Cheap Tissue Culture

[Sundrew]

Thanks BigBella, that timing will be great.


Here was the progress made today, I cut apart the plastic container to make my clean box. Here it is with the little spray bottle of bleach I will use to clean it and anything that enters it:

<a href="http://www.flickr.com/photos/84442298@N03/8504407899/" title="DSC02115 by Sundrew, on Flickr"><img src="http://farm9.staticflickr.com/8239/8504407899_a235dbf42c_z.jpg" width="640" height="424" alt="DSC02115"></a>


Here is the only 10-10-10 fertilizer I could find. It checked out from what I read on the back, I just don't know how to quantify this and add it to the gel mixture. Any suggestions?

<a href="http://www.flickr.com/photos/84442298@N03/8505517894/" title="DSC02116 by Sundrew, on Flickr"><img src="http://farm9.staticflickr.com/8108/8505517894_1d181296a1_z.jpg" width="480" height="640" alt="DSC02116"></a>

<a href="http://www.flickr.com/photos/84442298@N03/8505517934/" title="DSC02118 by Sundrew, on Flickr"><img src="http://farm9.staticflickr.com/8519/8505517934_1813fd2039_z.jpg" width="480" height="640" alt="DSC02118"></a>


By next weekend I intend to have ordered some agar and have the liquid mixed. Hopefully I'll have the pressure cooker and forceps next week as well. Then I will have everything I need.

 

[Sundrew]

I ordered some proper fertilizer today, as well as some agar. I should be mixing the media by this weekend. In my research, I came across another potential experiment for a cheap gel, corn starch. It would create a firm and soft surface. The problem would be in spotting contamination on the cloudy surface, but here is a modified version that is clear: http://www.amazon.com/Barry-Farm-Cl...UTF8&qid=1362107804&sr=8-1&keywords=clear+jel. I will probably be experimenting with normal cornstarch.

 

[Api]

Good luck, Drew. Man, I would love to get my hands on a coffee flavored burmannii.

 

[Sundrew]

Thanks. We'll see if it works...

 

[Sundrew]

Liquid nutrients done! The pressure cooker will be bought later today, as well as some corn starch to play around with as a gelling agent.

The materials:

<a href="http://www.flickr.com/photos/84442298@N03/8524368719/" title="DSC02169 by Sundrew, on Flickr"><img src="http://farm9.staticflickr.com/8511/8524368719_df3215392d_z.jpg" width="640" height="475" alt="DSC02169"></a>


The result:

<a href="http://www.flickr.com/photos/84442298@N03/8525484076/" title="DSC02165 by Sundrew, on Flickr"><img src="http://farm9.staticflickr.com/8369/8525484076_811876a03b_z.jpg" width="640" height="480" alt="DSC02165"></a>


It's currently in a pitcher in my freezer, until I use it next week.

 

[BigBella]

Drew,

Just remember that the media will all look like water when it is first removed from the pressure cooker; and that it will only gel once it completely cools. That may take a bit longer -- the gelling -- for the corn starch . . .

 

[Sundrew]

Thanks for the advice. Do you think I should wait a full day before opening and adding explants? Also, would you recommend adding the agar/corn starch and then dispensing to the jars, or dispensing to the jars first and then adding agar/corn starch individually? I read on FTC that the latter is a more reliable method.

 

[Vbkid]

Drew, mind sharing the recipe/proportions you used for the nutrient?

 

[Sundrew]

Everything can be found here: http://sundrew.blogspot.com/


But this was the recipe, as directed to me by BigBella:

Table sugar (sucrose): 1/8 cup
Distilled water 1 cup
10:10:10 fertilizer (1/4 teaspoon dissolved in one gallon of water). Use 1/2 cup of that mix
Inositol tablet (250 mg) -- use 1/2 of tablet
Vitamin tablet with thiamine -- use 1/4 tablet
Agar flakes (amount varies with manufacturer)

I diluted it by 1/2 because I'm working with CPs.

 

[BigBella]

Drew,

Mix everything together, since the quality of corn starch varies and it is better to have it completely dissolved, then having globs of it suspended in media. Also, autoclave it at the higher end, in terms of time. Gelling agents other than gelzan and agars can be "temperamental" at times, in terms of completely dissolving. I would do a small trial amount and see what happens.

Also, be damn well careful when futzing with pressure cookers . . .

