Peculiarities of sample preparation of samples of monadic forms of microalgae for scanning electron microscopy
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Abstract
In order to optimize sample preparation of monadic forms of microalgae for scanning electron microscopy (SEM), domestic and foreign guidelines were analyzed. The green microalga Dunaliella salina Teodoresco (strain IBSS-2 from the Collection of Hydrobionts of the World Ocean, FIC IBSS) was used to develop the technique, and the protocol was tested on the Black Sea (from Collection of the Black Sea cryptophytes, BS-Cry, IBSS). It was shown that it is reasonable reduce the final concentration (f. c.) of glutaric aldehyde (glutaraldehyde, GA) in the sample to 1 % (for D. salina) or to use stepwise fixation with Lugol’s solution (for cryptophytic algae) when fixing the material. When concentrating microalgae equipped with flagella, the mildest possible filtration is necessary (vacuum less than 0.2 atm); the sample should be washed only if necessary; further dehydration should be carried out in a bucket or plastic plate. The use of glasses coated with poly-L-lysine led to good results. It has been shown that there is no particular difference between «ethanol» and «ethanol-acetone» dehydration, but that the former method takes less time and does not require operation in a fume cupboard. The «critical point» drying (2.5–3 h) and sputtering (Au/Pd, 0.5–1.0 min), followed the regimes usually recommended in modern manuals. If it is impossible to carry out all stages of sample preparation in one day or in expeditionary conditions, it is possible to store samples for up to two weeks in fixative solution or in 75 % ethanol solution (in the process of dehydration). The proposed protocol for pre-microscopic sample preparation for SEM studies can be useful for studying surface structures and detailing morphological characteristics of unicellular algae equipped with flagella and has been successfully applied in taxonomic and biotechnological studies.
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References
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