalin solution. This can be achieved by the addition of marble chips(calcium carbonate) to formalin solutions which will tend to react with formic acid produced by oxidation of formalin to form a neutral solution, by using phosphate buffer or Neutral buffered formalin that will tend to remove excess hydrogen ions and balances the pH of the formalin solution and by passing the formalin solution through the mixed bed of iron exchange resins that will tend to remove both formate and hydrogen ions and thus maintaining a neutral formalin solution with which will bring not the formation of formalin pigments to the formalin fixed tissue.
* How do you remove it? [Remove the formalin pigment to the tissue by treatment of the tissue with saturated-alcoholic aqueous solution of picric acid]

#### Para-Formaldehyde

* What does it look like?
* How do you remove it?

#### Mercury Pigment

* What does it look like?
* How do you remove it?

#### Chrome Pigment

* What does it look like?
* How do you remove it?

#### Picrates

* What does it look like?
* How do you remove it?

### Dehydration

* What is the purpose of dehydration?
* What is the most common method of dehydration?
* How do you treat delicate tissues differently?
* List the six main dehydrating agents.
* Why would one not want to just use 100% alcohol to begin dehydration with?
* In the last jar of alcohol, you can add _______________________ to absorb any water that is left.

### Clearing

* What is the purpose of clearing the tissue that will be examined under the microscope?
* State the advantages and disadvantages of the following substances, which are commonly used as clearing agents:
** Xylene
** Toluene
** Benzene
** Cedarwood Oil
** Chloroform

### Impregnation

* What are the two functions of wax impregnation?
* What are the two advantages of vacuum impregnation?
* The most common melting point of wax used is ______ to ______ °C.
* Circle the correct answer: Harder wax has a higher / lower melting point.
* The consistency of solidified embedding material should be the same as that of the _____________.
* State the advantages, the disadvantages, and the main uses of the following substances, which are commonly used as embedding material:
** Paraffin Wax
** Cellulose Nitrate
** Synthetic Resins
** Freeze-Dry
** Gelatin
** Ester Wax
** Water Soluble Wax

### Embedding

* How does one orient the following specimens when embedding:
** Hairy or Calcified edges?
** Multiple sections?
** Tissues with lumens?
** Tissues with a long and short edge?
** Muscle tissue?
* What three main advantages do automatic tissue processors confer?
* The fixative most often used on bone is a substance known as ___________________.

### Decalcification

* Decalcifying fluid should be _________ to _________ times the volume of the tissue.
* What are the advantages, the disadvantages, the average time of use, and the fluid concentration for the following decalcification agents:
** Nitric Acid
** Formic Acid
** TCA (trichloracetic acid)
** EDTA (ethylenediaminetetraacetic acid)
* What temperature is generally used for decalcification?
* How is the temperature maintained?
* What are the two tests employed to see if decalcification is complete?
* What are the three steps for the chemical test for decalcification?
* What should be done if the final solution is cloudy?
* What should be done if the final solution is clear?

### Microtomy

* The usual thickness of sections for microtomy is approximately ________ micrometers (µm).
* What are the advantages and disadvantages of the following microtomes? Is one required to use a knife or does the block move along the microtome?
** Rotary
** Freezing / Cryostat
** Ultramicrotome
** Sliding
* Draw the shape and enumerate the main uses of the following knives:
** Wedge
** Planoconcave
** Biconcave
** Tooled Edge
* Explain the difference between the angle of clearance, the tilt, and the slant.

### Staining

* Define the concepts of progressive stain and regressive stain. Which is more commonly used in histological examinations?
* Compare the direct stain versus the indirect stain. How do they differ? How are they similar?
* Define the word lake as it refers to staining.
* What is an accentuator?
* What are trapping agents?
* List four general factors that affect staining.
* What steps are required to “bring a slide to water”?
* What is the purpose of bringing a slide to water?
* List the steps for an H&E Stain. What are the names of the chemicals used?
* What are the colours produced from an H&E Stain for the following structures?
** Nuclei
** Decalcified Bone
** Erythrocytes (red blood cells)
** Cytoplasm
* Why use Scott’s Tap Water rather than regular tap water to stain?