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Cheap Tissue Culture

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

EDIT: What a great 100th post!
 
  • #102
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.

<a href="http://www.flickr.com/photos/84442298@N03/8583529477/" title="DSC02237 by Sundrew, on Flickr"><img src="http://farm9.staticflickr.com/8098/8583529477_03c2ac75c1_z.jpg" width="640" height="480" alt="DSC02237"></a>


The aftermath.

<a href="http://www.flickr.com/photos/84442298@N03/8583529479/" title="DSC02238 by Sundrew, on Flickr"><img src="http://farm9.staticflickr.com/8367/8583529479_9c759b1dcd_z.jpg" width="640" height="379" alt="DSC02238"></a>


The river of bleach.

<a href="http://www.flickr.com/photos/84442298@N03/8583529437/" title="DSC02239 by Sundrew, on Flickr"><img src="http://farm9.staticflickr.com/8518/8583529437_5bce642aa9_z.jpg" width="640" height="480" alt="DSC02239"></a>


The best cutting.

<a href="http://www.flickr.com/photos/84442298@N03/8584629154/" title="DSC02241 by Sundrew, on Flickr"><img src="http://farm9.staticflickr.com/8243/8584629154_98e44d3917_z.jpg" width="640" height="480" alt="DSC02241"></a>


The cutting with terrible aim.

<a href="http://www.flickr.com/photos/84442298@N03/8583528617/" title="DSC02244 by Sundrew, on Flickr"><img src="http://farm9.staticflickr.com/8380/8583528617_f15b9008c7_z.jpg" width="640" height="480" alt="DSC02244"></a>


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)!

<a href="http://www.flickr.com/photos/84442298@N03/8583528537/" title="DSC02243 by Sundrew, on Flickr"><img src="http://farm9.staticflickr.com/8371/8583528537_8b589d0a9c_z.jpg" width="640" height="480" alt="DSC02243"></a>
 
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  • #103
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?
 
  • #104
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 . . .
 
  • #105
I have one jar left of media from the original batch and don't really have time to make/sterilize more. I'm going to be sterilizing D. burmannii seeds (very, very small). How long should I keep them in 10% bleach (I decided on this being to only sterilization)? I would normally do 10 minutes, but these are so small I'm thinking I should stick to 8 or so.
 
  • #106
Since maintaining sterility has been an issue so far, I would suggest ten to fifteen minutes in a 10% household bleach solution (actually the concentration of sodium hypochlorite -- bleach's active ingredient -- will only be about 0.006% at that dilution, based upon the average six percent contained in, say, Clorox), followed by two to three rinsings with sterilized distilled water; or even a rinse with H2O2 or peracetic acid.

Drosera seed may be tiny; but they are quite tough and can be filthy . . .
 
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  • #107
Well that was terrible. I did all the prep for the seeds, 10 minutes in bleach, 4 minutes in H2O2, and then two 10 minute washes in steril water. Then when it came to extricating the seeds...the filter paper turned to mush. I picked through it but could free and place in the jar only 1 seed of the 30-45 I had collected for this jar. Plus, the jar lid fell off and was open for a while as I tried to stick the seeds inside. It was rough.
 
  • #108
Well that was terrible. I did all the prep for the seeds, 10 minutes in bleach, 4 minutes in H2O2, and then two 10 minute washes in steril water. Then when it came to extricating the seeds...the filter paper turned to mush. I picked through it but could free and place in the jar only 1 seed of the 30-45 I had collected for this jar. Plus, the jar lid fell off and was open for a while as I tried to stick the seeds inside. It was rough.

Sorry to hear about that . . .

What happened? Did you manage to agitate the filter paper envelopes that entire time, in the bleach and H2O2 -- shake the daylights out of them during the process? Yeah, then the filter paper will turn to military grade toilet tissue in a NY minute; but I have routinely soaked seed packets in serial solutions upwards of twelve hours or more without incident; and only occasionally bothered to shake the bottles . . .
 
  • #109
I shook them in the bleach solution fairly hard, but then I noticed some seeds were slipping out of the filter paper. So from then on I did slow and steady stirring. The problem occurred when I tried to unfold the filter paper, and it just tore. Then I had the multiple layers of folds I had made to try and seal the seeds in to sort through. Eventually it all fused and the seeds were just locked in the pulp. I managed to get a few seeds on my hands, but then I could barely get them onto my tweezers and then flick them into the container. I was also trying to move really fast the whole time so I didn't compromise them from being in the open air.
 
  • #110
So.. not to raise an old thread but I was wondering how you guys sterilize your forceps inside the transfer container? I've read to dip them in 70% alcohol and use a medium flame after? Are there any other effective alternatives that don't use fire?

