Introduction to Histology
From Wikiversity
The purpose of histology is a method to examine tissues at a microscopic level. For example, if a patient is undergoing a biopsy, the doctor cannot just look at the lump and decide whether it is cancerous or benign. The tissue must be sliced into very thin sections to properly be mounted, stained and examined under the microscope. Tissues are generally sectioned in slices 5 to 10 micrometers thick. A typical cell is about 10 micrometers thick, so this size of section will 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. This process is not as simple as it may sound.
Fixing
The first step of processing the tissue is to "fix" it. The fixation step 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.
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.
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?
ARTIFACTS:
Formalin Pigment What does it look like? What is the reason for it? How do you prevent it? How do you remove it?
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 How do you remove it?
Picrates What does it look like? How do you remove it?
FIXATION
Primary fixatives contain ________________ _________________, while compound fixatives contain _______________________.
What two fixatives are used for electron microscopy (EM) ? 1 2
Fixative used should be ___________X the volume of tissue to be fixed.
Rate of Fixation is affected by: 1 2 3 4
How do fixatives prevent autolysis & putrefaction? (Their action on the tissue)
How to fixatives harden tissue?
One way to slow down autolysis is with temperature: (circle) 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 _______________________&_____________________ more rapidly than other tissues.
Explain coagulation of tissue proteins: Explain cross-linking of tissue proteins:
Fixative Advantages Disadvantages Main uses Formalin Acetic Acid Picric Acid Alcohol Gluteraldehyde Mercuric Chloride Potassium Dichromate Osmium Tetroxide Zenker’s Helly’s Bouins Carnoys
DEHYDRATION
What is the purpose of dehydration?
What is the most common method of dehydration?
How do you treat delicate tissues differently?
List the dehydrating agents
1 2 3 4 5 6
Why would you not want to just use 100% alcohol to begin 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?
Clearing Agent Advantages Disadvantages Xylene Toluene Benzene Cedarwood Oil Chloroform
IMPREGNATION
What are the 2 functions of wax impregnation? 1
2
What are the advantages of vacuum impregnation? 1 2
Most common melting point of wax used is ______ to ______ °C
Harder wax has a higer / lower melting point. (circle)
The consistency of solidified embedding material should be the same as ___________________________.
Embedding Material Advantages Disadvantages Main Uses Paraffin Wax Cellulose Nitrate Synthetic Resins Freeze-Dry Gelatin Ester Wax Water Soluble Wax
EMBEDDING
How do you 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 advantages do automatic tissue processors confer? 1
2
3
The fixative most often used on bone is _____________________________________.
DECALCIFICATION
Decalcifying fluid should be _________ to _________ X the volume of tissue.
Decalcification Fluid Concentration Used Time Adv/Dis/Misc Nitric Acid Formic Acid TCA EDTA
What temperature is generally used for decalcification?_____________________
How is the temperature maintained?__________________________________________
What are the two tests to see if decalcification is complete? 1 2
What are the steps for the chemical test for decalcification? 1
2
3
If the final solution is cloudy, ___________________________________________________
If the final solution is clear, _____________________________________________________
MICROTOMY
The usual thickness of sections for microtomy is ______________________µm
Microtome Use Knife or Block moves? Advantages Disadvantages Rotary Freezing / Cryostat Ultratome Sliding
Knife Draw the shape Use Wedge Planoconcave Biconcave Tooled Edge
Explain the difference between angle of clearance, tilt, and slant:
STAINING
Progressive stain vs. regressive stain. Define each, which is more commonly used?
Compare a direct stain vs. an indirect stain.
Define “lake” as it refers to staining.
Define “accentuator”.
Define “trapping agent”.
List four factors that affect staining 1 2 3 4
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, and name the chemicals used:
Results of H&E stain – what are the colours produced:
Nuclei:
Decalcified Bone:
RBCs:
Cytoplasm:
Why use Scott’s Tap Water rather than regular tap water to stain?
