Culture Cells from a Frozen Stock

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Culture Cells from a Frozen Stock We store all M. xanthus strains as frozen stocks in the -80C freezer. Each week you will need to make a new nutrient agar plate of cells that we can use for research. Note: do not attempt to make broth cultures straight from frozen stock as the risk for contamination is very high.

1. Remove the necessary CTTYE plates from the 4C fridge. Let it come to room temperature, or if pressed for time, place in the 37C incubator for 5 min.

2. Spray off your work area with 70% ethanol (spray bottles located around the lab) and wipe dry with a paper towel.

3. Label the plate on the bottom with the strain name, your initials, and the date. The date is important because you don’t want to use the same plate for more than ~7 days.

4. Find some p1000 tips (you do not need filter tips for this) and put those in your work station as well as a p1000 pipette.

5. Once everything is set up, you are ready to get your cells. You want to work quickly so that the stocks aren’t out of the freezer for more than 1 minute if possible so they do not thaw. Most stocks will be in the small -80C freezer in the lab in the box labeled “JAC” in red sharpie.

6. Remove the box from the freezer and locate the correct strain. Before transferring cells to the plate, make sure that you double check the label because once the cells are growing there is no easy way to verify that you cultured the correct strain.

7. Remove the cap from the tube and do not touch the rails or the undersize of the cap with your hands. Place the cap face down onto a clean section of the bench that you have not been touching with your gloves or sleeves.

8. Open the lid of the plate and place it face down onto a clean surface as well.

9. Use the p1000 pipette to select a tip from the box, then, only touching the upper of the tip, remove it from the pipette with your fingers and use it to scrape some of the cells up from the frozen sample in the tube.

10. Transfer the cells to the plate and throw the tip into the biohazard bag. While the bit of culture melts onto the plate, cover the plate and immediately return the tube and box to the freezer.

11. Grabbing a new tip, spread the thawed cells around the plate gently without scraping up the agar. Typically I will spread the culture over the surface leaving about a 1/2 inch around the edge of the plate uninoculated.

12. The plate can be put in the 32C incubator for 2-3 days for the cells to grow. After the plate has good growth, it should be transferred to the room temperature incubator.