Making Dicty Spore Stocks

Procedure

1. Suspend confluent cells in own growth buffer and move to 50 ml Falcon tube.
2. Remove 15ul to count w/ hemocytometer.
3. Spin the cells in Steenšs clinical centrifuge ­ preferably at RT but 4šC is ok too. 1500 rpm for 4 min.
4. Aspirate off media. Resuspend in 25ml MES Stravation Buffer.
5. Spin Again.
6. Resuspend in ____ml Mes Buffer. (You want to have 2x10^8 cells in 4mls of this resuspension)
7. Coat MES plates w/ 4mls of suspension by dripping with a pipette and swirling to coat evenly.
8. Let sit for 30 min.
9. Aspirate off excess liquid. (Try not to remove attached cells...tip plate and suck off from edge and not over media)
10. Incubate for 1-3 days in Dicty 27deg incubator/fridge until Dicty have grown fruiting bodies.
11. Add 500ul MES buffer to one sterile epi-tube. Label four more with the strain and date.
12. Rub plate surface with strerile loop. Collect dicty on loop and move to the tube with MES Buffer
13. Mix tube and transfer 100ul of this mixture to each of the other 4 tubes.
14. Store in a Dicty Stocks box in -80C freezer.