Making a Broth Culture from an Agar Plate

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Making a broth culture from an agar plate To do essentially all experiments with M. xanthus, we need to make overnight broth cultures in CTTYE. This allows us to control the cell density more carefully for phenotype assays.

1. Obtain a sterile flask with an overturned sterile beaker as a lid (cabinet under the incubators). It is crucial that you use a sterile flask for this. If one is not available, talk to Jess and we can figure out a solution.

2. Working by the station with the bunsen burner, sterilize the benchtop and p1000 pipette with 70% ethanol and wipe down until dry.

3. When ethanol is dry, light the bunsen burner. Use caution here as it makes a small flame that is hard to see/hear. Don't set any of your materials up so that you have to reach over the flame, and be sure to turn it off when you are done.

4. Using a 10mL serological pipet, transfer 10mL of CTTYE nutrient media to the flask and replace the beaker. (Use sterile technique here, which means to flame off the mouth of the bottle before you draw media from it, flame the mouth of the flask before you transfer media to it, and flame the mouth of the bottle and the inside of the cap BRIEFLY and QUICKLY before replacing the cap. Any tape on the bottle will catch if you hold it over the flame so please be careful of that).

5. Immediately replace the cover on the stock bottle of media so it is not at risk for contamination.

6. Using a p1000 pipette with a tip, gently scrape up some cells from the plate of M. xanthus, submerge the tip into the flask containing the media, and shake the pipette until the clump of cells transfers to the media. From here I like to pipette up and down gently to disrupt that large clump of cells into several smaller ones.

7. Place the beaker over the top of the flask, label the 'bottom' of the beaker with a sharpie to identify your culture, and wipe the bottom of the flask with a paper towel to ensure that it is completely dry.

8. With two small pieces of lab tape, tape the bottom edge of the beaker to the flask on each side. This will allow the beaker to stay in place while we shake the culture during incubation. Without the tape, the beaker will roll around and might fall off and shatter in the shaker.

9. Put the flask in the 32C tabletop shaking incubator that is technically in the Johnson lab (just on the other side of the balances, etc.--it is covered in foil). It can sit on the white sticky mat and will stay there as long as the entire flask is on the mat and it is completely dry.

10. Cultures need to shake overnight at about 160 rpm. After making a few liquid cultures, you will get a better feeling for how much inoculum you should add and how long it will take to be within the OD range that we want to use for phenotype assays.