- Media: Rich or Synthetic Complete
- Yeast cells on a plate (Yeast plates can be found in the 4 degree Celsius fridge. They should not be used if more than one month old)
- culture tubes
- test tube rack
- Inoculating loops (open and leave in sterile hood)
- agitating incubator at 30 degrees Celsius
- sterile 25 ml pipets
- Rainin GlassMaster
- Diluted Ethanol in a spray bottle.
1. Sterilize containers of materials with ethanol and place in the clean room hood (turn off UV light in the hood). Work in the Hood (make sure to sterilize your gloves).
2. Using the 25ml pipet and the Rainin GlassMaster remove the desire amount of media and place in culture tubes.
3. Using the inoculating loops add 1-3 colonies to each culture tube (number of colonies determines pace of growth). Spinning inoculating loops in media disperses cells well.
4. Cap the tubes, place lid on cell plate, remove everything from the hood.
5. Sterilize hood with ethanol, wipe the inside of the hood down to ensure sterilization. Turn on UV light.
6. Vortex the culture tubes.
7. Culture tubes go in the 30 degree Celsius agitating incubator on the second floor.
8. Incubation time will depend on amount of media, type of media, and number of colonies added.
9. Grow cells to log growth. Cell growth can be measured using UV/vis spectrophotometry.
- Take a 1ml sample from the culture tube for UV/vis.
- UV/Vis should be set to 600 nm.
- Use the media you are growing in as a blank.
- At .5 absorbance or greater cells are in log growth and ready to be harvested (Cells will die above an absorbance of 1).
10. Centrifuge culture tubes at 1000 g for 10 minutes at 4 degrees Celsius.
11. Remove supernatant leaving yeast cell pellet. Place on ice, or freeze in -80 degrees Celsius immediately.