TITLE: Autoclaving Erlenmeyer Flasks for Sterile Algal Cultures
AUTHORS: Jakub Nedbal

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes autoclaving the Erlenmeyer flasks for sterile microalgae culture. It describes the cleaning and preparation of flasks for autoclaving. In the second part, the protocol describes the method of autoclaving.</div></div>

[STEPS]
?. [Cleaning Culture Flasks]
Wash the dish washing area with 70 % denatured ethanol.
?. [Cleaning Culture Flasks]
Wash away all permanent marker labels and writing from the Erlenmeyer flasks using 70 % denatured ethanol.
?. [Cleaning Culture Flasks]
Wash used flasks with dish-washing liquid using vial cleaning brush. Scrub off any dried deposits of algae.
?. [Cleaning Culture Flasks]
Thoroughly rinse many times with copious amounts of hot and cold water to remove all detergent.
?. [Cleaning Culture Flasks]
Rinse off tap water from within the flask with a small amount of deionized water, swishing around the entire inside of the flasks. Repeat, if possible.This step might not be essential in areas with soft water supply. However, in hard water areas, using only tap water will lead to accumulation of limescale attracting cell debris that will get progressively harder to wash away without the use of acids.
?. [Cleaning Culture Flasks]
Let the flasks dry on a drying rack.
?. [Preparing Flasks for Autoclaving]
Once completely dry, seal each flask mouth with a cotton wool roll or ball.
?. [Preparing Flasks for Autoclaving]
Cut about 10 cm × 10 cm (4" × 4") piece of aluminium foil for each flask.If the aluminium foil is thinner than 24 µm, such as standard kitchen aluminium foil, make pieces larger and fold them over to create two or three ply thick pieces to seal the flasks with.
?. [Preparing Flasks for Autoclaving]
Seal the flask opening with the aluminium foil covering the cotton wool.
?. [Preparing Flasks for Autoclaving]
Cut a small piece of autoclave tape and stick it to the top of the aluminium foil. Do not tighten the aluminium foil with the autoclave tape, as it would make it hard to remove later.
?. [Preparing the Autoclave]
The procedure below is specific to the autoclave and the conditions in our lab. This protocol is not universally transferable and it must be redesigned for local equipment, conditions, and safety regulations.Use this protocol only as a guidance to develop your own protocol.Beware, this type of autoclave does not have a vacuum pump. It is therefore not suited for autoclaving bags of waste, sealed vessels, pipette tip boxes, and any containers into which the steam will have problem penetrating efficiently.
?. [Preparing the Autoclave]
Prepare all the autoclave equipment as listed in the Materials section above.
?. [Preparing the Autoclave]
Prepare a tidy space on solid desk.Place a strong lab jack or other heat-resistant support away from the edge of the desk.
?. [Preparing the Autoclave]
Pour hot tap water into the autoclave up to 1 cm (½") below the heating spiral seal, as in the image below.Ensure no water reaches the electrical connector on the outside of the autoclave while filling it up.You can also use cold tap water, but then the autoclaving will take longer to finish.
?. [Preparing the Autoclave]
Place the autoclave on the heat-resistant support.
?. [Preparing the Autoclave]
Insert the autoclaving container and load it with the prepared conical flasks. The autoclave will fit nine flasks comfortably, and will not fit more than ten.
?. [Sealing the Autoclave]
Place the lid over the autoclave, aligning the fixing loops with the screws on the autoclave body.
?. [Sealing the Autoclave]
Orient the autoclave so that the pressure release valves are pointing towards the wall, and definitely not into the room.The released steam could injure someone if the valve point in the direction where people may happen to be.
?. [Sealing the Autoclave]
Screw all five sealing wing nuts gently.
?. [Sealing the Autoclave]
Start tightening the wing nuts in small steps at a time. Create and observe a tightening step order, in which neighboring wing nuts are never tightened after one another. For clarity, wing nuts are labeled A−E, in the picture below. The tightening sequence could be, for instance,  A−C−E−B−D−A−C−E−B−D−…Keep tightening the wing nuts, repeating the same sequence, until all wing nuts are fastened hand tight. The autoclave comes with a tightening spanner, but this was never required.
