This method can help answer key questions in the Klebsiella field, such as which genes are used to regulate capsule and associated virulents. The main advantages of this technique are that it provides a means to physically separate capsulated and non-capsulated strains, and it can also be used for rapid comparisons between strains of capsule production. Though this method was developed to study capsule regulation in Klebsiella, it can also be applied to different bacterial species.
Generally individuals who are new to this method will struggle because pouring the gradients and adding the cells to the gradients can be quite difficult. Visual demonstration of this method is critical, as we show the techniques that are difficult to master, providing suggestions and tips to help the user. To begin, select a single colony from a stock plate with a sterile loop, and inoculate 10 milliliters of an appropriate broth.
Incubate the culture overnight. Then transfer the culture to a 15 milliliter tube, and centrifuge. Next, discard the supernatant, and re-suspend the pellet in two milliliters of PBS.
Repeat the centrifugation, and discard the resulting supernatant. Then re-suspend the pellet in two milliliters of PBS. To create the density gradient dilutions, combine density gradient medium with PBS.
Then aliquot 600 microliters of each gradient dilution into individual two milliliter tubes. Take up 100 microliters of cells with a 200 microliter pipette, and place the pipette tip on the side of the tube just below the meniscus of one of the many gradients. Aspirate the bacterial cells onto the gradient very slowly, taking care not to mix the interface.
Centrifuge the prepared tubes for 10 minutes at 8, 000 g's. After this, transfer the tubes to a rack to visualize the minimum gradient dilution required to retain the cells just above the gradient layer. First, transfer one milliliter of the most dilute density gradient dilution into a five milliliter round-bottom tube.
Using a syringe with a 1.5 inch needle, take up one milliliter of the next most concentrated density gradient. Place the needle at the bottom of the tube and inject the contents of the syringe very slowly. There must be a clear interface at each step of the density gradient.
If the layers mix, you will not achieve clean separation of your bacteria. Remove the needle from the gradient gently so as not to disturb the interface between the gradient dilutions. Then place the tube in a rack.
Carefully add 600 microliters of the prepared cells to the top of the gradient in the tube. Then place the tube in a tube adapter, and weigh the tube with the adapter to ensure balance. After this, place the tube adapter in a fixed angle rotor within a bench-top centrifuge, and centrifuge the tube for 30 minutes at 3, 000 g's.
Finally, after centrifugation, remove the tube carefully, place it in a rack, and photograph the results. In this protocol, density gradient centrifugation was used to separate bacterial strains based on how much capsule they produce. Though the exact results depend upon the bacterial species, most strains will migrate to a single location within the gradient.
Application of this method to a bacterial mutant library should produce a major band above the gradient, a less dense band through the uppermost layer of the gradient, and a minor acapsular fraction at the bottom. While attempting this procedure, it is important to remember that the gradient interfaces should not be mixed, and the gradient should not contain air bubbles. Following the procedure, validation methods such as the uronic acid assay or microscopy should be performed to examine capsule amounts.
This is especially important when using this technique for new strains. Don't forget that working with Hazard Group 2 pathogens can be dangerous. Precautions such as corrective protective equipment, adhering to risk assessments and disposing of cultures correctly should always be taken while performing this procedure.
