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JDW

Joined
Jul 12, 2012
Messages
80
Location
Mishicot, WI
Sigh, here I go on another expensive crazy project. I'm starting a small tissue culture lab. The goal of this project is to breed orchids and maybe some other plants, experiment with different procedures, and produce plants to trade/sell. Eventually if things go well I will turn it into a on-the-side hobby business. I bought all of the equipment and supplies I'll need last month, and hope to start planting this weekend!

Early September I bought wood, a HEPA filter, a fan, and everything else to build a laminar flow hood from scratch. This is the filter: 0-007-4-19-06-su-52-00-e0988 flanders hepa air filter
IMG_0261.jpg

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It cost $166 including shipping. To push air through the filter I got a furnace blower fan from a junkyard for $20.
LamFan.jpg

Which is something I'm stuck on. I cannot find out how to wire it! There isn't a wiring diagram on the motor, and I've searched for hours on the internet trying to find the answer. The model number shows zero results and searching for the wire colors hasn't turned anything up either. Anyone know how to do this? I tried a few combinations but can't get the thing to power up.

Anyways, here are three boxes of wood.
Work area
LamWorkBox.jpg

Top is 1/4" plexiglass and bottom and sides are coated in 2-part epoxy resin.
Filter housing
LamFilterBox.jpg

The opening is the same dimensions as the filter area. To install the filter I will run a thiiiick bead of silicone sealant around the inside of the box opening, and set the filter on so it lines up. I may attach it with a few bolts, dunno yet.
Fan housing
LamFanBox.jpg

The two openings are for 20"x20" furnace filters. The fan box will sit on top of the filter box, and the work box will hang off the front of the filter box. All I have to do yet is get the fan working, cut a hole in the top of the filter box and mount the fan, attach the three boxes together, and make a stand.

Once I finish the laminar hood I can get to work planting a few hundred nepenthes seed from ebay. If I can't finish my hood this weekend I'll have to break down and use this one I bought.
KipsHood.jpg

I was at an orchid show and bumped into a woman from the orchid society near me. We got talking and she told me a member of the club is getting out of TCing and selling all his equipment. Wish I found this out BEFORE I bought almost everything I needed :censor:. But he had a lot of containers and I didn't have any yet, so I went and picked up all his stuff. 300 quart-size milk bottles, thirty 500mL erlenmeyer flasks, 12 Zuma flasks, all the small equipment/tools, a flow hood, tons of dry media, and thousands of unused orchid seed. All for $500.

After buying and sorting through all that junk, I made up some flasks. I have 20 milk bottles of 25% MS+vit and a few to try out the orchid seed. How I prepared my media is first measure out ingredients for one liter of media. The MS salts came in 1L size packets. I dissolved this in 100mL of RO/DI water to make a 10x concentrate. To make a liter of 25% strength MS to go into flasks I first measured out one liter of water and brought to a boil on the stove. To the boiling water I added 25mL of my MS stock solution, 26g of table sugar, and 6g of agar. I stirred this for 10 seconds, turned of the heat and continued stirring until everything was fully dissolved. I measured the pH, though I hadn't calibrated my meter, and it showed 5.2 so I didn't bother adjusting it. After leaving the media to cool for five minutes I put 100mL of media into 10 milk bottles. The bottles are set on their side which gives a large surface of media about 1/2" thick. To sterilize the flasks I used a pressure cooker. I stacked 5 milk bottles on their sides inside the pressure cooker, closed it and turn the burner on high. It took about ten minutes to get up to 15psi. Once it reached 15psi I set a timer for 15 minutes. When the timer went off I came back and turned off the burner, and left it to cool. After a week none of my flasks are contaminated. Oh, the jars are sealed with rubber stoppers that have a hole in the middle stuff with cotton.
TcBottles.jpg


My first project is going to be some nepenthes seed. I ordered 15 different kinds from albermarlesounds on Ebay. N. mirabilis var. echinostoma, ampullaria red w/ green lip, amp red with dark spots, and green with red lip, inermis, jacquilinea, glabrata, jamba, maxima, adrianii, eustachya red, rafflesiana nivea, hirsuta red, aristolochiodes, and albomarginata red. I haven't ever grown nepenthes from seed so I'm hoping I get something out of this :/. I also got two pods of N. singalana x (ventricosa x alata) from a member here. To hedge my bets I went and sowed some of each variety the normal way. Here are trays under four t5's.
NseedTrayOpen.jpg

NepSeeds.jpg


For putting the seed in TC I will try sterilizing with bleach. Ten percent for 10 minutes or so. The flasks I have prepared already don't have Plant Preservative Mixture (PPM) in them and most people have trouble sterilizing nepenthes seed with bleach without killing them, so I will make some flasks with PPM and try some both ways. Luckily the batch of junk I bought came with a few vials of PPM :-D
 
Joined
Jun 6, 2008
Messages
3,014
Location
SF, CA
Quite the effort there. I too built a laminar flow hood a few years back, after using a far bulkier and expensive commercial version.

