1. Preparing Agar Plates As demonstrated by Andrew Corcoran

2. Use an appropriate flask  Use a flask that is twice the volume of the media you will make  Typically a 1 L flask to prepare 0.5 L batches

3. Measure out the water

4. Add a spin bar and spin

5. Weigh out the appropriate amount of media

6. Add the powder  Some powders are very fine

7. Make sure all the powder gets hydrated Wad of dry media

8. Heat until there is a hint of boiling  Avoid boiling over!  Agar will partially dissolve and get a little clearer  Wear a “Clavie”

9. Remove the spin bar with the wand Wand Spin Bar

10. Apply an aluminum foil lid

11. Use autoclave tape  Turns color after heating  Tear off tape and apply to counter (easier to write on it)  Include initials and media type

12. Apply to flask

13. Always place media into autoclave tray  Secondary containment  Spills in the autoclave clog vents

14. Operating autoclave  First rule – always make sure there is no pressure in the autoclave! Chamber pressure – Should read zero Jacket Pressure - There should always be pressure here

15. Make sure previous cycle is finished Chamber pressure – Should read zero Autoclave status

16. Open the door  Lefty loosey  Pins will retract  Crack 1 inch. Allow excess steam to vent  Open slowly  Remove contents carefully (contents may be superheated) Pins

17. Place tray in center of rack  Slide out metal shelf (avoid reaching into autoclave) Shelves

18. Close the door  Righty tighty  Make sure pins engage  Door closed light will be lit  Turn until hand tight

19. Press flask icon  Fast exhaust – Scissors  Fast exhaust with drying – Gown  Slow exhaust - Flask

20. The control panel Fast exhaust Fast exhaust/dry Slow exhaust Service – don’t touch Start button Cycles are pre- programmed. There is no need to change settings!

21. Press start

22. Steam pressure will rise

23. After autoclaving place flask in 55 o C waterbath Tape has darkened

24. Pouring the plates  Start by disinfecting the work area

25. Stack plates 4 high (right side up)

26. Label plates on the bottom  Media type  Date  Label the bag they will go in

27. Wipe away water from flask

28. Remove lid and begin pouring plates from the bottom of the stack

29. After the first plate is filled cover and begin filling the second (and so on)

30. Avoid moving or jostling plates Agar on the lid - faux pas! Condensation will form on lid of plate

31. When done, put hot water in flask

32. Pour into special trash can

33. Stack the plates  This prevents condensation on most plates  Also provides more work room

34. Any questions?