FlytrapCare Carnivorous Plant Forums

Hi everyone,

I normally disinfest my explants and seed with 5,000 ppm NaDCC, 5-10m for seed, or under 2m for leaf and stalk tissue.
A while ago I was enquiring about using GA3 for recalcitrant seeds. I normally use a 500ppm GA3 solution for 24-48h, followed by the disinfestation protocol above. Someone suggested using a combined solution of 500ppm GA3 AND 500ppm NaDCC for 24h to disinfest my seed.
I know 24h soaks with low concentration disinfestant are used with tissue, but how well do they work on seed? Is it possible to combine GA3 treatment and disinfestation? Will the seeds not imbibe too much NaDCC?
This brings me to the question of, if the imbibed NaDCC is not harmful, why rinse it at all? I have seen it listed as an ingredient in medium used to inhibit microbial growth...

Is anyone doing this regularly? Is seed germination affected by a long soak in disinfestant (presumably they imbibe a negligible amount in a 5m soak)?

Thanks!

Hi,
do you have good experiences with NaDCC ? I have tried it many times in high concentrations 10g/L for a few minutes and also in low concentrations 300mg/L for a day or so. I have found that short sterilization in NaDCC is not effective at all for me. I was not able to disinfect the seeds or explants with stronger concentration even after 20-30 minutes.I had contaminations.
I had better success with lower concentrations 300-600ppm for 12 - 48hours.
I dont rinse the NaDCC.

Well, I have good success with my standard protocol of 5 ppt NaDCC (5g/L), for 5-10 minutes. I've had 3 instances of contamination in about 80 flasks the last 6 months. However I do use PPM in the medium at 1/2the recommended dose.

PPM can certainly completely hide bacterial contamination for many, many months.

Matt wrote:PPM can certainly completely hide bacterial contamination for many, many months.

You got that right!

goldslinger wrote:

Matt wrote:PPM can certainly completely hide bacterial contamination for many, many months.

You got that right!

Hah, yep! The good news is that the tissue still grows fairly well and you can still get plants out of it even if the cultures are contaminated.

Matt wrote:Hah, yep! The good news is that the tissue still grows fairly well and you can still get plants out of it even if the cultures are contaminated.

Yea, I've deflasked about 75 infected B-52'S that are 'preemies,' and getting tired of doing that. Gave a few away to some of Our Younger members, still have or traded some others.

I have been put a red piece of tape on them so I don't accidentally multiply them and the problem since I have multiple plants of origin of the same Cultivar. This way I hope to weed them out eventually out of the stock. I keep them on another shelf, but I probably should bag them, too.

I would like to at least try to get them as big as possible.

Yeah, I have a lot of tissue that's mildly contaminated as well. Those cultures grow much more slowly and usually stay quite small. They're a pain. I often end up running them down the garbage disposal if they're too small. I too am clearing out all contaminated tissue and moving away from using PPM when initializing so that I know for a fact I have a clean start.

Matt wrote:I too am clearing out all contaminated tissue and moving away from using PPM when initializing so that I know for a fact I have a clean start.

I think that is a sound plan. There for awhile I was throwing out about 25% of My multiplication explants, almost exclusively B-52'S, and that's how I know it was indigenous, because same clean hood procedures for all, so why just the B-52'S? Had to be internal. Since I had no Mother plant of origin label for those cultures, some latent still got replated after throwing out those, but it's getting better now.

Now, I won't replate any cultures that don't look right, not just the Ones that have the obvious spots of bacteria. If the leaves are curly, yellowing, not growing good, etc. I will just grow it out.

I am now down to about 3% contamination rate and I just replated about 40 jars in the same Day; will know in a couple of weeks how that did. I washed My hands and forearms (no scrubbing), wore a hair net and lab coat, alcohol'd My hands and forearms, put rubber gloves on; sprayed alcohol on everything going in, etc. I reaked of alcohol when finished. My wife said She could smell it on my breath.

My laminar flowhood is still in the garage. I'm gonna have to change some things before I can hire 4 Men to carry it to My room and build a cart.

Gary

goldslinger wrote:Now, I won't replate any cultures that don't look right, not just the Ones that have the obvious spots of bacteria. If the leaves are curly, yellowing, not growing good, etc. I will just grow it out.

That sounds like a good plan Gary.

goldslinger wrote:I am now down to about 3% contamination rate and I just replated about 40 jars in the same Day; will know in a couple of weeks how that did. I washed My hands and forearms (no scrubbing), wore a hair net and lab coat, alcohol'd My hands and forearms, put rubber gloves on; sprayed alcohol on everything going in, etc. I reaked of alcohol when finished. My wife said She could smell it on my breath.