One thing that I would suggest is to move away from the rubber stopper with the holes -- regardless of the amount of cotton jammed into them. It is very difficult to manage a sterile culture in that environment -- from personal experience. Even the commercially available "vented" lids have their own set of problems.

Also, ten percent bleach for ten minutes will all but nuke the Nepenthes seed; and the low-land species should be soaked in a four to eight percent PPM solution (to MS salts) for upwards of twenty-four hours, since they are generally covered in fungal and mold spores . . .
 

JDW

Joined
Jul 12, 2012
Messages
80
Location
Mishicot, WI
Hey BigB I was hoping you'd see this. So the hood you use now you built yourself? If I didn't buy the stoppers with the bottles I would have gotten ones without holes. The guy I got them from has used them for years and they worked. The stoppers are all pretty old and stiff, so I might have to buy new ones soon anyway. Have you been able to sterilize any nepenthes seed with bleach? What rates and time worked if any? And what is the idea behind the ppm/ms instead of just ppm/water? I didn't get as much done with my hood today as I wanted, so I think I'll set up the small one I bought and get some cultures started this weekend. If I have to soak my seed 24hrs I'll have to start tomorrow! Oh and I was wondering if you've ever tried transfering material from a PPM culture to a non-PPM one, does growing in PPM for a while kill off all the bad stuff? Thanks for you help!
 
Joined
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Messages
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Location
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Have you been able to sterilize any nepenthes seed with bleach? What rates and time worked if any? And what is the idea behind the ppm/ms instead of just ppm/water? I didn't get as much done with my hood today as I wanted, so I think I'll set up the small one I bought and get some cultures started this weekend. If I have to soak my seed 24hrs I'll have to start tomorrow! Oh and I was wondering if you've ever tried transfering material from a PPM culture to a non-PPM one, does growing in PPM for a while kill off all the bad stuff? Thanks for you help!

Sterilizing Nepenthes seed with bleach has always been hit and miss with me over the years; some species respond well enough, while others are toast given the same treatment. The PPM solution -- four or eight percent -- is mixed with 1:2 MS solution (in lieu of simply water) that hasn't had sucrose added and has not been adjusted for pH. The rationale behind that is that the very acidic environment of that mix contributes to the sterilization of the seed's exterior, without doing it any harm. I have never lost seed with that method. Check the manufacturer's web site for other specifics; also seek out the fine TC forum on www.flytrapcare..com, where there has been plenty of discussion about PPM, biocides; and / or biostatic compounds.

In terms of your question about transference, I frequently re-plate sterile cultures, both to some PPM-laden media and to those without. If sterile technique is pursued, PPM is not always necessary or cost effective . . .
 

JDW

Joined
Jul 12, 2012
Messages
80
Location
Mishicot, WI
Got some done this weekend. Didn't finish my hood, and the smaller one I bought was too heavy for me to move inside so I improvised.
30gGlovebox.jpg

Took a 30 gallon aquarium laying around and taped plastic over the front. I sprayed down the inside with 20% bleach and wiped it down. I flasked up the more desirable nep species in the PPM media, I only made 10 flasks of that. The nep seeds soaked overnight in 10% PPM mixed with 20% MS salts solution instead of water. I also flasked some Drosophyllum seed. To sterilize that I soaked it in 10% bleach for 15 minutes. The Drosophyllum is on 20%MS without PPM (I dyed my PPM media blue so I know what it is).
allflasks.jpg

NepSeedOnPPM.jpg

Dlusi.jpg

On Sunday my brother stopped in, so I had him help me drag the flow hood inside and flasked up the rest of the nep seed. I put the 7 remaining packets of seed on 20%MS in the milk bottles. To sterilize this nepenthes seed, since it wasn't going in a media containing PPM, I soaked them in 5% bleach for 15 minutes. We'll see if that worked in a few weeks. And since I had extra of N. amp and N. mirabilis var. echinostoma, I did some of those on P273 media which is used for orchid seed.

