Redneck Orchid Flasking

[John D.]

I am also using the 3M Micropore, 3 pieces, 2 outside one inside. No problem so far with shrinkage of the tape in the pressure cooker or contamination as long as the lids are tight. I also use the PTFE filter disks with equal success.

[vmax3000]

Gin,
I used this product called Sun Cap Closures (18mm) that I picked up from Sigma Aldrich. They are autoclavable, Royal, and I drilled a hole through the lid of (of all things) a pace picante jar. Those things actually work well! Since I now no longer have access to a laminar flow hood and an autoclave, I would love to learn how to do this with my pressure cooker. Where did you learn how? I have a seed pod coming due on my B. nodosa. Thanks for any feed back, guys! Love OB!

[Royal]

Pressure cookers work very well for sterilization. I use my mother-in-law's p-cooker from the 70's. Works like a charm. I work in a glove box that I made - it's really rough and way too small. Kind of a pain, but it gets the job done. Oh, what I would do for a LF hood!

There is lots of info on the web on this topic. I just did a search for orchid seed germination -- that's how I found the OB! Check out the Orchid Seedbank Project and Lotte and Thomas. They both have some good info to get you started. Good luck!

[Gin]

I have the same problem the box is to small it is a 10 gal . aquarium maybe a 20 would be better, yard sale
I read and read a lot of different sites , tried to sort out what I might be able to do
vmax ...I use the pressure cooker with 2 or 3 inches of water in the bottom 15 lbs. pressure for 15 minutes .
Thanks for the info. on the tape could be I used the wrong type or not enough of it . Gin

[OzPhal]

G'day Gin,

I'm a microbiologist by trade - and also have a love of Orchid Culture

I looked at your sterilisation cycle with interest... the standards from a microbiological perspective are:

121oC for a hold time of 30mins. What that means is that any autoclave will take time to get up to operating temperature - when it does reach operating temperature then it needs to maintain that temperature for 30mins minimum (unless it's a dry heat autoclave which isnt the case with a pressure cooker). You could do one of two things:

1) modify your pressure cooker to incorporate a temperature gauge and, once it reaches 121oC, start your 30min timing
2) Purchase some autoclave tape which changes colour when the cycle has met the required temperature/sterilisation times - i would definitely extend your sterilisation time though

15mins will kill of most vegetative cells but you'll find that the media wont get to sufficiently high temperatures to potentially kill some of your mold or bacillus spores. I would suggest erring on the side of caution - 15mins for the most part may work but you may also partially sterilise some of the spores and these may germinate a few weeks down the track once they have recovered.

Also, once you have completed your cycle let the pressure cooker cool down to room temperature without taking the lid off. This will take sometime but it will ensure that only sterile air is sucked in to the jars during the cool down period. It will also prevent the media from boiling. If you find that it is boiling and you take the lid off and there's media halfway up the side of your jars then it means that the pressure cooker hasnt sealed properly. Always leave your caps loose (slightly) though as there is nothing worse than exploded glass jars in your autoclave/pressure cooker.

Hope i'm not coming across as a know all coz i'm certainly far from that - i hope i've helped...

Originally Posted by Gin

I have the same problem the box is to small it is a 10 gal . aquarium maybe a 20 would be better, yard sale
I read and read a lot of different sites , tried to sort out what I might be able to do
vmax ...I use the pressure cooker with 2 or 3 inches of water in the bottom 15 lbs. pressure for 15 minutes .
Thanks for the info. on the tape could be I used the wrong type or not enough of it . Gin

[vmax3000]

Hi All,
Just plated my B. nodosa pod, yesterday. We'll have to wait and see if my aseptic technique even exists. There was an extra laminar flow hood in the Molecular Bio lab that no one's been using, so the director turned it on, exposed a plate and cultivated it....she got nothing. So, if I get anything besides plants, we'll know who the culprit is !
Regardless, it was a nice break from writing!

[quiltingwacko]

Gin you are the Julia Child of flasking

[John]

Hi Gin, For what it's worth, I have been using the Nexcare Spot bandaids on canning jar lids for some time now without any contamination problems. I used to put one on the inside and one on the outside but found that the inside one would sometimes fall off and into the media. It doesn't seen to hurt anything but looks bad. I just punch holes in the lids with a nail. In your picture, it looks like you have vented lids which means that the lids will not make a seal because they need a vacuum. I put the foil on the outside of the jars before they go into the pressure cooker to help keep the lid area sterile. After the jars cool, I write on the foil the media type, date of prep and later with the seed or plant name.
John

[Gin]

Thanks John , next time I will put the foil on the outside , the mother flasks were not vented and did not go bad . So far the seedlings are doing fine I took them out of the mother flask into moss a couple of weeks ago . Vmax dont forget to tell us how things went with the nodosa seeds . Gin

[vmax3000]

I am sad. This morning, I looked at the three flasks I had plated on Wednesday and two of them have a white fluff growing in them.... Those of you who do this more often, remind me. What are the various methods of contamination, again?? I am holding out hope for the third flask, but not so much, if I didn't sterilize the pod enough. I soaked it in hydrogen peroxide...it was dry. I have never used a dry pod. When I used a green pod, I had great success for the first and second flasks, but my culture molded when I placed them in the third flask.... still haven't confirmed my aseptic technique, apparently. Any ideas from the more experienced crowd? Thanks for the thread!