3% contamination is a very respectable number Gary for not using a LFH! Your routine is very similar to my routine minus the lab coat and hair net. Last month I think I did somewhere around 500 cultures without any PPM. I got contamination in one of them. I'm really proud of that. Unfortunately it was in a larger culture of Cephalotus tissue. I planted some of it out (I'm a bit anxious to see how it does) and the rest that wasn't touched by the contamination I replated. Hopefully some of them stay clean, but I'm expecting to see more contamination in those cultures.

I'm not sure what the deal is here in Ashland, but I think the air is relatively clean compared to Boulder. I've initialized hundreds of pieces of VFT leaves and flower stalks. I've had very little contamination (I'd guess 5%) and that's without using PPM, which is an incredibly low contamination percentage for initialization (not replating). In Boulder, I could never have gotten that and that was growing my plants indoors. Here in Ashland, I keep them in my greenhouse. It's to the point now where I'm very confident that I can initialize a VFT from a leaf, which used to be a daunting task for me (flower stalks were always fairly easy). I'm sure some of it has to do with my improved skill level, but it also has to do with the clean tissue. I got some flower stalks from Steve this spring and he packaged them up well and kept them clean when he harvested them and I still got 100% contamination.

goldslinger wrote:My laminar flowhood is still in the garage. I'm gonna have to change some things before I can hire 4 Men to carry it to My room and build a cart.

Hah, wow! I know those things are huge. I still would like to get one someday, but I feel like I'm doing quite well working in my 30 gallon aquarium. It's cramped and I despise the smell of alcohol, but things are working out OK for now.

Matt wrote:Hah, wow! I know those things are huge. I still would like to get one someday, but I feel like I'm doing quite well working in my 30 gallon aquarium. It's cramped and I despise the smell of alcohol, but things are working out OK for now.

I think You have it figured out.
My aquarium is very small; only a 20 Gallon; and am always accidentally touching the sides with the hands, tweezers, etc. so have to respray or soak. With the contamination issues experienced, I always feel a sense of urgency when dividing a clump that sometimes don't inspect it good enough (doesn't help with the condensation on the glass) and I kinda of hack it to pieces, leaves falling off everywhere.

I was gonna make a glove box with a 35 or 50 gallon aquarium when that e-bay auction came up.

This conversation got Me curious. I took 6 used cultures that I had in a tub (used up the new ones) that the transfers were done in the hood, so should be good. I wiped down the LFH unit still on the floor out in the garage with 10% bleach solution (spraying on the cloth to wipe the hepa grate). I labeled 3 cultures with red tape for outside control, and 3 with green tape for inside.
I then turned the blower and U.V. light on and let it run for 30 minutes, then turned off the U.V. light and switched on the regular light. I put the 3 red labeled on top of the clear hood, and the 3 green ones in the mid section on the inside. uncapped them all and waited 20 minutes, then capped them all back up. We will see in a week or two. If it works alright, I need to install snaps and get a cover for the front.

Yea, just the HEPA is 30 x 40 x 6. It will make a good air scrubber just running in My small room, lol!

I'm anxious to get it in the room, but I'm still working on it off and on. I'm embarrassed to tell You what I've spent on this hobby; pretty much have all of the toys. I've curtailed My flying to compensate some.

I'm just tired of breathing alcohol. I tried 10% bleach, but the smell is even more intolerable, and there is a slimy residue that eventually turns into a chalky mess on everything. It also is more corrosive on My tools, it seems, and I feel like I have a head cold for two days afterwards. Maybe I'm overkilling it, but I have alot of work at stake, and still don't have ample jars of some cultures to fall back on if contaminated.

Gary

goldslinger wrote:With the contamination issues experienced, I always feel a sense of urgency when dividing a clump that sometimes don't inspect it good enough (doesn't help with the condensation on the glass) and I kinda of hack it to pieces, leaves falling off everywhere.

Yes, I have that sense of urgency too. That's why my Cephalotus culture got contaminated; it was exposed to the air for too long while I was dividing. But in most cases, I use fairly small jars and I have a lot of tissue to work with (I don't usually divide until I can make at least a dozen subcultures) so if a few of them get contaminated, that's fine. But I've been lucky here in Ashland with almost no contamination so far. The same was not true in Boulder.

I'm interested in hearing the results of your HEPA filter test on your LFH. That sounds like a good way to see if the HEPA is still good.

goldslinger wrote:I'm embarrassed to tell You what I've spent on this hobby; pretty much have all of the toys. I've curtailed My flying to compensate some.