So in total I have 19 flasks of nepenthes seed done... and 6 more species in the mail :blush:.
 

lance

Class 5 Nepenthes hoarder
Joined
Jul 25, 2011
Messages
1,306
Location
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How interesting! Hope everything stays sterile for ya ;). I look forward to seeing the results.
 

JDW

Joined
Jul 12, 2012
Messages
80
Location
Mishicot, WI

JDW

Joined
Jul 12, 2012
Messages
80
Location
Mishicot, WI
Got some contamination. Most of the non-PPM 25% MS bottles starting growing stuff. Three with fungus, one bacterial. There are still two that are clean. These are the ones that were soaked in 5% bleach for 15 minutes. One of ten of the PPM containing flasks got mold, the N. ampullaria Hot Lip. And one of the two flasks on p273 media, also 5% bleached for 15 min. All the contaminants appear to have come from the seed and not the media or outside. Now I don't have a flask of N. amp Hot Lip :( Here's the best pic I could get, mold in a non-PPM bottle.

IMG_0281.jpg
 
Joined
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Messages
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Mold issues are quite common with lowland Nepenthes; so much so, that they are typically soaked upwards of twenty-four hours in a PPM solution. Nepenthes ampullaria are among the worst in that respect. You may still be able to "rescue" that culture, if the mold hasn't spread too far. Simply remove the seed under sterile conditions; re-soak them; and re-introduce them to new media. I have had to occasionally do that with Heliamphora seed, which have a large surface area to "clean"; and it's always a balancing act between sterilizing the seed and nuking it.

Contamination may be occurring from a number of factors, including the lids or the amount of time that the media was sterilized. Considering how recently you made the media, it may have not been sterilized long enough; or mold and bacteria were introduced during the transfer of seed. PPM is supposedly not temperature labile during autoclaving; but I have noticed that batches kept for a third less time under sterilization were more resistant to later contamination.

You'll probably have far more success when you have your hood set-up completed . . .
 

JDW

Joined
Jul 12, 2012
Messages
80
Location
Mishicot, WI
Three more cultures contaminated today. I opted to remove the seeds and plant them in soil because I don't have a hood set up right now. I can't show you what I'm seeing because my camera doesn't want to focus through the glass of the bottles, but it's clear the contamination is coming from the seeds. I'm assuming because my sterilization treatments didn't work well. I have more seed in the mail and for that I want to try putting some of each variety in PPM and several different treatments of bleach. With my 5% for 15 minutes I could see the "wings" of the seed were much lighter in color while the embryo area stayed dark. BigBella, do you know if the wings are projection on the testa, or the entire coat itself? I wonder if the the embryo area has a second layer of protection, where I could bleach the seeds longer so the outside gets sterilized yet not damage the embryo. Also, with the last seeds I did, I put way too many per jar and couldn't spread them out well. With the contamination in the PPM cultures it appears that the molds started growing on a large clump of seed where not all of them were touching the media.

I also wonder if there's some way to remove the wings, they are annoying....
 

JDW

Joined
Jul 12, 2012
Messages
80
Location
Mishicot, WI
Things are going ok still. I have one flask of the non-ppm media that is still uncontaminated, and 5 of the 10 ppm containing flasks. There hasn't been any germination in the flasks yet but the amps and glabrata in soil are coming up like weeds. I got more seeds in and flasked up. Aristolochiodes, sumatrana, giant wavy maxima, red albomarginata, bicalcarata, and naga. All 10 of the new flasks are uncontaminated after two weeks. This time soaked the seeds in 10% ppm for over 24 hours as a disinfectant.
 

JDW

Joined
Jul 12, 2012
Messages
80
Location
Mishicot, WI
Still no germination of in vitro nepenthes. But one Drosophyllum did!!
DrosoTC.jpg


Of the seeds sown in soil about 250 have germinated and I transplanted 100 into a plug tray. Mostly ampullaria but I'm excited to see some inermis and jamba coming up. Last night I made 10 baby food jars of 2ml/L BAP and .25ml/L NAA to try doing callus culture of the droso and some nepenthes seedlings.
 
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What variety of ampullaria are you germinating? because I would be very interested in some in the future if they are ones I don't have.
 

JDW

Joined
Jul 12, 2012
Messages
80
Location
Mishicot, WI
What variety of ampullaria are you germinating? because I would be very interested in some in the future if they are ones I don't have.

