We are preparing for our upcoming experiments testing the growth rate of e.coli in different water types. When working with e.coli all the equipment must be sterile. Here at KochLab we use a Tuttnauer 3545M autoclave to sterilize our equipment and mediums.

Autoclaves use high pressure steam to sterilize equipment, read more at Wikipedia. Here is our process for solid items (liquid items follow the same procedure with some setting changes as noted in parentheses):

  1. Add water to the autoclave reservoir through the opening on the top of the machine.
  2. Take out the tray and put all the items to sterilize on the tray. Place a foil seal over the top of any open container. Place a piece of autoclave tape on each item. When the white lines on the tape turn black, the item is autoclaved properly.
  3. Turn on the autoclave and turn the bottom dial to Fill Water. When the water has filled to the front lip, turn the Fill Water dial off.
  4. Place the tray of items in the autoclave, close and lock the door. Tighten the lid… REALLY TIGHT! The autoclave is cooking things under heat and pressure.
  5. Set the temperature to 273° F (250°F) and the timer to 45 min (60 min). The orange Heat light should be on.
  6. After the timer sounds, wait about half an hour. Check the autoclave and make sure the white pressure arrow is at zero.
  7. If the white pressure arrow is at zero, set the dial to 0 (zero, turn counter-clockwise) and loosen the door so that it is open about 1 inch.
  8. Set the dial to Dry and the timer to 30 min. The orange Dry light should be on.
  9. After the timer sounds, turn the knob to 0, open the door, and, using heat resistent mits, remove the sterilized items.
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