Also, how long do these materials (MS, BAP, and PPM) stay good for? The site I'm going to order from states to put them in the fridge immediately after arrival, which makes it seem like they expire quickly. However all the other sites I look at with the same materials don't state to put it in the fridge?
 
  • #111
My forceps are soaked in 70-99% isopropyl alcohol and flamed before each use, occasionally cooled in either the sterile TC media to be used; on a tool rack in front of the laminar flow hood; or, in sterilized water. You could always use a glass bead sterilizer, in lieu of an alcohol lamp; but that wouldn't fall under the rubric of "cheap tissue culture," not by a long shot. Alternatively, you could autoclave them before each use; but that would be time-consuming and a one-shot deal -- especially if a number of transfers were required.

The only way to fully ensure sterility; to ensure that no toxic alcoholic residue remains on the tools; is to consign those dirty buggers to the holy purifying flames . . .
 
  • #112
Oh.. Right. Alcohol Lamp. Haha I was thinking lighter, the lamp seems like a better idea. Thanks for the info!

Could you also comment on the longevity of MS, BAP, and PPM? Why does only one source state to refrigerate upon arrival?
 
  • #113
To keep it cheap, I just used 90% alcohol. I was nervous about using a lighter for sterilization so close to alcohol fumes.
 
  • #114
Oh.. Right. Alcohol Lamp. Haha I was thinking lighter, the lamp seems like a better idea. Thanks for the info!

Could you also comment on the longevity of MS, BAP, and PPM? Why does only one source state to refrigerate upon arrival?

Refrigerated, most media and PGRs are good for about a year, give or take. Some compounds are inherently unstable and will eventually degrade; and far more than one source requires refrigeration. "Store at 2-8 degrees celsius," is printed upon every package that I have . . .

To keep it cheap, I just used 90% alcohol. I was nervous about using a lighter for sterilization so close to alcohol fumes.

The general idea is to flame the forceps away from, say, an open bottle or beaker of alcohol, using a Bunsen burner; a lighter; or, an alcohol lamp.

Denatured alcohol, available at hardware stores, as a fuel and solvent, is generally used for the lamps . . .
 
  • #115
The general idea is to flame the forceps away from, say, an open bottle or beaker of alcohol, using a Bunsen burner; a lighter; or, an alcohol lamp.

It's a good thing my mom noticed the huge puddle of alcohol and bleach inside the work area, I had my finger on the lighter when she stopped me. Not my best moment :blush:
 
  • #116
^ Hahaha good thing your mom was around. I'm a little nervous about using an open flame in an environment sprayed with alcohol. I'll switch to using a 10% bleach solution to sterilize inside the transfer aquarium. Found an interesting article from Stanford stating it's more effective anyways. Apparently 70% alcohol needs a longer contact time (like 10+ minutes) to be effective. Being used as a spray and dissipating quickly doesn't provide that.

One last question: What method/products do you use to disinfect explants? I'm particularly interested in Cephalotus explants but there are so many methods out there! Sorry for all the questions, I attempted micropropagation many years ago and failed miserably so I've been a little scarred on my retry. It was definitely a good learning experience though.
 
  • #117
One last question: What method/products do you use to disinfect explants? I'm particularly interested in Cephalotus explants but there are so many methods out there! Sorry for all the questions, I attempted micropropagation many years ago and failed miserably so I've been a little scarred on my retry. It was definitely a good learning experience though.

The method for cleaning explants varies a bit, depending upon where they are grown -- outside; in a terrarium; or, a greenhouse. It generally begins with a thorough cleaning under running water, followed by a multi-hour soak in a PPM solution; and then, placing them in media. Sometimes, bleach solutions are also used; so too, alcohol dips; and, also, peracetic acid.

There are any number of protocols, given at the tissue culture forum at flytrapcare.com. I suggest you read through the many threads . . .
 
  • #118
It's a good thing my mom noticed the huge puddle of alcohol and bleach inside the work area, I had my finger on the lighter when she stopped me. Not my best moment :blush:

:nono: messy messy messy
 
  • #119
Attempt #2 is in motion! I have nine vials full of the remainder of my medium, though this time I tried agar in three of them. It does make for a much clearer gel. I just sterilized and added some burmannii seeds to one vial as a practice, and it went very well. To better secure the seeds, I stuck seeds to my forceps and then added them directly to the media. Hopefully my lighter made the tweezers sufficiently sterile.
 
  • #120
FIRST GERMINATION WOOHOOOOOOOOOO!!!! :boogie:
 
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