Preparing Agar Preparing Agar PlatesPlatesAs demonstrated by Andrew As demonstrated by Andrew CorcoranCorcoran

Use an appropriate flaskUse an appropriate flask

Use a flask that is Use a flask that is twice the volume of twice the volume of the media you will the media you will makemake

Typically a 1 L flask Typically a 1 L flask to prepare 0.5 L to prepare 0.5 L batchesbatches

Measure out the water Measure out the water

Add a spin bar and spinAdd a spin bar and spin

Weigh out the Weigh out the appropriate amount of appropriate amount of mediamedia

Add the powderAdd the powder

Some powders are very fineSome powders are very fine

Make sure all the powder Make sure all the powder gets hydratedgets hydrated

Wad of dry media

Heat until there is a hint Heat until there is a hint of boilingof boiling

Avoid boiling over!Avoid boiling over! Agar will partially dissolve and get a little clearerAgar will partially dissolve and get a little clearer Wear a “Clavie”Wear a “Clavie”

Remove the spin bar with Remove the spin bar with the wandthe wand

Wand

Spin Bar

Apply an aluminum foil Apply an aluminum foil lidlid

Use autoclave tapeUse autoclave tape

Turns color after Turns color after heatingheating

Tear off tape and Tear off tape and apply to counter apply to counter (easier to write on it)(easier to write on it)

Include initials and Include initials and media typemedia type

Apply to flaskApply to flask

Always place media into Always place media into autoclave trayautoclave tray

Secondary Secondary containmentcontainment

Spills in the Spills in the autoclave clog ventsautoclave clog vents

Operating autoclaveOperating autoclave

First rule – always make sure there is no First rule – always make sure there is no pressure in the autoclave!pressure in the autoclave!

Chamber pressure –

Should read zero

Jacket Pressure -

There should always be pressure here

Make sure previous cycle Make sure previous cycle is finishedis finished

Chamber pressure –

Should read zero

Autoclave status

Open the doorOpen the door

Lefty looseyLefty loosey Pins will retractPins will retract Crack 1 inch. Allow Crack 1 inch. Allow

excess steam to ventexcess steam to vent Open slowlyOpen slowly Remove contents Remove contents

carefully (contents carefully (contents may be superheated)may be superheated)

Pins

Place tray in center of Place tray in center of rackrack

Slide out metal shelf (avoid reaching into autoclave)Slide out metal shelf (avoid reaching into autoclave)

Shelves

Close the doorClose the door

Righty tightyRighty tighty Make sure pins Make sure pins

engageengage Door closed light will Door closed light will

be litbe lit Turn until hand tightTurn until hand tight

Press flask icon Press flask icon

Fast exhaust – Fast exhaust – ScissorsScissors

Fast exhaust with Fast exhaust with drying – Gowndrying – Gown

Slow exhaust - FlaskSlow exhaust - Flask

The control panelThe control panelFast exhaust

Fast exhaust/drySlow exhaust

Service – don’t touch

Start button

Cycles are pre-programmed. There is no need to change settings!

Press startPress start

Steam pressure will riseSteam pressure will rise

After autoclaving place After autoclaving place flask in 55 flask in 55 ooC waterbathC waterbath

Tape has darkened

Pouring the platesPouring the plates

Start by disinfecting the work areaStart by disinfecting the work area

Stack plates 4 high (right Stack plates 4 high (right side up)side up)

Label plates on the Label plates on the bottombottom

Media typeMedia type DateDate Label the bag Label the bag

they will go inthey will go in

Wipe away water from Wipe away water from flaskflask

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

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

Avoid moving or jostling Avoid moving or jostling platesplates

Agar on the lid - faux pas!

Condensation will form on lid of plate

When done, put hot When done, put hot water in flask water in flask

Pour into special trash Pour into special trash cancan

Stack the platesStack the plates

This prevents condensation on most platesThis prevents condensation on most plates Also provides more work roomAlso provides more work room

Any questions?Any questions?