Generate St Curve for OD600 to Cells/mL conversion

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1. Obtain O/N M. xanthus cultures at different densities (ideally relatively equally spaced to achieve close to 0.2, 0.4, 0.6, and 0.8 OD600.

2. Measure the absorbance of these samples and convert values to OD600 by factoring in the path length. Record these values.

3. Melt down CTTSA in the microwave, using caution not to boil it over. Heat in increments of 30 seconds once it starts melting.

4. Aliquot 4mL of CTTSA into as many tubes as is needed for your experiment, one 4mL tube per plate. Make sure these tubes are sterile! Put these tubes in a water bath or dry bath set to ~50C until you are ready to use.

5. Read this step completely before working with your cells. You want to have all tubes labeled before you start. Pipet 500uL of each of your O/N cultures into a labeled 1.5mL microcentrifuge tube. We want to serially dilute our culture of ~10^8 cells to 10^3 cells. This means performing a 1:100 dilution, another 1:100 dilution, and then a 1:10 dilution. Draw out if you need a visual! Make dilutions into CTTYE, and always work with at least 10uL volumes for dilutions like this to improve accuracy.

6. Once you have your 10^3 dilutions for each culture, you will mix your cells into soft agar and pour over the surface of warmed CTTYE plates. For the 5/26 experiment, you will plate two plates from each diluted culture.

7. Remove 4 CTTSA tubes from the heat block to let them cool for about 1 minute. To 2 tubes of CTTSA, add 100uL of diluted culture each, vortex briefly, and pour over the surface of the CTTYE plate. Do the same to the other 2, adding 50uL of culture. Repeat for each culture density.

8. Wait 15 min for the CTTSA to solidify over the plates, and then invert plates and place them in the incubator for ~2-3 days.