Separation of Bacteria by Capsule Amount Using a Discontinuous Density Gradient

Abstract

Source: Feltwell, T., et.al. Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient. J. Vis. Exp. (2019)

This video demonstrates the use of a discontinuous density gradient to separate bacterial strains based on capsule amount. By centrifuging a transposon mutant library through the gradient, bacteria with different capsule densities localize to distinct layers. The separated fractions can then be collected for downstream analysis.

Protocol

1. Adding Prepared Cells to Gradients and Separation by Centrifugation

1. Add 600 µL of prepared cells to the top of the gradient in the tube very slowly and without mixing the interface.
2. Place the tubes in tube adapters and weigh the combined adapters and tubes to ensure they are balanced.
   1. Place the tube adapters in a fixed-angle rotor within a bench-top centrifuge. Centrifuge for 30 min at 3,000 x g.
   2. After centrifugation, carefully remove the tubes, place them in a suitable rack, and photograph the results as a record.
3. Recover the bacterial fractions for uronic acid quantification or DNA extraction.
   1. If downstream applications are not required, dispose of the samples/gradients via the appropriate liquid biological waste route in the laboratory.
      NOTE: It is important to validate the separation for new species/strains of bacteria. Individual fractions from the gradient should be examined by microscopy, by uronic acid assay, or another suitable quantitative assay to confirm the capsule phenotype of each fraction. If the aim is to separate based on aggregation or cell size, independent appropriate assays should be used.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Percoll	GE Healthcare	17-0891-01
Centrifuge 5810R with Rotor A-4-81 and 500ml buckets	Eppendorf	5810 718.007
Adapters for 15 ml tubes	Eppendorf	5810 722.004
Fixed angle rotor F-34-6-38	Eppendorf	5804 727.002
2.6 - 7 ml tube adapter	Eppendorf	5804 739.000
Centrifuge 5424 including Rotor FA-45-24-11	Eppendorf	5424 000.460
5 ml polypropylene round bottom tube	Falcon	352063