Micro-techniques Flashcards
What are the requirements to study tissues under the microscope?
- The tissue sample must be stained
- The tissue sample must be cut very thinly
What are the methods of tissue preparation for a light microscope?
- Paraffin technique
- Freezing
What is the method of tissue preparation for an electron microscope?
- Electron microscopic preparation
What is the aim of tissue processing (LM)?
To put tissue in a substance (wax) that allows it to be thinly sliced in a correct arrangement.
What are the types of fixatives we can use for paraffin blocks?
- Acetic acid
- Formaldehyde
- Ethanol
- Glutaraldehyde
What is the main fixative commonly used in the paraffin technique?
Formaldehyde
Aim of fixation
- To prevent autolysis and bacterial attack
- To fix tissues os they will change shape and volume during processing
- To prepare tissue for staining
Aim of dehydration
To remove water from tissues to allow tissues to be placed in the paraffin wax
What is the common dehydrating agent and why?
Alcohol, because it is a hydrophilic substance (it attracts water)
How are specimens dehydrated?
in a graded ethanol series from water through
10%
20%
50%
95%
100%
of ethanol to prevent shrinking
Aim of clearing?
removing alcohols and permitting tissue infiltration with paraffin wax
Extra info:
Clearing is performed after dehydration to replace the ethanol content of the tissue with xylene. This process makes the tissue transparent, which renders the specimen suitable for microscopy.
Clearing
a process of replacing the dehydrator with a substance that is miscible with embedding medium or paraffin wax.
Some clearing agents?
- Xylene
- Chloroform
- Benzene
- Petrol
Aim of embedding?
Tissues are surrounded by a paraffin wax which when solid will provide external support for the tissue to be cut.
What is the most important step in embedding?
Correct orientation of tissue in mould
How are paraffin blocks cut?
by a microtome
Microtome
machine that cuts paraffin blocks using a metal knife, 6u (micrometer) in thickness
Mounting
sections spread on hot plate, mounted on glass slides
Staining
Special dyes are used to stain histological sections (2D slices of tissue from a 3D piece of tissue) and make them ready for microscope examination
what are the two commonly used dyes for paraffin wax?
- Haematoxylin (Hx)
- Eosin (E)
Reactions of stains
- Acidic - Eosin stain
- Basic - Haematoxylin stain
- Neutral - Leishman stain
Physical stain
No reaction in fatty tissues; the stain is SUDAN III
Vital stain
The staining of living tissues inside the body
What is the vital stain used for?
To stain phagocytic (immune cells) as TRYPAN BLUE
Metachromatic stain
Staining tissues a colour different from the original stain. (ex: red stain shows blue when staining a tissue)
-Toluidine blue staining of mast cells
Mast cells
Phagocytic cells
-Type of white blood cell
-Uses phagocytosis to engulf:
bacteria
foreign particles
Dying cells
Usage: To protect the body
Histo-chemical stain
Staining tissue to identify components of the cell as glycogen and protein
Freezing technique
-Water-rich tissues are hardened by freezing and then cut
-sections are examined by (LM)
-Much faster than paraffin and is used in operations to acquire a quick diagnosis
Which technique is faster paraffin or cryosection?
cryosection
what is a synonym for freezing technique?
cryosection
What is an electron microscope used for?
To show ultrastructure (deep magnification of the structure of cells)
Fixation (EM)
uses glutaraldehyde and osmium tetraoxide
Embedding (EM)
Tissues are inserted in Epon (type of resin) in gelatin capsule
Cutting (EM)
-capsules are cut by ultra-microtome
-using glass/diamond knife
-section measurement 50-100nm
Mounting
sections mounted on metal grids
Staining
by heavy metals (lead nitrate and uranyl acetate)
Blasais
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