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Goals of Specimen Preparation Observe specimen near natural state as possible. Preservation of as many features as possible. Avoid artifacts (changes, loss or additional information ). Preparation of Biological Samples. Fixation and washing/rinsing
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Observe specimen near natural state as possible.
Preservation of as many features as possible.
Avoid artifacts (changes, loss or additional
A process which is used to preserve (fix) the structure of freshly killed material in a state that most closely resembles the structure and/or composition of the original living state.
Usually in form of paraformaldeyde powder or 37% to 16% aqueous solution
pH (Isoelectric point)
Total ionic strength of reagents
Length of fixation
Method of application of fixative
Solution containing a weak acid and its salt.
Serves to hold pH steady during the fixation process.
Paraffin Sectioning for Light Microscopy
Making the water trough
Tape or plastic
a) Cutting edge
b) Knife angle (45o)
Loop - assist picking up sections
Eyelash tools - assist with section manipulations
Formvar, Carbon, Collodion
-Used when sections or samples are smaller than support of grid.
-100 mesh or less, slot grids
-Fragile or very thin sections
Avoid when possible because:
Usually has holes or uneven thickness
Added thickness affects clarity and contrast
Formvar and carbon
Positive staining - forms a complex with specimen
Negative - stain and specimen do not interact and specimen remains electron transparent
1) Improved resolution
3) Unique information
2) Limited surface topography
1) High density to provide high contrast
2) High solubility and minimal reaction to sample
3) High melting and boiling point (beam stable)
4) Precipitant formed is extremely fined grained
Stains commonly used:
Phosphotungstate, sodium tungstate, uranyl acetate and uranyl nitrate
Small grid and support film (formvar, paraloidin. Sometimes carbon added.
Thin suspension of sample and excess removed.
Dry then add negative stain and remove
Factors affecting staining:
concentration of stain
pH of stain
- Dry and view.
Negative stain of purified RhMV virus labelled with anti-RhMV and detected with anti- rabbit conjugated to 10 nm gold. Bar = 100 nm.
Photograph provided by Fred Gildow Lab, Department of Plant Pathology, Penn State.