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Thread: JDW's Tissue Culture

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    JDW's Avatar
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    JDW's Tissue Culture

    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



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

    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

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

    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

    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.

    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 . 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.


    My first project is going to be some nepenthes seed. I ordered 15 different kinds from **************** 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.



    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

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    BigBella's Avatar
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    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 . . .
    “Sì perché l'autorità dell'opinione di mille nelle scienze non val per una scintilla di ragione di un solo . . ."

    -- Galileo "Biff" Galilei

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    JDW's Avatar
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    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!

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    BigBella's Avatar
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    Quote Originally Posted by JDW View Post
    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 . . .
    “Sì perché l'autorità dell'opinione di mille nelle scienze non val per una scintilla di ragione di un solo . . ."

    -- Galileo "Biff" Galilei

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    JDW's Avatar
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    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.

    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).



    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 .

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    An effort should really be made to seal those containers with something like Parafilm (http://en.wikipedia.org/wiki/Parafilm), to prevent any contamination -- especially given how much sugar and fertilizer is contained in TC media . ..
    “Sì perché l'autorità dell'opinione di mille nelle scienze non val per una scintilla di ragione di un solo . . ."

    -- Galileo "Biff" Galilei

  7. #7
    Class 5 Nepenthes hoarder lance's Avatar
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    How interesting! Hope everything stays sterile for ya . I look forward to seeing the results.


    In addition to growing plants, I design and build RC planes powered by Tesla batteries. Check out my progress at www.chargedplanes.com

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    JDW's Avatar
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    Quote Originally Posted by BigBella View Post
    An effort should really be made to seal those containers with something like Parafilm (http://en.wikipedia.org/wiki/Parafilm), to prevent any contamination -- especially given how much sugar and fertilizer is contained in TC media . ..
    Everything sealed up now with: Vessel Wrap

    Quote Originally Posted by lance View Post
    How interesting! Hope everything stays sterile for ya . I look forward to seeing the results.
    Thanks and ditto

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