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Thread: JDW's Tissue Culture

  1. #9
    JDW's Avatar
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    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.


  2. #10
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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 . . .
    “Sì perché l'autorità dell'opinione di mille nelle scienze non val per una scintilla di ragione di un solo . . ."

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  3. #11
    JDW's Avatar
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    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....

  4. #12
    JDW's Avatar
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    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.

  5. #13
    JDW's Avatar
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    Still no germination of in vitro nepenthes. But one Drosophyllum did!!


    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.

  6. #14
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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.
    NCSU's Carnivore Nut
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  7. #15
    JDW's Avatar
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    Quote Originally Posted by mcmcnair View Post
    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.

  8. #16
    JDW's Avatar
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    I lied, just got permission to use the pics from **************** ebay listings where I bought the seed.

    Red w/ dark spots

    Hot Lip

    Red w/ green lip

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