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Thread: Cheap Tissue Culture

  1. #97
    Sundrew's Avatar
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    No it was funny. I don't mind at all.

  2. #98
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    double post
    Last edited by Sundrew; 03-21-2013 at 03:08 AM.

  3. #99
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    I'm going to plant some explants today. Does anyone know if TC will work on a root tip emerging from the pot of my N. ampullaria? Also, how do you transfer wet seeds from sterilization into a vessel without having them stick to the forceps/other seeds?

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    Quote Originally Posted by Sundrew View Post
    I'm going to plant some explants today. Does anyone know if TC will work on a root tip emerging from the pot of my N. ampullaria? Also, how do you transfer wet seeds from sterilization into a vessel without having them stick to the forceps/other seeds?
    Nepenthes explants are extremely difficult to sterilize properly, since an endosymbiotic alga lives within its tissues; and can easily overrun a culture vessel. Lowland species are even more difficult, for that reason.

    In terms of transferring seed, I use pouches of filter paper and simply use some forceps to remove them from the wet surface; ans sticking is seldom a problem. Also, innoculating loops are useful and I believe both pokie22 and I offered to send you some, along with standard media . . .
    “Sì perché l'autorità dell'opinione di mille nelle scienze non val per una scintilla di ragione di un solo . . ."

    -- Galileo "Biff" Galilei

  5. #101
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    I'm done! Full explanation with pictures tonight, but right now I just feel like celebrating!

    EDIT: What a great 100th post!

  6. #102
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    Today was a great day. I planted the sterilized cuttings in the jars, and it all went pretty well. I cut off four of the developing leaves of my D. burmannii (hopefully it isn't damaged that badly), washed them in hot soap and water, and proceeded with the following procedure. In each of the steps I shook the jar vigorously.


    INSIDE HOOD

    1. Dunk explants in 91% alcohol for 10 seconds. Skip this for seeds.
    2. Dunk in 3% H2O2 for 4 minutes.
    3. Dunk in 10% bleach solution for 7 minutes. Put seeds in here for 4 minutes.
    4. Place in sterile jar of water and rinse for 5 minutes. Repeat.
    5. Pull samples out of jar with sterilized forceps.
    6. Loosen cap on TC jar.
    7. Place explants in jar extremely quickly.


    STERILIZATION:

    1. Using tiny spray bottle, spray 10% bleach solution on every surface.
    2. Generously spray jars with 10% bleach solution.
    3. Wash hands with soap and water, then wipe with 70% alcohol.


    To prepare the steril water, I filled loose jars with distilled water and pressure cooked them for 25 minutes. For the bleach solution, I used 2/3 cup of extra-concentrated bleach in 9 1/3 cups of tap water. I figured any impurities in the tap water would be washed off in the final rinse. If I were to do this again, I would use flat forceps the whole time, for this I used surgical forceps with teeth that I am sure damaged the cuttings. I used flat forceps for the final transfer, because I did not want the cuttings to stick.


    Here are some pictures from today:

    I was about to try and sterilize forceps with a lighter, then realized I had these covering every surface. An oxygen source, two potent fuels, and a reactive poisonous vapor source.

    DSC02237


    The aftermath.

    DSC02238


    The river of bleach.

    DSC02239


    The best cutting.

    DSC02241


    The cutting with terrible aim.

    DSC02244


    The jars' new home (I realized this was close to the lights and moved them to my basement's stone floor where they will stay in the 60s)!

    DSC02243
    Last edited by Sundrew; 03-24-2013 at 05:27 AM.

  7. #103
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    That was fast. I found my first contamination in I tink 3 of the 4 jars today. It is in the form of a white fuzzy mold on the leaves. I do not think this is necessarily bad news. My media is ok, so the cheap materials were not the problem. Secondly, I was able to recognize contamination, which I will chalk up as a win. So I need to improve upon my sterilization of explants, and I may try this all again tomorrow or the next day with some seeds and see how they turn out. I think I should add some kind of wetting agent to the bleach solution, any suggestions? I've heard of these Tween products, but is there anything readily available that does not need to be ordered online?

  8. #104
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    A small -- very small -- amount of antibacterial hand dishwashing soap is quite effective. I would also suggest using peracetic acid -- a combo of hydrogen peroxide and acetic acid (white vinegar) during the sterilization process. Recipes are readily available online . . .
    “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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