Tissue Processing Flashcards
To study tissues, one must
prepare thin and translucent
histological sections or
tissue slices that can be
studied with the aid of a ______
microscope
In order to make a histology
slide, the tissue specimen
undergoes various steps of
_________
tissue processing
STEPS IN TISSUE PROCESSING
- Fixation
- Decalcification
- Dehydration
- Clearing
- Infiltration
- Embedding
- Cutting
- Sectioning
- Sectioning
- Mounting
Since cellular decomposition begins immediately after the death of a human/patient, tissues must be ______ to
prevent alterations in their structure caused by decomposition.
fixed
After the specimen is surgically removed from the body, it should be preserved or fixed with a ______ to prevent
the decay of the tissue.
fixative
- Small pieces of tissue are placed in solutions of chemicals that cross-link proteins and inactivate degradative enzymes, which preserve cell and tissue structure.
Fixation
- To preserve tissue structure and prevent degradation by enzymes released from the cells or
microorganisms, pieces of organs are placed as soon as possible after removal from the body in solutions
of stabilizing or cross-linking compounds called fixatives.
FIXATION
WHY IS FIXATION IMPORTANT?
- Avoid tissue destruction by digestive enzymes (autolysis) or through bacterial degradation.
- Terminate cell metabolism.
- Hardens the tissue by cross-linking or denaturing proteins.
- Kill pathogenic microorganisms such as bacteria, fungi, and viruses.
Commonly used fixative in fixation
Formalin or formaldehyde
37% - 40%
● Only done in specimens such as bone and calcified tissues (and arteries w/ atherosclerosis)
DECALCIFICATION
decalcifying agent
Nitric acid
Done by successively bathing the specimen in a mixture of ethanol and water from 70% to 100%.
Dehydration
In dehydration,_______removes water out of tissue.
Alcohol
before infiltration, the fixed tissue must undergo dehydration by having its water
extracted gradually by transfers through a series of increasing ethanol solutions, ending in ________which removes all water.
100% ethanol
ASCENDING GRADES OF
ALCOHOL
50%
70%
90%
100%
● Removal of the dehydrating agent by immersing the specimen in the solvent that the alcohol and embedding medium is miscible.
CLEARING
- Alcohol is removed in organic solvents in which both alcohol and paraffin are miscible.
Clearing
T or F|Clearing agent are highly volatile
True - it becomes vapor once exposed to heat
Clearing Agents
Xylene and Toluene
Between Xylene and Toluene, which is more commonly used?
Xylene
After the clearing procedure, the tissue is placed in a melted paraffin
in an oven set at __ -__degree celsius.
52-60 degree Celsius
The fully cleared tissue is then placed in melted paraffin in an oven at 52°-60°C, which evaporates the clearing solvent and promotes _______ of the tissue with paraffin
INFILTRATION
WHAT IS USED IN iNFILTRATION?
melted paraffin wax
is allowing it to harden in a small
container of paraffin at room temperature.
Embedding
- makes use of a plastic solution
which hardens tissue by cross-linking
polymakers.
○ Eliminates the need to use oven and paraffin; little tissue distortion
Plastic resins
avoids the higher temperatures needed with paraffin, which helps avoid tissue
distortion.
Plastic embedding
After the specimen is hardened, it is trimmed into appropriately sized blocks.
○ _____ is removal of excess paraffin.
Cutting
In sectioning, the block is mounted in microtome and cut with a _______.
steel knife
Sectioning is done with the aid of _____
microtome
sections are placed on glass slides and stained for light microscopy or on metal grids for electron-microscopic staining and examination
MOUNTING
- Most cells and extracellular material are completely colorless, and to be studied microscopically tissue
sections must be (dyed).
STAINING
Application of color to the tissue to highlight structures.
Staining
Most commonly used
stain:
Hematoxylin and Eosin
T or F| Hematoxylin and Eosin are insoluble in water
FALSE - These dyes are water soluble
blue;
basic dye;
usually stains nucleus and RNA-containing portion of cytoplasm.
Hematoxylin
red;
acidic-dye;
usually cytoplasmic components and collagen.
Eosin
Tissues with negative charges/acids are readily stained with
basic dyes - basophilic
Tissues with positive charges are stained with
acidic dyes - acidophilic
- stains DNA in the cell nucleus, RNA-rich portions of the cytoplasm, and the matrix of cartilage, producing a dark blue or purple color.
Hematoxylin
- stains other cytoplasmic structures and collagen pink.
Eosin
considered a counterstain, which is usually a single dye applied separately to distinguish additional features of a tissue.
Eosin
Turnaround time for Standard Routine Tissue Processing
5 DAYS
- is a faster but more expensive way to do biopsy however it is by case basis.
Frozen Section Biopsy
Turnaround time for Frozen Section Biopsy
15 MINUTES
○ This method is rapid.
○ Routinely done in hospital to study specimens during surgery.
Frozen Sections
In Frozen Sections, ____ and ____ are best preserved in this method
Lipids and Enzymes
Fixative agent for frozen section
Liquid Nitrogen
