Plate Colonies Using CTTSA

1. Warm up plates you’re using to 32°C. 2. Warm up a heat block to 55°C and place sterile test tubes into it. Have an equal number of test tubes to plates you’re pouring. 3. Determine how many cells you want to plate to determine the volume of cells you add to the CTTSA later. If you want to be able to count colonies, usually try to have ~200 - ~500 cells be added to the CTTSA. 4. Melt CTTSA in the microwave. This usually takes 3-4 minutes, depending on the amount of soft agar in the bottle. Once it begins to boil, take it out using the heat glove. 5. Using sterile technique, insert a 25 mL serological pipette into the CTTSA and take out the number of plates * 4 mL (for example, with 2 plates take up 8 mL). If you have more than 7 plates, take 28 mL of CTTSA and repeat this step. 6. Distribute 3-4 mL of CTTSA into each test tube, putting the caps back on each test tube. 7. When you are ready to plate cells, take the test tubes out of the heat block and let them sit for ~3 minutes. The outside should be warm but not hot. 8. Gently vortex the cells you’re plating, then add them to the soft agar. 9. Gently vortex the soft agar, then carefully pour them onto the plate. Slowly swirl the plate to make sure the soft agar equally covers the surface. Let it sit until the soft agar doesn’t move around too much. To be safe, you can cool the plates for 15 minutes. 10. Incubate them for 2-5 days at 32°C, checking to see when the colonies grow.