Spore Assay
1. Harvest overnight M. xanthus culture during log growth phase, wash twice in TPM, and concentrate to %x10^9 cells/ml. You will likely need several mL of culture to plate out a spore assay. Each large TPM plate will need 4 groups of 5 20uL spots (400uL per plate, where one plate = 1 replicate). You may wish to use the 15mL conical tubes and the centrifuge in superlab to prepare cells.
2. While you are washing and preparing cells, leave TPM plates open to dry by flame or under the hood so that drying time is lessened. DO NOT leave flame unattended.
3. Divide your plate into quadrants and in each quadrant, spot 5 20uL spots of cells at the proper density, just far apart enough so that your cell spots do not run into each other. Leave plates open until completely dry.
4. Cover plates, invert them, and store in the incubator for 5 days at 32C.
Day 2 (Read ahead to make sure you have the time set aside)
1. Remove plates from the incubator and image if desired.
2. Turn heat block to 50C in preparation, and check that you have enough sterile water to complete the below protocol. For each plate you prepared, you will need 9mL of sterile MiliQ water in a 15mL falcon tube (pre-filling and pre-labeling these tubes is recommended).
3. Using a bent metal spatula (one should already be prepared for you in the lab, sterilize in between by dipping in 70% ethanol and running through the flame), gently scrape 3 of your 4 groups of cell spots from each plate together into one small area, careful so as not to disrupt the surface of the agar. Use the spatula to transfer the cells to the pre-filled 9mL of sterile water.
4. Repeat step 3 for each of your plates.
5. Important; WEAR EAR PROTECTION WHEN USING SONICATOR. Turn on the sonicator and sterilize the probe with isopropyl alcohol. Wipe excess with kimwipe and rinse in an empty tube of sterile water so there is no excess alcohol getting into your sample.
6. With the sonicator set to an intensity of 18.5, and using a 'blank' tube of 9mL of water without cells, determine where the probe should be within your sample so that your output is 20 Watts for 10 seconds. Then use that same probe placement for all samples. Tip: use the gradations on the falcon tube for this, and try to keep your hand placement on the tubes consistent as well.
7. Sonicate each sample at 20 Watts for 10 cycles each (1 cycle: 10 sec on, 30 sec off) for a total of 100 active seconds. This should deliver 2000J to your sample total. Sonicate just one sample at a time, sterilizing the probe with more isopropyl alcohol in between samples.
8. Incubate samples at 50C for 2 hrs.
9. While sonicated samples are incubating in the heat block, prepare your tubes of CTTSA. Each tube that you currently have will be split into three for technical replication. (Ex. if you have three tubes from the sonicator, you will dilute and plate those out on 9 total plates at the end.
10. Melt down the CTTSA in the microwave, make 4mL aliquots for each tube that you need, and keep warm in a water bath or heat block until you need them. You can also prep tubes for serial dilution (see below), and take out the CTTYE plates you will need so they can come to room temperature.
11. Once your samples are done heating, vortex to break up any clumps and you are ready to serial dilute.
12. From your well-mixed tube of collected spores, take 100uL of sample into 900uL of sterile water, a 1:10 dilution. Repeat for another 1:10 dilution, changing tips in between and ensuring that samples are well mixed. Then transfer 10uL into 4mL of CTTSA and pour over a room temp CTTYE plate (with Kan if cells are resistant). [Note: three is potential that if we get the spore assay working well, another 1:10 dilution may be necessary, but start here]
13. Allow CTTSA to solidify for 15min and put plates in incubator at 32C for 5 days. Count colonies and then back calculate from your serial dilution to determine the number of spores recovered.