Prepping a Submerged Culture

1. Obtain 60 mm petri plates and overnight culture, and wipe down the bench with 70% ethanol. Make sure that you are setting up nearby the incubator.

2. Turn on the spectrophotometer, and while waiting, label the lid of the petri plate with initials, strain, date, and any treatments.

3. Measure the absorbance of your overnight culture using the spectrophotometer, and calculate the density according to the protocol "Measure Absorbance of M. Xanthus Culture"

4. In a conical tube, dilute the overnight culture in CTTYE to 2x10^7 cells/ml so that you have approximately 10 ml of diluted culture per plate. The solution does NOT need to be made all at once (ex. if your dilution calls for 34 ml of CTTYE to 1 ml of overnight culture, you can make 35 ml of diluted culture, fill your plates, and then dilute more culture as needed).

5. Using a serological pipette, add 8 ml of the diluted culture to each petri plate.

6. Carefully move the plates to a 32C, ambient CO2 incubator and ensure that the plate is level. Remember, the cells SHOULD NOT be shaking, because the cells will settle at the bottom of the petri plate to create a semi-adherent layer.

7. After 24 hours of incubating, aspirate the CTTYE off with a 10 ml serological pipette and replace with the same amount of MMC (and any dyes, etc).

8. Proceed with imaging/time lapse!

Tips and tricks!

a. When moving the CTTYE plates into the incubator, wait to put the lid on the plates until AFTER you place them in the incubator.

b. Aspirate from the "edges" of the plate

c. Remove about half the CTTYE from the plate while it is still inside the incubator

d. After aspirating half of the CTTYE off, hold the plate at an angle to aspirate as much of the remaining CTTYE as possible.

e. When adding MMC to the plate, pipette up 9 or 10 ml of MMC but only add 8 ml of it to the edge of plate. When the pipette is emptied entirely, the solution is ejected with greater force and will disrupt the layer of bacteria.

f. When needing to transport, take the shortest path, pre-open doors, and walk slow! Also, use a tray!

g. Originally, it only took about 24 hours in MMC to result in stable fruiting bodies, but as of Spring '24, it seems that it can take up to 48 hours for stable fruiting bodies to form.