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Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.
1. ? Prepare a 5% (v/v) sterile methanol solution in fresh ATCC medium 1174 without agar.?
2.?? Harvest cells from a culture which is at or near peak density by adding 3.0 ml methanol solution to the slant and washing cells into suspension.
3. Adjust the concentration of cells to 2 x 106/ml with fresh methanol solution.
4.? Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).? The time from mixing of the cell preparation and the methanol solution, before the cooling cycle begins, should be no greater than 15 min.
6.?? Place vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If freezing unit can compensate for the heat of fusion, maintain rate at -1 C/min through heat of fusion. At -40°C plunge ampules into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus.? Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen.? (The cooling rate in this apparatus is approximately -1°C/min.) ??
7. Store ampules in either the vapor or liquid phase of a nitrogen refrigerator. Frozen preparations stored below
??? ?-130°C are stabile indefinitely. Those stored at temperatures above -130°C are progressively less stabile as the storage temperature is elevated.? Shelf life must be determined empirically for storage temperatures above
-130°C.
8.?? To establish a culture from the frozen state place an ampule in a water bath set at 35°C. Immerse the vial to a level just above the surface of the frozen material. Do not agitate the vial.
9.?? Immediately after thawing, do not leave in the water bath, aseptically remove the contents of the ampule and add to a centrifuge tube containing 5 ml of ATCC medium 1174 without agar.? Centrifuge at 300 x g for 5 min.
10.Remove most of the supernatant and then resuspend the pellet.? Place culture on ATCC medium 1174 slants and incubate on a 15° horizontal slant at 50-100 μEinsteins/m2/s irradiance at 25°C.? Maintain under a 14/10 h light-dark photoperiod.
Avron M, Ben-Amotz A. Production of glycerol, carotenes and algae meal. US Patent 4,199,895 dated Apr 29 1980
Ben-Amotz A, et al. Chemical profile of selected species of microalgae with emphasis on lipids. J. Phycol. 21: 72-81, 1985.
Ben-Amotz A, Avron M. On the factors which determine massive Beta-carotene accumulation in the halotolerant alga Dunaliella bardawil. Plant Physiol. 72: 593-597, 1983.
Ben-Amotz A. Accumulation of beta-carotene in halotolerant algae: purification and characterization of beta-carotene-rich globules form Dunaliella bardawil (Chlorophyceae). J. Phycol. 18: 529-537, 1982.
Ben-Amotz A, et al. Massive accumulation of phytoene induced by norflurazon in Dunaliella bardawil (Chlorophyceae) prevents recovery from photoinhibition1. J. Phycol. 23: 176-181, 1987.
Ben-Amotz A, Avron M. The wavelength dependence of massive carotene synthesis in Dunaliella bardawil (Chlorophyceae)1. J. Phycol. 25: 175-178, 1989.
Ben-Amotz A, et al. Stereoisomers of á-carotene and phytoene in the alga Dunaliella bardawil. Plant Physiol. 86: 1286-1291, 1988.
Ben-Amotz A. New mode of Dunaliella biotechnology: two phase growth for B-carotene production. J. Appl. Phycol. 7: 65-68, 1995.
Ben-Amotz A, et al. Use of the beta-carotene rich alga Dunaliella bardawil as a source of retinol. Br. Poult. Sci. 27: 613-619, 1986. PubMed: 3815129
Nucleotide (GenBank) : U91900 Dunaliella bardawil phytoene synthase mRNA, complete cds.
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