Histology and Its Methods: Part II Flashcards
“Doing histology” requires a _________ with a specimen and a microscope
Slide
What is the major problem facing scientists studying tissues?
As soon as a tissue is removed from a living organism, it begins to degrade due to bacterial destruction and cellular self-digestion
What two processes contribute to cellular degeneration?
- Bacterial destruction
- Cellular self-digestion
To study tissues in their most natural states, what must occur?
Steps must be taken to inhibit destruction and preserve the tissue
Why must tissues and organs be sectioned for study?
Because tissues and organs are too thick for light from a light microscope to pass directly through them
What are the major steps to preparing a tissue for study?
The tissue sample is
- Removed
- Fixed
- Dehydrated
- Cleared
- Infiltrated
- Embedded
It can take anywhere from ___ hours to ___ days to prepare a slide from tissue fixation to observation
12 hours
2.5 days
What factors result in the significant differences in time regarding tissue preparation?
Tissue size, embedding medium, and staining method
A _________________ is a solution of stabilizing or cross-linking compounds that inactivate degrading enzymes
Fixative
What’s a common fixative for light microscopy?
Formalin
What’s a common fixative for electron microscopy?
Glutaraldehyde
After fixation, what occurs during the preparation of tissues for study?
Dehydration
How does tissue dehydration occur?
Water is gradually extracted by transfers through a series of more concentrated alcohol (ethanol) solutions, eventually ending in 100%
What occurs during clearing?
Ethanol is replaced by an organic solvent of alcohol and embedding medium, resultuing in a translucent appearance
What’s the step called when a tissue is placed in melted parafin at 58 degrees celsius or snap frozen in liquid nitrogen?
Infiltration
After infiltration, what occurs during tissue slide preparation?
Embedding
________________ is the process by which the tissue is placed in a small mold contraining melted parafin and then allowed to harden
Embedding
Hardened block with tissue and surrounding embedding medium is trimmed and placed for sectioning in a ___________________
Microtome
During sectioning, tissues are sliced according to needs. Sections are sliced between 3 and 10 micrometers for _______ microscopy and less than one micrometer for _________ microscopy
Light
Electron
Why must tissue sections be sliced smaller for electron microscopy than light microscopy?
Because electrons have a much smaller wavelength and can’t penetraten through thicker sections
What’s the final step of tissue preparation?
Sections are placed on glass slides and stained for light microscopy or metal grids for electron microscopy
What’s the most common type of stain?
Hematoxylin and eosin
Hematoxylin stains cellular components with a negative charge, like nucleic acids that have an affinity for positively charged (basic) dyes, otherwise called _________________
Basophilic
Which H&E staining component stains negative cellular components?
Hematoxylin
Cellular components with a positive charge, like proteins with amino groups, stain with eosin, making these cellular components ______________
Acidophilic
Hematoxylin binds ___ charges; eosin binds ___ charges
Hematoxylin stains _____; eosin stains _____
Negative; positive
Purple; pink
Which component of H&E staining serves as the counterstain?
Eosin
Why are counterstains useful?
Because staining procedures often label only specific structures, rendering parts of the cell invisible
________________ staining contains three colored components to allow for greater distinction among extracellular tissue components
Masson trichrome
In Masson Trichrome staining, what types of cellular components are stained?
Extracellular matrix
In Masson trichrome staining, collagen-rich ECM stains _____, cytoplasm stains _____, and nuceli stain ______
Blue
Pink
Red/purple
___________________ stains negatively charged, sulfate-containing glycans, usually mucins or glycosaminoglycans
Alcian blue
_____________ stains neutral glycans
Peridic acid Schiff
_______________________ uses solutions of silver salts to visualize ECM fibers and nervous tissue elements
Metal impregnation
What type of stain is shown?
Masson trichrome
What type of stain is shown?
Alcian blue
What type of stain is shown?
Metal impregnation
DAPI stains __________________ the color ________
Nucleic acids
Blue
In _______________ sections, specimens are cut along the direction of the organ
Longitudinal
In _____________ sections, specimens are cut perpendicular to the length of the organ
Cross/transverse
In _________________ sections, specimens are cut at an angle between cross and longitudinal sections
Why are sections often examined in parallel (serial sections)?
Because sections are limited to two-dimensionality, representing only a part of a larger three-dimensional structure
Distortion in preparatory steps may result in structural abnormalities called _______________, structures seen microscopically that may differ from structures present when tissue was alive
Artefacts
What are five problems that can result during tissue preparation and in turn distort slide interpretation?
- Shrinkage, resulting in artificial spaces
- Loss of molecules, like lipids, glycogen, and other low molecular weight substances often lost during fixation
- Wrinkles of a section
- Stain precipitates
- Differential staining (it’s impossible to stain all tissue components on a slide)
