Preparing Agar Plates As demonstrated by Andrew Corcoran

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

Measure out the water

Add a spin bar and spin

Weigh out the appropriate amount of media

Add the powder  Some powders are very fine

Make sure all the powder gets hydrated Wad of dry media

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

Remove the spin bar with the wand Wand Spin Bar

Apply an aluminum foil lid

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

Apply to flask

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

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

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

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

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

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

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

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!

Press start

Steam pressure will rise

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

Pouring the plates  Start by disinfecting the work area

Stack plates 4 high (right side up)

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

Wipe away water from flask

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

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

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

When done, put hot water in flask

Pour into special trash can

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

Any questions?