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Cheap Tissue Culture

Hello TF,

I'm pursuing a new cheap project! Recently, I was assigned a project in school where we have 7 weeks to pursue anything we want, and then we give a presentation on the process and what we have learned by the end. It's an awesome opportunity, and I have decided to move up my summer plans of attempting Tissue Culture. I am a student, and do not have access to any of the $15,000 laminar flow hoods or other equipment typically used. I'm not even going to try doing this with the typical MS agar and growth regulators. I'm going cheap, with a budget of $50. So, I'm going to have to get inventive and be careful, because I know how sensitive this practice is, and I by no means expect to be successful. But I'm going to try very hard to be, and hope I get it right the second time around :). What follows is where I am in the process of acquiring materials.


Today I bought most of the supplies I think I will need. These include:
  • A plastic box as a pseudo sterile hood as seen on the HTC webpage
  • 91% isopropyl alcohol
  • 70% isopropyl alcohol
  • Hydrogen peroxide (3% I believe)
  • 8.5% bleach (It was the best I could find, this was the super concentrated version too)
  • Inositol tablets
  • Multivitamin pills
  • Spray bottle to sterilize surfaces
  • Distilled water
  • Table sugar
  • Coffee filters to hold samples during sterilization


As per very nice instructions from BigBella, I still need for my media:
  • Agar
  • 10:10:10 fertilizer


In terms of equipment, all I need are:
  • Baby food jars
  • Long forceps
  • Pressure cooker


For those that have taken the time to read this far, I really appreciate that. There are a few questions I still need answered. They are the following:
  • Are there alternatives to agar that I can also buy in a supermarket? I was informed that agar may not be available for a while around me. I could always order it if there is no alternative.
  • Is a 10% bleach solution 10% sodium hypochlorite or 10% of the bleach solution mixed with water (so would I just buy a 10% sodium hypochlorite version of Clorox Bleach, as opposed to mixing 10mL of the 8.5% Clorox Bleach I bought in 90mL of water)?
  • Is there any (preferably cheap) alternative to PPM I can use in the media to try and prevent unwanted growth? Any ideas with which I can experiment?


Thank you so much for any help you can offer, and I will update this thread with the process and results.
 
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What equipment do you have access to? Balance, pressure cooker, pipets....?? Where are you located? I could send you some stuff to play with.
 
I am fairly certain my school will let me borrow digital scales and graduated cylinders. I have a lot of pipettes, and I could probably get some nice glass graduated ones from school. I don't have access to things like an alcohol lamp or hot bead sterilizer. I'm located on the East Coast of the US. I'd be very grateful for anything you'd like to send me, as long as it is safe. I'm trying not to mess with the more powerful hormones and chemicals before I have a little experience with the cheap stuff.
 
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Have fun with this project Drew. I bet the science department will have alcohol lamps, we had plenty at my high school.
 
Thanks Drew, I'm really excited for it. And if all goes well I should have a lot more of my brown D. burmanniis to spread around. I'll ask my science teachers if they have any lamps to spare.
 
Just make one yourself:

736079067-Jelly-Jar-Alcohol-Lamp.jpg
 
Cool Peat! What kind of alcohol do you use? Did you use anything special for the wick?
 
I can give you some gelrite, MS + vits powder, homemade PPM (use this judiciously as my results are not in but it is better than nothing), loops, small vials...etc. I do have baby food jars but they are heavy to ship, and personally I hate them. It is probably better to find a local free source than get them from me.

As for the alcohol lamp, just use a lighter. You can dip/soak your instruments in alcohol and flame when necessary. You should have an adjacent bottle of sterile water to cool off the instrument before use.
 
@Drew, I didn't make that. But it seems simple enough to whip up. I found the pic on an, uhhhh, shroom forum.. :p
 
  • #10
Pokie that would be awesome! I currently have an email out to everyone I know who has a baby, so we'll see if I can get any jars that way. What do you mean by a loop? How did you make your own PPM? That's so cool! I think using a lighter would be easier, thanks for the recommendation.

@Peat: :lol:
 
  • #11
Please refer to my thread on Nepenthes TC. A loop is for manipulating seeds/sprouts and can be used in lieu of tweezers which can be frustrating in some instances when the seeds stick. There are ones that are made of metal so that one can reuse them by flaming/autoclaving or there are the plastic disposables that have been gamma irradiated and intended for one time use. You might want to the look for a local "freecycle" mailing list as that is where I procured my baby jars from. As for the PPM, the patent is in the public domain.

Send me a PM with your address and I will gather some stuff for you. Let me know if you want the MS media or not. If so, do you want me to preweigh out some samples for some specified amount (ex. 100ml recipe..etc).

Jen
 
  • #12
That thread was very informative. Although I do not have the same access to equipment, I think I can follow much of the same procedure. I'll PM you soon, I really appreciate this.
 
  • #13
For anyone who would like to read a little more in depth as to what I'm doing: I have to maintain a blog for this project. You can see it here: http://sundrew.blogspot.com/
 
  • #14
So, I assume that you will you have access to an autoclave or pressure cooker; and that that information on your blog, wasn't simply an illustration of the typical micropropagation process?

Spores of microorganisms can survive very high temperatures, even sustained boiling, at normal atmospheric pressure. Generally, media has to be autoclaved at 121°C and 1 atm; or 15 psi; or 6.8 kg · 6.5 cm−2 for twenty-five minutes or more (depending upon the volume of the media) . . .
 
  • #15
Anybody who does canning would have a pressure cooker.

My mother calls it jamming.........
 
  • #17
So, I assume that you will you have access to an autoclave or pressure cooker; and that that information on your blog, wasn't simply an illustration of the typical micropropagation process?

Spores of microorganisms can survive very high temperatures, even sustained boiling, at normal atmospheric pressure. Generally, media has to be autoclaved at 121°C and 1 atm; or 15 psi; or 6.8 kg · 6.5 cm−2 for twenty-five minutes or more (depending upon the volume of the media) . . .

Yes, I am in the process of finding a suitable pressure cooker, I should have one by this weekend. Sorry if this was not as detailed as your or pokie's great instructions, but I have to write it out in a way that my teachers and peers can understand. And thank you for all your help BigBella! Did you see your shout out?
 
  • #18
Yes, I am in the process of finding a suitable pressure cooker, I should have one by this weekend. Sorry if this was not as detailed as your or pokie's great instructions, but I have to write it out in a way that my teachers and peers can understand. And thank you for all your help BigBella! Did you see your shout out?

Great -- though ensure that the cooker can reach or exceed 15 psi; not all pressure cookers are capable of doing so. Good luck with the project and keep us posted . . .
 
  • #19
How long does it typically take to see initial growth on an explant? I won't expect seed germination, but over the course of a few weeks (3-5), can I expect to see some kind of growth? If, of course, there is no contamination.
 
  • #20
How long does it typically take to see initial growth on an explant? I won't expect seed germination, but over the course of a few weeks (3-5), can I expect to see some kind of growth? If, of course, there is no contamination.

It could take as little as three to four weeks; and that could be dependent upon the age of the leaf; how "savagely" it was sterilized; the temperature at which the culture are kept; and the concentration of the media, etc.

Also, should the leaf experience phenolic bleeding (a darkening of the jello, not associated with contamination -- and that seems likely), keep moving it within the culture to a clear area, until that process stops . . .
 
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