?. [Operating the Autoclave]
Plug the power supply lead into the autoclave.Make sure the lead and the autoclave electrical connector are completely dry before this step.If the electrical connections are wet, let them dry out over a long period of time and/or consult H&S officer to agree the best ways to proceed.
?. [Operating the Autoclave]
Plug the power plug leading from the autoclave into the Countdown Timer.
?. [Operating the Autoclave]
Plug the Countdown Timer into a mains socket.
?. [Operating the Autoclave]
Turn on the mains socket using the switch provided.Pressing the delay button on the Countdown Timer twice, to set it to ½ hour.Two blue lights will light up, meaning the autoclave is on and the 30-minute countdown has started.
?. [Operating the Autoclave]
Keep attending to the autoclave while it is heating up and the pressure is building up.Observe the pressure gauge regularly.The autoclave takes about 15 minutes to heat up to the operating temperature, when starting with hot tap water. It will take longer with cold water.
?. [Operating the Autoclave]
Once the autoclave reaches a pressure of 0.18−0.19MPa, the safety valve will release the steam. This lowers the pressure (and temperature) inside the autoclave sufficiently for the safety valve to close again and allow the autoclave to start heating itself up.
?. [Operating the Autoclave]
The autoclave is designed to cycle between the process of heating up and releasing the pressure (and temperature) as long as the power remains on.This leads to cycling of temperature and pressure inside the autoclave, helping the penetration of steam into any closed vessels.The release of steam also warms up the room. The released steam may cause damage to skin or eyes if they get exposed. Large buildup of steam in the room may trigger the fire alarm. To minimize risk of the above events, the autoclave is manually turned on and off, as described below.
?. [Operating the Autoclave]
Mark the time, when the autoclave reaches pressure of 0.18 MPa or releases the steam for the first time.Keep autoclaving for 15−20 minutes after this pressure has been reached.
?. [Operating the Autoclave]
Observe the pressure gauge. When it shows pressure of 0.18 MPa, turn the power OFF at the mains socket.
?. [Operating the Autoclave]
Keep observing the pressure gauge until the pressure drops down to 0.15 MPa.
?. [Operating the Autoclave]
Switch off the autoclave at the power socket to prevent the safety valve from releasing the steam.
?. [Operating the Autoclave]
This will take about 2:10 minutes. At that point, turn the power ON at the mains socket.
?. [Operating the Autoclave]
Return to the previous step of observing the pressure gauge and waiting until the pressure raises back to 0.18 MPa. This takes about 45 seconds. Turn OFF the switch at the power socket again, once the pressure is reached.
?. [Operating the Autoclave]
Keep repeating the two steps above until 15−20 minutes pass since the pressure was first reached, or the Countdown Timer runs out and turns itself off. It takes five to seven ON-OFF cycles.
?. [Operating the Autoclave]
Switch off the power at the socket for the final time.Unplug the Countdown Timer from the socket.Do not temper with the autoclave until it cools down.
?. [Unloading and Preparing the Autoclave for Storage]
Keep the autoclave standing and wait for it to cool down and depressurize itself, ideally overnight.You could use the pressure release valve to lower down the temperature rapidly. However, this is potentially dangerous and is not advisable. It requires physically tempering with the hot autoclave full of hot water and will inevitably release large amounts of hot steam.
?. [Unloading and Preparing the Autoclave for Storage]
Once the autoclave has cooled down to the room temperature, loosen the nuts. Use supplied spanner if they are too tight to loosen them by hand.Sometimes the autoclave lid is sealed by the underpressure formed inside. Pry it open using the supplied spanner.
?. [Unloading and Preparing the Autoclave for Storage]
Remove the lid and the content.
?. [Unloading and Preparing the Autoclave for Storage]
Empty the autoclave and let it dry before storage.
?. [Unloading and Preparing the Autoclave for Storage]
?. [Unloading and Preparing the Autoclave for Storage]