Centrifugation - the very, very basics.

The basis for all centrifugation-based techniques is the difference in density between the object to be analyzed and the medium in which it is suspended. If the object is less dense than the medium, it will float. If it is denser, it will sink. Centrifugation simply speeds the sinking process. Bacterial cells are denser than standard growth media. If a culture of bacteria is left on the bench top undisturbed, the bacteria will fall out of solution (they will sediment). The rate of sedimentation is determined by the difference in the object's density compared to the density of the medium and the earth's gravitational field. The rate of sedimentation will also be influenced by its size. A small particle, such as a phage, will remain in suspension (even though it is even denser that a bacterium), due to Brownian effects (first discovered by Robert Brown in 1827 and explained in molecular terms by Albert Einstein in 1905. The constant jostling of the surrounding molecules is enough to overcome the effects of gravity, and keep phage suspended in solution. To overcome these Brownian effects, and speed the process of sedimentation, we can increase the effective gravitation field by using a centrifuge. A typical laboratory centrifuge produces a centrifugal force of ~10,000g. More specialized (and rather more expensive) instruments can produce centrifugal forces of up to ~600,000g.

The equation describing sedimentation rate is n = d^2 (po-pm) x g/18n where n is the sedimentation rate d is a function of particle size and shape po is the density of the particle pm is the density of the medium g is the strength of the gravitational/centrifugal field n is the viscosity of the medium. The effective centrifugal field is a function of the diameter of the centrifuge's rotor and the speed at which it spins. Depending upon the situation, particles can be separated on the basis of size (and to some extent shape), density, or a combination of both. In our studies, we exploit the size different between an intact bacterium, a T4 phage, and the subcellular debris that remains after cell lysis. A short 5 minute centrifugation at 5,000 g in a table top centrifuge is enough to pellet intact bacteria, and leave phage in solution. A longer spin of 10 minutes at 10,000 g will remove most cellular debris, while leaving the phage in solution. T4 phage are large and they can be pelleted by centrifugation, but this takes ~20 minutes at 35,000g