This article outlines a method for the long-term cryopreservation and revival of cyanobacterial strains. The process involves the use of cryoprotectants to stabilize cells during freezing and provides a protocol for reviving the strains for future studies.
Begin by inoculating a loop of a cyanobacterial strain onto a growth medium. Incubate to allow the cells to grow to the desired density.
Wash the cells by centrifugation and discard the supernatant containing residual metabolites and debris.
Resuspend the pellet in a small volume of growth medium to create a concentrated suspension.
Mix an aliquot of this suspension with glycerol, a cryoprotectant that stabilizes the membrane and prevents ice crystal formation during freezing.
To another aliquot, add dimethyl sulfoxide, a cryoprotectant that penetrates cells and inhibits ice nucleation.
Store the samples at low temperatures, where metabolic activity halts and cells remain viable for extended periods.
To revive, scrape a small amount of the frozen biomass onto an agar plate without antibiotics and streak to reduce stress and support gradual recovery.
This method enables the long-term cryopreservation and revival of cyanobacterial strains for future studies.
To store the strains long term, set up a fresh culture of the strain by inoculating a loop full of cells into 30 to 50 milliliters of BG11 medium. Grow the culture for three to four days to an OD750 equal to 0.4 to 0.7.
After using BG11 to wash the cells once, resuspend the pellet in approximately two milliliters of BG11. Add 0.8 milliliters of the concentrated cells to one tube. Then add 0.2 milliliters of 80% filter sterilized glycerol.
To another tube, add 0.93 milliliters of concentrated cells and 0.07 milliliters of DMSO. Store both tubes at negative 80 degrees Celsius. To revive the strains, remove the tube and use a blunt-end toothpick to scrape off some cells onto an agar plate without antibiotics. Then use a sterile loop to streak out as normal.
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