Flasking questions - Autoclave step

[wetfeet101b]

Hello Everyone,

I have a few questions regarding the autoclave (or pressure cooker) stage of preparing the plates for flasking.
I have never attempted to create any flasks before so please bear with me

1. I have heard that the microwave oven can now be used as a substitute to an autoclave or pressure cooker to sterilize the flasks. Is this true? And has anyone tried it with any success?

2. When placing the flasks in the autoclave, do you need to wait until the agar solution has hardened? or do you sterilize it while the agar is still in liquid form?

3. Also when in the autoclave, do you leave the jars open? or do you use the jar cover and close it tight before autoclaving? or do you just need to put a "soft" cover to prevent the water/steam from entering the jars?
I am concerned that if the jar covers are tightly closed, the pressure difference between the inside and outside of the jars could cause it to break - or is the pressure range not that great so this is not an issue?

Right now I do not have laboratory grade flasks rated for the autoclave, but I do have plenty of the Starbucks cold coffee bottles and some old canning jars from fruit jellies (the ones with the two-part covers).


Thanks, and Happy Growing.
John

[magooba]

I watched a BBC program once, some Plant Doctor; Steven (I think) who aided a young woman that had an enclosed system in a a large jar. Somehow it got infected and everything died... His Remedy (in short): Get new (suitable) plants, sterilise jar (using stisilant of choice), Micro-wave Media @ 7Min/Kg. But don't take my word for it ! lol

[cindiras]

Canning jars in a presure cooker should be o.k., I would think. You would want the lid on loosly so presure can excape from the jars, but tightened enough that when the lids cool, they seal to the glass jar.

[Becky15349]

Hi John,

I did a great deal of tissue culture in grad school, so I'm going to take a stab at answering some of your questions. I'm not an expert, and if you would like to direct you to an expert, I will more than willingly provide contact information for some individuals you could ask.

I've never heard of someone using a microwave instead of an autoclave, but I imagine you could, if the microwave is capable is reaching the same temperature as the autoclave, for the required sterilization period. I believe it was an hour to several hours in an autoclave, if I remember correctly...and the temperatures were exceedingly high. I'm just not sure a microwave is capable of achieving this...and if it fails, you will definitely get bacterial growth. Bacteria loves agar

In school, we actually sterilized test tubes, instruments, etc before we added agar. Once items were sterile, they were brought covered into the HEPA hood, along with vials of agar, and we poured agar into the test tubes in the sterile environment of the hood. We then sealed the test tubes and set them aside for future use. We always poured agar when it was in liquid form, because it really does become jello-like and cannot be "re-poured" once it has "set" and hardened.

In the autoclave, test tubes and the like were generally put onto a large metal rack upside down without the rubber test tube plugs. Since you are using jars and screw-on covers (I presume) I would sterilize them separately, simply because they were not designed to withstand a huge temperature increase and you might have a mess on your hands

I'm sure there are loads of other people out there doing non-laboratory type of orchid flasking, so I hope they offer their opinions. Like I said, if you need more advice from an expert, let me know

~Becky

[John D.]

I use a pressure cooker. The jars should be clean but don't have to be presterilized. Prepare the agar and pour into jars screw lids on or put stoppers in loosely. Put into pressure cooker hot. The agar does not need to set before sterilizing. If everything is hot and you bring the temp up at a reasonable rate the jars will be the same temp and pressure inside as out so they shouldn't break. Hold at 15# pressure for 15 minutes and lower temp slowly, don't open until back to 0#. You'll have more trouble with lids coming off if you vent the pressure early because the higher pressure inside the jar will raise the stopper or lid. At 0# and while still hot open pressure cooker and tighten lids if necessary. Be carefull of steam and hot water but you must not let them cool and suck in unsterile air as the jars cool. Or if you have a HEPA filter flasking hood put the pressure cooker in it to cool completely venting it at 0# tighten lids/stoppers in the sterile hood when cool.
Hope this helps.
John

[GrumpyBear]

i read somewhere about using some specific kind of cotton as a type of filter that would let air in but keep bacteria out (you had to have a certain thickness of the cotton for it to be effective, i'll look that all up again when my flasking time comes...) if anyone has heard of this my question is this: since this filter is by design so air can move through it can you then keep the lids tight when you pressure cook?

[John D.]

Yes the vented flasks will equalize, The flasking patches (PTFE filter disks) work well and help prevent moisture loss, using a small hole and three layers of 3M Micropore tape works also. I have not seen problems with contamination with either method. You can tighten unvented flask lids but have to be carefull to check them when you open the cooker, if you open early the higher pressure inside the flask will lift the lid or stopper.

[The Peloric Orchid]

I just got back from working in a lab this summer, and we actually did a LOT of tissue culture.

After you mix your agar solution, put it in the autoclave for 20-25 minutes (The doctor used 25, but the grad student only used 20. It takes 15 minutes for sterilization to occur, but the autoclave takes time in reaching the necessary temperature and pressure).

We used flasks which were sealed with aluminum. Take a sheet of aluminum, place it over the top, and pound it (gently enough to not break the flask, but firmly enough to create a tight seal. That way, when you take the flask out it will remain sterile and the seal will prevent it from spilling in the autoclave).

After the timer went off, we turned the autoclave off, waited for the pressure to drop, took the flask out, and poured the media into the dishes/jars. These were either put in sterile bags and then in the walk in fridge, or they just sat around long enough to firm up in the flow hood.

I'm willing to bet it will work with a tight seal, but test it first to see if it works.