Separation of Bacteria by Capsule Amount Using a Discontinuous Density Gradient

Begin with a tube containing a pre-set discontinuous density gradient, prepared using non-toxic colloidal silica particles coated with polyvinylpyrrolidone.

The gradient comprises three non-overlapping layers arranged in order of increasing densities from top to bottom.

Take a bacterial transposon mutant library, a pooled suspension of bacterial strains with random transposon-induced mutations.

A subset of these mutations disrupts genes that regulate the capsule, a polysaccharide layer surrounding the bacterial cell wall, resulting in strains with varying capsule amounts.

Add the bacterial library to the top of the gradient very slowly, without mixing the interface.

Centrifuge the tube at a moderate speed.

The gradient facilitates the differential migration of bacteria based on their capsule amounts.

Hyper-capsulated strains, being less dense due to their hydrated polysaccharide layer, localize near the top.

Weakly capsulated strains settle in the middle, and non-capsulated, denser strains migrate to the bottom.

The separated bacterial strains are ready for downstream analysis.

Carefully add 600 microliters of the prepared cells to the top of the gradient in the tube. Then place the tube in