Electron Microscopy Fixation

Microscopy Methods in Microbiology
Microroscopy Methods in Microbiology Part 2
Microroscopy Methods in Microbiology - Introduction
Observation of Microbes in the Living State
Observation of Microbes in Smears
Dyes and Staining
Useful pH Range and Colour Change of Some Indicator Dyes
Factors Affecting Staining
Microscopy and Micrometry
Microscopy and Micrometry - Introduction
Behaviour of Light
Light Theories
Resolving Power
Refraction and Reflection
Bright Field Microscopy
Use of Bright Field Microscope

Electron Microscopy Fixation

	Electron Microscopy Fixation: The primary objective of fixation is the rapid killing and preservation of the specimen with the minimum destruction and maximum capacity to withstand the subsequent steps. In addition, some fixatives ( chromium and uranium salts and lead compounds) have got staining ability. Fixation for electron microscopy differs in several ways from that applied to the light microscopy, though, some of the reagents are similar. The most important of these is the use of buffer to stabilize the pH. The process of fixation is usually carried out at lower temperature. Several new fixatives have been currently employed which include osmium tetroxide, potassium permanganate, certain aldehydes, acrolein etc.
Osmium tetroxide s the most widely used reagent yielding satisfactory results. The penetration of this fixative is slow, but it reacts rapidly with proteins, unsaturated fatty acids and phospholipids. The procedure for fixation of microorganisms described by Kellenberger et al (14 ) is given here. Mix 30 ml of suitable suspension with 3 ml of Kellenbergers fixative (S₁) and centrifuge at 1800 X g for five minutes. Resuspend the pallet in one ml of Kellenbrgers fixative containing 0.1 ml of tryptone medium (S₂), Incubate overnight at room temperature and then add 8 ml of Kellenberger s buffer (S₃) and centrifuge at 1800 X g for five minutes. After removing the supernatant, resuspend the pallet in distilled water and use this suspension for direct electron microscopy.
Glutaraldehyde-osmium tetroxide is also a commonly used fixative. Prepare 4% solution of glutaraldehyde and 2% solution of osmium tetroxide in phosphate buffer (pH 7.0). Take about 10 ml of glutaraldehyde fixative in a centrifuge tube and keep the specimen for 2-15 hours. Remove the material by centrifugation and. wash with buffer three times. To the sediment, add osmium tetroxide solution to cover it and incubate at 0-5°C for five hours. Remove the cells, wash with buffer and dehydrate it with series of alcohol.

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