FlytrapCare Carnivorous Plant Forums

Hi there

I'm planning on culturing some CPs for the first time and was hoping someone could help me with a few questions. My starting point is some Darlingtonia seeds. I have experience working with cell cultures, sterilizing equipment, making media and working in a laminar flow hood, but this would be my first time doing micropropagation.

My first question is what makes an ideal culture flask and lid? I was going to use small jam jars or baby food jars with their original lids and use tape to seal them up. If I do that, should I have an air vent stuffed with non-absorbent cotton wool to allow for gas exchange?

Also, once the darlingtonia seeds are in the media, how often should you transfer to fresh media and what source and intensity of light should I use? Is there an optimum temperature for Darlingtonia as well?

Excited about giving this a go and would be very grateful for any advice you can give.

Cheers,

Mark

mlong wrote:My first question is what makes an ideal culture flask and lid? I was going to use small jam jars or baby food jars with their original lids and use tape to seal them up.

That should work fine.

mlong wrote:If I do that, should I have an air vent stuffed with non-absorbent cotton wool to allow for gas exchange?

No, that's not necessary. Some air will move in and out of the vessel anyway.

mlong wrote:Also, once the darlingtonia seeds are in the media, how often should you transfer to fresh media

Every 6 to 8 weeks or so seems to be best for most species.

mlong wrote:what source and intensity of light should I use?

Low light levels are fine. I use T8 bulbs, two per shelf (20"x48"), which is a bit much actually.

mlong wrote:Is there an optimum temperature for Darlingtonia as well?

All cultures grow best between 72°F and 82°F.

Brilliant, cheers Matt.

I'm a TC noob but here's my input. I went with baby food jars. I got them on craigslist. Unfortunately the cheapest way I could get them was with food in them. I hated to waste the food but I didn't want to eat/use craigslist food. I choose baby food jars because they were the cheapest and there are polypropylene, snap on caps available that fit them and I wanted to be able to allow light in through the top. In hindsight, I don't think the clear tops were crucial as lots of light gets in through the sides. Lots of microprop is done in test tubes tilted at an angle to allow light in. I got a bag of about 100 large centrifuge tubes (also polypropylene) on amazon.com for really cheap that would probably work as culture vessels too (I'm just using them for sterilizing, and storing solutions). That would have been cheaper than the baby food jars, but the cultures would probably have to be transferred and divided more frequently. They would also need stands to hold them but baby food jars can just sit on their own and be stacked. I'm happy with the baby food jars.

I've been using parafilm to seal my vessels. It hasn't been great. It tends to tear or crack on the edges of the caps after a while. Plastic wrap or tape would probably work just as well, though tape might leave some sticky glue behind.

I think vented caps may allow you to let the culture grow longer before needing re-plated, but I don't see how a culture can be safely vented unless it is in sterile air. I would also be weary of it allowing evaporation of water from the media. I just do this at home as a hobby and left a few jars with seeds that didn't grow sitting out without the parafilm seal. After a while, not only did the media reduce down to a thin hard layer, a couple jars had ants manage to squeeze in past the phytocaps!

Ants!? Yikes, hadn't really thought of that happening!

I do actually have some sterile 50 ml centrifuge tubes so could give them a go as well. In the past I've cut holes into a block of polystyrene to stand the tubes upright. From what you and Matt have been saying I shouldn't worry about additional ventilation too much. I've worked with cell cultures in liquid media before where we've stoppered flasks with bungs made from cotton wool wrapped in bandage, which seems to keep microbes and spores out of the flasks. I'm guessing this ventilation may be more important in liquid cultures though, as we can see the biomass increase 30 x in 2 weeks in some cases.

Thanks for your help!