I've actually spent very little money on my TC stuff so far. The things that I have spent the most money on are the baby food jars and phytocaps...hah. Some of the hormones I bought were also expensive, but they last a very long time and I've not had to purchase any for a while now. However, I've spent a lot of money on greenhouses this year and trays for plants. I just got myself another greenhouse today, a 10'X12' from Harbor Freight. I got a great price on it though, so I'm really happy about that. I can't wait to get it set up, but I'll likely have to wait until my wife comes back home to help me.

goldslinger wrote:I'm just tired of breathing alcohol.

I hear that. That's my number one reason for wanting a LFH. I can only do tissue culture for 4 or 5 hours at the most before I start feeling ill. I've cut down a lot on how much and how often I spray it, but it's still horrible.

Matt wrote:I've actually spent very little money on my TC stuff so far.

Yea, most of what I bought isn't needed, just wanted.

Matt wrote:I just got myself another greenhouse today, a 10'X12' from Harbor Freight.

Is this it?
http://www.harborfreight.com/10-ft-x-12 ... 93358.html

Are the panels ridgid or flexible? It says polycarbonate; that's tough stuff.

I would like a Greenhouse; if it can tolerate 60mph winds, hail, and such. I feel it might have to be the army barracks style, You know, rounded for wind to go around, but I would love to have that Victorian style. I like all plants; My latest kick is orchids. Also, isn't there the expense of fans, heaters, benches, etc? I would have to bury a power cable or go expensive solar or something. It gets over 100 degrees here in Summer, so a cooler would be in order. I can put it near a tree for part shade for part of the Day.

About the contamination levels, maybe humidity, rainfall levels, etc. can play a role in contamination levels, if that's a clue.

Gary

goldslinger wrote:Yea, most of what I bought isn't needed, just wanted.

I totally get that! Unfortunately my income has dwindled steadily over the last few years and my wife isn't working, so we have to be pretty careful with how much we spend right now. In the past, I'd have bought all the "toys" as well.

goldslinger wrote:Is this it?
http://www.harborfreight.com/10-ft-x-12 ... 93358.html

Yep, that's the one! On sale for $699.99 and I used a 20% coupon, so I got it home for $559.99. I'm pretty excited about that!

goldslinger wrote:Are the panels ridgid or flexible? It says polycarbonate; that's tough stuff.

I would like a Greenhouse; if it can tolerate 60mph winds, hail, and such.

The panels are polycarbonate and very tough. The frame is very weak aluminum. There are several places on the web that discuss strengthening and reinforcing it and I plan to do that. I actually have the 8'x10' already and it's pretty tough. The 10x12 isn't as tough, so it takes a bit of extra work to make it "weather resistant" including reinforcing it with steel pipe and screwing the panels in. But our weather here in Ashland isn't anything like you'd experience in Kansas. I think that you're right about needing a rounded, army barracks style one there. This greenhouse would almost certainly be destroyed by the wind in less than a year there in Kansas.

goldslinger wrote:Also, isn't there the expense of fans, heaters, benches, etc?

Yeah, I've gone the cheap route on that as well. I build my benches using this technique:
http://www.hammerzone.com/archives/work ... w20xl.html
So they cost around $20 a piece. It doesn't get terribly hot here normally, so a single 20" high velocity fan ($30 at Wal-Mart) works well for my 6x8 and 8x10 greenhouses, though I might need two for the 10x12. When it gets over 90F, I use a shade cloth to help with controlling the temperature.

As for heating, I'm only heating one greenhouse right now, but I'll probably heat the 10x12 as well. It doesn't get terribly cold here in the winter either, so I just used a small milkhouse style portable heater and I was able to keep my small greenhouse around 40F this winter. The 10x12 might require something a bit larger, but I only plan on growing temperate CPs in it, so if it gets down into the low 20s on occasion (which is rare here in Ashland), that's fine.

goldslinger wrote:About the contamination levels, maybe humidity, rainfall levels, etc. can play a role in contamination levels, if that's a clue.

Yeah, I've thought about that some. The wind in Portales is ridiculous. I'm thinking that, coupled with the fact that Steve regularly takes his plants out of the greenhouse, means that they're exposed to more dust and dirt in the air than mine are here in Ashland. Also, perhaps there are just places in the country that have more mold in the air. I don't know.

Matt,
Your greenhouse looks real good. I hope I have a big yard to buy one.

Goldslinger,
If you hate smell of alcohol you can buy a 3M 8577 mask for about $6 at Walmart or around $4 at the following link. This mask against organic solvents include alcohol. I use glove box too. After I move everything into glove box I seal it with tape and spray inside with alcohol. I use a "sealed" glove box with long gloves so I don't smell anything. When open box I wear mask. All I smell is something like beer.

link:
http://www.boss-safety.com/shop/particu ... 0_563.html
http://cgi.ebay.com/3M-8577-P95-Respira ... 27aedbeb99

Kinh