Development Assay on Agarose Plates

1. Measure the density of an overnight M. xanthus culture using the spectrophotometer. Before spinning and prepping your samples, calculate the volume of TPM starvation buffer that you will use to bring your overnight culture to a concentration of 5x10^9 cells/mL that we typically use for starvation assays.

If you take 1mL of your overnight culture at the density that you calculated, you can use the C1V1=C2V2 calculation.

(1mL)(O/N culture cells/mL) = (5x10^9 cells/mL)(V2), where V2 is the volume of TPM you will use to resuspend the cells.

2. Add 1mL of overnight culture to a 1.5mL microfuge tube and centrifuge at max speed for 1-2 minutes.

3. Decant off supernatant and pipet off the excess.

4. Wash pellet in 100uL of TPM starvation buffer, making sure to thoroughly resuspend the pellet with the pipet.

5. Spin at max speed for 1-2 minutes, remove supernatant, and repeat this wash step.

6. Remove all traces of supernatant without disturbing the cell pellet, and then resuspend the pellet in the volume of TPM that you calculated in step 1. Be very thorough with the resuspension and make sure that you disrupt all clumps of cells. Try to do this without vigorous vortexing if possible.

7. Plate 5-10uL droplets onto the surface of a DRY 1.0% agarose TPM plate (typically for a 60mm plate you would plate 3-5 droplets per plate with enough space in between that they will not run together).

8. Leave the lid off of the plates until the droplets are completely dry, then replace the lid, invert, and proceed to incubation and/or imaging.