Fluorescein Diacetate (FDA) Vital Staining

1. Stored vials should be transferred from the Cryostore to a small Dewar containing liquid nitrogen and transferred to the laboratory for thawing.
2. Vials are thawed by placing in a pre-heated water bath (40^oC) and agitate until the last ice crystal has melted
3. On thawing rapidly transfer to a laminar flow cabinet and wipe the outside of the vial with 70% (v/v) ethanol.
4. Using a disposable plastic pipette transfer the 1-mL of each of 3 thawed samples to 9-mL of fresh sterile BBM+V.
5. Incubate under standard conditions for 24 h prior to performing FDA assessment.
6. Prepare a standard FDA stock solution. Fluorescein Diacetate (FDA) (Sigma, F 7378). FDA [0.001% (w/v)] is prepared by first dissolving 25 mg of FDA crystals in a few drops of acetone and making up to the final volume (25 ml) with methanol.
7. Decant 3 x 1-mL aliquots from step 4 into test-tubes and add 50-μL of FDA stock solution to each tube in turn.
8. Incubate at room temperature for 1-5 min.
9. Observe under an excitation-fluorescence (Cells with functioning esterase activity cleave the stain which then fluoresces intensely yellow/green under UV illumination; non-viable cells appear colourless or red due to the autofluorescence of chlorophyll.)
10. As a control use non-cryopreserved cells. To test stain efficiency.
11. Count 50-100 cells and record FDA + ve as bright green fluorescent cells.
12. Viability was expressed by number of FDA positives (under fluorescence) against the total number of cells observed within the field(s) viewed.

% viability = (No. post-treatment FDA + ve) / (Total No. of cells observed in field(s) of view assayed) X 100.

Note; if control (step 10) had less than 100% viability then this should be factored into the absolute post-thaw viability levels e.g. If control samples had viability levels of 80% and post-thaw viability levels were 80%, then in reality 100% of the cells capable of surviving have survived. This approach will allow direct comparison with the CFU data.

References

DAY, J.G. (1998): Cryo-conservation of microalgae and cyanobacteria. - CryoLetters 1 (suppl.): 7-14.

DAY, J.G. (1999): Conservation of algae. – In: BENSON, E.E. (ed.): Plant Conservation Biotechnology: Chapter 8, 111-124. Taylor and Francis, London.

DAY, J.G., M.M. WATANABE, J.G. MORRIS, R.A. FLECK & M.R. MCLELLAN (1997): Long-term viability of preserved eukaryotic algae. - J. Appl. Phycol. 9: 121-127.

DAY, J.G., R.A. FLECK & E.E. BENSON (1998a): Cryopreservation of multicellular algae: problems and perspectives. – CryoLetters 19: 205-206.

DAY, J.G., M.M. WATANABE & M.F. TURNER (1998b): Ex situ conservation of protistan and cyanobacterial biodiversity: a CCAP – NIBS collaboration 1991 - 1997. - Phycol. Res. 46 (Suppl.): 77-83.

FLECK, RA, DAY, JG, RANA, KJ, BENSON, EE (1997) Use of cryomicroscopy to

visualise freeze-events on cryopreservation of the coenocytic alga Vaucheria sessilis. CryoLetters 18, 343-354.

FLECK, RA, DAY, JG, CLARKE, KJ, BENSON, EE (1999) Elucidation of the metabolic and structural basis for the cryopreservation recalcitrance of Vaucheria sessilis,

Xanthophyceae. CryoLetters, 20, 271-282.

MORRIS, G.J. (1978): Cryopreservation of 250 strains of Chlorococcales by the method of two step cooling. - Br. Phycol. J. 13: 15-24.

MORRIS, G.J. (1980): Cryopreservation. An introduction to cryopreservation in culture collections. Institute of Terrestrial Ecology. 27. - Cambridge, UK.

MORRIS, G.J. & J.J. MCGRATH (1981): Intracellular ice nucleation and gas bubble formation in Spirogyra. - CryoLetters 2: 341-352.

TAYLOR, R. & R.L. FLETCHER (1999): Cryopreservation of eukaryotic algae: a review of methodologies. - J. Appl. Phycology. 10: 481-501.

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