Density centrifugation

Density centrifugation can be used in conjunction with other isolation and algal treatments to improve the purification of algal strains from unwanted algal and protozoan contaminants. The technique can also be used for the separation of subcellular particles and the isolation of algal viruses.

Percoll, a silica colloid, is diluted to a range of concentrations to produce a density gradient. An algal culture is then added before centrifugation. Centrifuging at low speeds, cells of varying densities will settle to the density band to match their individual density while the Percoll gradient remains stable. Percoll has a low osmolality and therefore doesn’t produce a significant osmolality gradient itself. However, care is needed to prepare isopycnic gradients, particularly for stepwise gradient preparation, as variations in osmolality can cause cells to shrink or swell. Density gradients can be produced either by the stepwise addition of pre-prepared gradient mixtures to the centrifuge tube or by the self-generation of gradient through the high-speed centrifugation of a uniform Percoll mixture. Due to the complexities in the formation of self-generating density gradients from the physics of centrifugation with effects from rotor angles, the non-recommended use of swinging bucket rotors and the variation of Percoll viscosity with osmolality, only stepwise density gradients will be covered in this method.

NOTES ABOUT HANDLING PERCOLL:
Refrigerate after opening.
To resterilise, autoclave at 120°C for 30min.
Prepared gradients should remain stable for several weeks.
Centrifuging at <10,000 x g will quickly cause the Percoll to pelletise at the bottom of the tube.
Not suitable for acidic media types as at pH < 5.5 the Percoll may begin to gel.

1. Equipment

  • Percoll
  • 15 mL centrifuge tubes and rack
  • Sterile media/stock solutions
  • Centrifuge
  • Sterile 1 mL pipettes and micropipettes
  • Bunsen burner or small flame
  • Density marker beads (optional)
NOTE: Density marker beads can be used to define density bands within the gradient. These added as a 1 mL mix in a separate but identical gradient to the sample and can be used to counter-balance the centrifuge. Small variations in density can occur with marker beads where the ionic strength of the media varies from the calibration solutions and thus this should be considered in any interpretation. They can also help with method development.

2. Method – Marine cultures

  • A Stock Isotonic Percoll (SIP) solution that is 50% Percoll is prepared by mixing an equal part of Percoll to an equal part of double strength evaporated media or seawater.
  • Using the 50% SIP solution, prepare stepwise Percoll gradients for 10%, 20%, 30%, 40% and 50% Percoll.
  • Starting with the most concentrated gradient and working to the most dilute, carefully pipette 1 mL of each layer into a 15‑mL centrifuge tube.
  • Add 1 mL of culture on the surface.
  • Centrifuge at 1500 rpm for 45min
  • Use a micropipette to gently remove the target algal cells from the interface that they have collected at.
  • The Percoll is washed from the cells at a ratio of 5 part media/seawater to 1 part Percoll/cell mix by centrifuging at 2500 rpm for 2 min and collecting the pellet of cells at the bottom of the tube.
  • Repeat the rinse 2-3 times.
  • The Percoll gradient separation may need to be repeated 3 times to effectively remove contamination.

3. Method – Freshwater cyanobacteria cultures

  • As cyanobacteria tend to float, a Stock Isotonic Percoll (SIP) solution that is 90% Percoll is prepared by mixing 9 parts of Percoll to 1 part of 10x concentrate of media.
  • Using the 90% SIP solution, prepare stepwise Percoll gradients for 0%, 20%, 50%, and 80%-90% Percoll.
  • Starting with the most concentrated gradient and working to the most dilute, carefully pipette the gradients following quantities: 2 mL of 80%-90%, 5 mL of 50%, 1 mL of 20% and 1 mL of 0% into a 15‑mL centrifuge tube.
  • Add 1 mL of culture on the surface.
  • Centrifuge at 1500 rpm for 45min
  • Use a micropipette to gently remove the target algal cells from the interface that they have collected at.
  • The Percoll is washed from the cells at a ratio of 5 part media to 1 part Percoll/cell mix by centrifuging at 2500 rpm for 2 min and collecting the pellet of cells at the bottom of the tube.
  • Repeat the rinse 2-3 times.
  • The Percoll gradient separation may need to be repeated 3 times to effectively remove contamination.

References

GE Healthcare (2007) Cell Separation Media: Methodology and applications, Sweden 80pgs

Cho J.-Y., Choi J.-S., Kong I.-S., Park S.-I., Kerr R. G. and Hong Y.-K. (2002) A procedure for axenic isolation of the marine microalga Isochrysis galbana from heavily contaminated mass cultures, J. App. Phycol. 14: 385-390

Lavoie, A., Mouget J.-L. and de la Noue J. (1986) Measurement of freshwater micro-algal cell density with Percoll density gradients, J. Microbiol. Meth. 4: 251-259

Price C. A. and Reardon E. M. (1978) Collection of dinoflagellates and other marine microalgae by centrifugation in density gradients of a modified silica sol. Limnol. Oceanog. 23: 548-553

Whitelam G. C., Lanaras T. and Codd G. A. (1983) Rapid separation of microalgae by density gradient centrifugation in Percoll, Br. Phycol. J. 18: 23-28