Diseases and Management of Crops under Protected Cultivation

(Diseases and Management of Crops under Protected Cultivation)
Aims of Fixation
To preserve the structure of cells and tissues with minimum or least alteration from the
living state.
To protect them against alterations during embedding and sectioning.
To prepare them for subsequent treatments such as staining and exposure to the electron
beam
Commonly used Fixatives
Glutaraldehyde
Paraformaldehyde Primary fixative
Acrolein
Karnovsky’s Fixative (Glutaraldehyde + Paraformaldehyde)
Osmium tetroxide
Secondary fixative
Some other compounds are also there which have the ability to partially fix or stain the
cellular constituents e.g. Chromium salts, Uranium salts, lead compounds and Phosphotungstic
acid (PTA).
Procedure of Fixation and Block Making
Primary fixation
1-2mm sq thick samples + 2.5% glutaraldehyde made
in 0.1M sodium phosphate buffer (pH 7.4) 2-24 hours at 4°C
Washing
Rinse thoroughly with 0.1 M sodium phosphate buffer (pH 7.4) to wash away excess fixative
Secondary fixation
Osmium tetroxide (1% solution) is commonly used, acts as electron dense stain reacts principally
with lipids.
Washing
Rinse thoroughly with 0.1 M sodium phosphate buffer (pH 7.4) to wash away excess fixative
Dehydration
Ethanol or Dry acetone is used to completely dehydrate the tissue.
Clearing
Xylene, Toluene or epoxy propane is commonly used.
Infiltration
Infiltration is done by gradually decreasing the concentration of clearing agent and proportionately
increasing the concentration of embedding medium.
Infiltration is carried out with liquid resins.
Embedding
Embedding is done in the embedding medium using a gelatin or beam capsule
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