$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
1. Preparation of Bacterial Strains or Mutant Libraries
1. Streak out the strains to be tested on appropriate agar plates. These are stock plates for the experiment.
1. Incubate the plates overnight at the desired temperature to achieve single colonies. For this experiment, culture K. pneumoniae (NTUH-K2044 and ATCC43816 strains) on Luria broth (LB) agar at 37 °C, and S. pneumoniae (23F wild type and 23F Δcps) on blood agar in a humidified candle jar at 37 °C.
2. Pick a single colony from a stock plate (step 1.1) to inoculate 10 mL of appropriate broth using a sterile loop or cocktail stick. For screening of random mutant libraries, inoculate the broth with 10 μL of the random mutant library stock (TraDIS Library).
1. Incubate K. pneumoniae strains in low salt LB media at 37 °C with shaking, and S. pneumoniae strains in brain heart infusion (BHI) media, statically at 37 °C.
2. Transfer the overnight culture to a 15 mL tube and centrifuge in a bench-top centrifuge for 10 min at 3,200 x g in swing out buckets with 15 mL tube inserts and aerosol tight lids.
3. Discard the supernatant and resuspend the pellet in 2 mL of 1x phosphate-buffered saline (PBS).
NOTE: Dispose of supernatant via the appropriate liquid biological waste route in the laboratory. The purpose of the centrifugation and resuspension steps (1.2.2 and 1.2.3) is to concentrate the bacteria for easy visualization on the gradient. Bacterial cultures can be loaded directly onto the gradient if preferred. Heavily capsulated strains may not form a tight pellet. If this occurs, remove as much supernatant as possible without removing any bacterial cells, add 1x PBS to a final volume of 5 mL, and resuspend the pellet. Continue the protocol with step 1.2.5.
5. Centrifuge the tubes as described in step 1.2.2.
6. Discard the supernatant and resuspend the pellet in 2 mL of 1x PBS. Cells are now ready to use.
NOTE: The density of the bacterial cell suspension is not critica,l but needs to be sufficient for visualization in the gradient. A minimum OD600 (optical density at 600 nm) of 4 is suggested.