Improved methodology for identification of cryptomonads: combining light microscopy and PCR amplification

Area of Science:

• Marine Biology
• Phycology
• Microscopy

Background:

• Cryptomonads are unicellular, biflagellate algae crucial in aquatic ecosystems.
• Their fragile nature complicates preservation for accurate identification from natural habitats.
• Existing preservation methods, like Lugol's solution, often fail to maintain cellular integrity.

Purpose of the Study:

• To develop an improved methodology for preserving cryptomonad cells.
• To evaluate the efficacy of various cytological fixatives for cryptomonad identification.
• To assess preservation quality for both light microscopy and molecular analyses.

Main Methods:

• Tested glutaraldehyde, formaldehyde, and Lugol's solution as fixatives.
• Optimized glutaraldehyde concentration and assessed the need for osmotic adjustments or buffers.
• Evaluated preservation of cell morphology, color, volume, and key taxonomic features.
• Extracted DNA for 18S rDNA amplification and phylogenetic analysis.

Main Results:

• 2% glutaraldehyde provided superior preservation of cryptomonad cell morphology, including delicate structures like flagella and the furrow/gullet complex.
• Glutaraldehyde-preserved cells retained original color and shape, unlike those in Lugol's solution.
• Cell density remained stable for at least 43 days in 2% glutaraldehyde.
• DNA extraction and 18S rDNA sequencing were successful from glutaraldehyde-preserved samples, yielding accurate phylogenetic data.

Conclusions:

• 2% unbuffered glutaraldehyde is an effective fixative for cryptomonads.
• This method enables reliable identification using light microscopy and molecular techniques.
• The optimized protocol enhances cryptomonad research in ecological and taxonomic studies.