Pseudokirchneriella subcapitata (Korshikov) Hindak (ATCC^® 22662^™)

1. Harvest cells from a culture that is at or near peak density by centrifugation at 700-800 x g for 5 min.
2. Adjust the concentration of cells to 2 x 10⁶ - 2 x 10⁷/mL in fresh medium.
3. While cells are centrifuging prepare a 14% (v/v) solution of sterile DMSO in fresh medium.
4. Mix the cell preparation and the 14% DMSO solution in equal portions. Thus, the final concentration will be 10⁶ - 10⁷ cells/mL and 7% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution to the beginning of the freezing process should be no less than 5 min and no greater than 15 min.
5. Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
6. Place the vials in a controlled rate freezing unit.  From room temperature cool at -1°C/min to -40°C.  If the freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through the heat of fusion.  At -40°C plunge 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. The frozen preparations should be stored 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. Vials should not be stored above -55°C.
8. To establish a culture from the frozen state place an ampule in a water bath set at 35°C. Immerse the vial just 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 16 x 125 mm screw-capped test tube containing 5 mL of ATCC medium 625.
10. Incubate the culture at 50-100 µEinsteins/m²/s irradiance at 25°C.  Maintain under a 14/10h light-dark photoperiod.

Mason CP, et al. Isolation of chlorine-containing antibiotic from the freshwater cyanobacterium Scytonema hofmanni. Science 215: 400-402, 1982. PubMed: 6800032

Skulberg OMAlgal problems related to the eutrophication of European water supplies, and a bio-assay method to assess fertilizing influences of pollution on inland watersIn: Skulberg OMAlgae and manNew YorkPlenum Presspp. 262-299, 1964

ASTM International Standard Practice for Algal Growth Potential Testing with Pseudokirchneriella subcapitata Pseudokirchneriella subcapitata. West Conshohocken, PA:ASTM International;ASTM Standard Test Method D 3978-04.

ASTM International Standard guide for conducting static 96-h toxicity tests with microalgae. West Conshohocken, PA:ASTM International;ASTM Standard Test Method E1218-04.

British Standards Institution Water quality - Freshwater algal growth inhibition test with unicellular green algae; [BS EN ISO 8692:2004]. London, UK:British Standards Institution;British Standard BS 6068-5.10:2004.

ISO Water quality -- fresh water algal growth inhibition test with Scenedesmus subspicatus and Selenastrum capricornutum. Geneva (Switzerland):International Organization for Standardization/ANSI;ISO ISO 8692:1989.

Standard Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates. West Conshohocken, PA:ASTM International;ASTM Standard Test Method E 1706-05.