Red with dark spots, Green w/ red lip (Hot Lip), and Red w/ green lip. Sry don't have pics. The seedlings only have one true leaf with itty bitty pitchers so it's gonna be a year or so before I start trading them.
 

JDW

Joined
Jul 12, 2012
Messages
80
Location
Mishicot, WI
I lied, just got permission to use the pics from albermarlesounds ebay listings where I bought the seed.

Red w/ dark spots
AlbmarlAmpRed.jpg

Hot Lip
AlbmarlAmpHotLip.jpg

Red w/ green lip
AlbmarlAmpRedGrnLip.jpg
 
Joined
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Messages
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Location
SF, CA
With my 5% for 15 minutes I could see the "wings" of the seed were much lighter in color while the embryo area stayed dark. BigBella, do you know if the wings are projection on the testa, or the entire coat itself? I wonder if the the embryo area has a second layer of protection, where I could bleach the seeds longer so the outside gets sterilized yet not damage the embryo. Also, with the last seeds I did, I put way too many per jar and couldn't spread them out well. With the contamination in the PPM cultures it appears that the molds started growing on a large clump of seed where not all of them were touching the media.

I also wonder if there's some way to remove the wings, they are annoying....

Your best bet is to leave the seed intact; you don't ever want bleach solutions, etc to come into contact with the embryo; just sow fewer in number per vial. I admire your courage, since you've also managed to choose some of more difficult to sterilize seed of the genera -- and without the benefit of a laminar flow hood. The lowland species are notoriously difficult to clean and may take an excess of twelve hours in a 4% PPM-MS solution; or else, serial bleach-alcohol treatments may eventually work.

Another method is to actually encourage the mold and fungal spores to grow, by "hardening" -- soaking the seed in ten percent solutions of sucrose for twelve hours or more; then, successive washes with alcohol, sterile water, H202; and then air-drying in a sterile environment.

Given the time involved and the effort, PPM is cheaper and simpler in the long-run . . .
 

JDW

Joined
Jul 12, 2012
Messages
80
Location
Mishicot, WI
Your best bet is to leave the seed intact; you don't ever want bleach solutions, etc to come into contact with the embryo; just sow fewer in number per vial. I admire your courage, since you've also managed to choose some of more difficult to sterilize seed of the genera -- and without the benefit of a laminar flow hood. The lowland species are notoriously difficult to clean and may take an excess of twelve hours in a 4% PPM-MS solution; or else, serial bleach-alcohol treatments may eventually work.

Another method is to actually encourage the mold and fungal spores to grow, by "hardening" -- soaking the seed in ten percent solutions of sucrose for twelve hours or more; then, successive washes with alcohol, sterile water, H202; and then air-drying in a sterile environment.

Given the time involved and the effort, PPM is cheaper and simpler in the long-run . . .

The issue I have about the wings is that the soaked seeds stick together and it is hard to spread them out evenly on the media. Using a forceps to pull the seeds out of the soaking solution I get 10-25 at a time, and trying to spread them out with a spatula or glass rod they just roll in a mass and don't separate. What I had to do is stir them so they aren't all floating at the top and grab one or two to place on the media. This took way too long for sowing 500 seeds!

Honestly I didn't know at the time that lowland seed is more difficult to sterilize. I just bought what I like and was available. But for me there hasn't been a difference. Only the first 10 milk bottles were done in a clean box. Everything after that I did inside a flow hood.


Just to note, my 10% bleach soak for 10 minutes didn't kill the seeds. I rescued some bleached ampullaria seed from contaminated flasks and placed them on soil. They are now sprouting just fine.
 
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Messages
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The issue I have about the wings is that the soaked seeds stick together and it is hard to spread them out evenly on the media. Using a forceps to pull the seeds out of the soaking solution I get 10-25 at a time, and trying to spread them out with a spatula or glass rod they just roll in a mass and don't separate. What I had to do is stir them so they aren't all floating at the top and grab one or two to place on the media. This took way too long for sowing 500 seeds!

First of all, the prospect of even sterilizing five hundred Nepenthes seeds at one time is a tall order, even under most commercial settings (I usually deal with a maximum of one hundred at any given time) -- the chances for contamination being far higher than doing serial smaller batches; and the method I have found most helpful for manipulating that "annoying" seed, is to soak them in the sterilization solutions within filter paper packets affixed with plastic paper clips (which can be color-coded for different species, as seen below). The seed is far easier to handle individually, while still attached to the damp paper.

With that method, my contamination rate remains below two percent . . .

TCPAPERS.jpg
 
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