Fixing Samples Flashcards
What are the three types of plane of section?
Cross section
Oblique section
Longitudinal section
What is the difference between perfusion and immersion?
Perfusion: inject fix into bloodstream, then remove organ (faster and better)
Immersion: submerge organ into fix
What are the steps to fix a sample for light microscopy?
Chemical fixation with Bouin’s fluid (perfusion or immersion)
Dehydration (add more ethanol)
Clearing with xylene (solvent for paraffin, not aqueous)
Add paraffin
Cut sample into slices with microtome
Mount sample on glass slide
Remove paraffin
Rehydrate (add more water)
Staining with hematoxylin and eosin
Light microscopy
What are the steps to fix a sample for electron microscopy?
Fix sample with gluteraldehyde (perfusion)
Dehydration with ethanol
Clearing with propylene glycol (solvent for epoxy)
Infiltration and embedding with plastic
Cut sample into slices using ultramicrotome (cuts thinner slices than microtome)
Mount sample on copper grid
Stain with lead citrate
Use electron microscope
What is the chemical fix used for electron microscopy?
Gluteraldehyde
What is the stain used for electron microscopy?
Lead citrate
What is the chemical fix used for light microscopy?
Bouin’s fluid
What are the stains used for light microscopy?
Hematoxylin and eosin
What is Bouin’s fluid made of?
Acetic acid
Picric acid
Formalin (formaldehyde)
Water
How thin does the microtome slice the sample compared to the ultramicrotome?
Microtome: 1-5 micrometers
Ultramicrotome: 5 nanometers
What kind of structures does hematoxylin stain?
Stain basophilic structures purple
Ex: cell nucleus
What kind of structures does eosin stain?
Stains eosinophilia structures bright pink
Ex: cytoplasm and collagen
What is an electron lucent structure?
The electron passes through a burns a hole in the plastic
What is an electron dense structure?
Electron bounces off stain and doesn’t pass through
What is resolving power or resolution?
Measurement of distance between two separate dots that you can distinguish as separate.
