Preparation of agar medium for flasking

[Afid]

Happy to help! They have some other orchid related stuff too (mostly agar and micronutrients and stuff). I also got some terrestrial flasking mix from them as well, I am excited to try it out at some point (probably next fall as soon as there are seeds to be found in my yard).

[cricketerry]

Sorry this is a but late for your first attempt. When sterilising the jars and media in a pressure cooker put the lids on the jars but do not screw them down tight. You want air to escape from the jar as it heats up. Also you do need something in the pressure cooker to lift the jars out of the water.The sterilisation takes place in the steam above the water not in the water itself.

[Afid]

No worries, I appreciate the advice and still have enough left over to try again over the weekend!

[camille1585]

I guess you have answers to your questions, but having prepared and autoclaved lots of media in the past for my work, I thought I'd chip in. Yes, you can mix agar cold. It's important to swirl it around well until completely dissolved. Lid should be screwed on loosely, just enough to keep it from coming off. If it's not a screw on type you can leave it loosely open and put foil over it. There should be a stand or something to keep the jars off the bottom and not in contact with the water. You can also chuck in any (metal) tools you plan on using for flasking in order to sterlize them as well, just wrap them in aluminium first.

if you have extra media, usually the best way to store it is already autoclaved and gelled. Why? Because once you have your nutrients in solution, it is a paradise for microorganisms. Sterlized, solidified agar medium stores rather well in a cool dry place.

As to your medium not gelling, it's likely that water splashed into the jars. Gelling is also pH dependent, but even if your pH really high it would still gel, just more slowly than at a lower pH. And silly question, are you starting your timer when the pressure cooker is steaming, or before? Ideally you want a full 20min at 121°C, so starting when the pressure cooker is 'whistling' at full strength/power (if that's how it's said in English!).

As to your plastic flasks, if they are proper, microbiology/tissue culture grade flasks, most can be autoclaved but it's best to check the product description first.

[Afid]

Originally Posted by camille1585

I guess you have answers to your questions, but having prepared and autoclaved lots of media in the past for my work, I thought I'd chip in. Yes, you can mix agar cold. It's important to swirl it around well until completely dissolved. Lid should be screwed on loosely, just enough to keep it from coming off. If it's not a screw on type you can leave it loosely open and put foil over it. There should be a stand or something to keep the jars off the bottom and not in contact with the water. You can also chuck in any (metal) tools you plan on using for flasking in order to sterlize them as well, just wrap them in aluminium first.

if you have extra media, usually the best way to store it is already autoclaved and gelled. Why? Because once you have your nutrients in solution, it is a paradise for microorganisms. Sterlized, solidified agar medium stores rather well in a cool dry place.

As to your medium not gelling, it's likely that water splashed into the jars. Gelling is also pH dependent, but even if your pH really high it would still gel, just more slowly than at a lower pH. And silly question, are you starting your timer when the pressure cooker is steaming, or before? Ideally you want a full 20min at 121°C, so starting when the pressure cooker is 'whistling' at full strength/power (if that's how it's said in English!).

As to your plastic flasks, if they are proper, microbiology/tissue culture grade flasks, most can be autoclaved but it's best to check the product description first.

Thanks for all the help! I will try to flask them when I have free time tomorrow and I think it will go perfectly this time around! I checked the PH of my media and it seemed to be at the perfect level without any adjustment. As far as time, I have been doing 25 minutes starting once it has reached full pressure, is this too long? Thanks again everyone for all the help!

[Afid]

I just checked on the newest batch, they have gelled perfectly!! Thanks for all the help everyone!!

[SunnySmiles]

I realize I'm a bit late and you didn't actually have troubles with the pH in the end - but maybe useful for future reference?
I've never done this for orchids, but when we do micropropagation at uni, pH adjustments are made with either hydrochloric acid if you want a lower pH or potassium hydroxide if you need to raise pH. And you really just want a tiny amount as they have quite a strong effect. Maybe someone more experienced can confirm if they are used in orchid propagation as well

[hobibotanik]

Originally Posted by neophyte

ps: assuming you are using a medium that contains plant supplements, how would you know that these additives are not destroyed by the pressure cooker? just wondering.

let me make a contribution. Most of Plant Growth Regulators and medium supplements can stay effective after sterilization except Vitamins and antibiotics.

Of course, the ideal addition time is after sterilization but it has contamination risk.

you can find very useful information about them storage and sterilization conditions at here.