 

[Sundrew]

Ok, I'll be sure to do a few trials. It'll be really cool if it works! In terms of pressure cookers, I got a little nervous when this guy behind me in line at Walmart said that he "Hated the aluminum pressure cookers because they leak." Not the kind of comment I was looking for.

 

[BigBella]

Ok, I'll be sure to do a few trials. It'll be really cool if it works! In terms of pressure cookers, I got a little nervous when this guy behind me in line at Walmart said that he "Hated the aluminum pressure cookers because they leak." Not the kind of comment I was looking for.

The cookers don't leak if properly maintained. Just make sure that the o-ring is intact, and, above all, clean. Just don't go into another room on a "Mackintosh workout" and not pay attention to it. Read the manual too . . .l

 

[Sundrew]

I have yet to put it together, so I'll be sure to read the manual and make sure I know how to maintain it. I don't need any explosions with glass bottles as shrapnel. Thanks again for all your advice.

 

[swords]

What you do not want to skimp on is your sterilizer, go and get a proper pressure pot with a regulator valve on it.

When I was growing edible mushrooms those cheapie "pressure pots" from Walmart with the tippy thing on top didn't work very well, I had to toss about 5 of every 10 cultures due to contamination. Get the heavy duty 21 quart steel can with clamps and the pressure gauge on top for about $90 (Mills Fleet Farm or other place selling the real canning gear) and you'll have almost flawless sterilization for any media, metal tools, etc. You can skimp on your sterilization unit and possibly get away with it if you're lucky but since sterilization is the #1 thing you need in TC why not spend the extra dough and get what you really need so you don't have to toss as many cultures. Tossed / contaminated cultures will eventually cost you more than buying proper equipment in the long run.

 

[DonH]

Is your project a guide to cheap tissue culture or tissue culture using commonly available supplies? Most of the basic materials (vessels, MS salts+vitamins, agar, PPM, hormones) can be purchased from Kitchen Tissue Cultures for ~$50. Used at reduced strength for carnivorous plants, you can get 3 liters of stock solution with 1 packet of MS. A 9 gram packet of agar is enough for 3 liters of 1:3 MS and you have enough PPM and PGRs to play with to start.

When you break down your expenses, how much does it come to? I know shipping will add to the price of purchasing from KTC but that can be offset some by the price of gas used to collect your supplies.

Your project is very interesting because you're using commonly available supplies at your local supermarket and garden center. I look forward to seeing your results.

 

[Sundrew]

I kind of intend for the project to be both, if it works. At the moment, it is just my attempt at tissue culture using cheap home materials. When given this project in school, I was debating how I should go about tissue culture. I thought that it would be more of a learning experience, even if it does not work the first time, to try and make a formula myself using home materials that accomplish the same functions as the "prepared" substances. Of course I'm relying heavily on the recipe given to me by BigBella and everyone's advice, but it is much more fun to me if I prepare everything myself. So, while I considered the extremely well thought out and helpful kits provided by HTC, I thought that I could learn more by mixing it myself. And if it worked, than I would have a procedure that nearly anyone could perform with very simple materials. In the future, once I have my procedure down and am used to steril protocol, I may purchase some more expensive materials.

As for cost, here is the tally at the moment of everything I had to buy that was not already in my home. I will find the receipts to correct this later:

Fertilizer: $6
Inositol: $11
Agar powder: $5
Distilled water: $2
Sterilization chemicals, spray bottle, and plastic box for sterile area: $25
Pressure cooker: $35

The rest of the materials on my list I either already had, or am borrowing or scavenging. All in all, it is still pretty cheap, especially considering that I would have had to by the last two regardless of whether I had gone with the kit instead. If I were to use all of what I had purchased, I could probably end up with 4-10 liters of nutrient liquid, depending on how much distilled water I buy. Everything else will last for many more uses than even that.

Thanks for your comment! I am also very excited to see if it works, hopefully I will have something interesting to present in April.

 

[Sundrew]

I had a little bit of progress today. I was playing around with corn starch as a gelling agent. I wasn't aware of the fact that if you cooked corn starch it becomes a very nice gel, so imagine my surprise when I microwaved it on a whim and got a fantastic medium to use for TC! I tried a few combinations, and 5 teaspoons of cornstarch well mixed into 1/2 cup of water seems to do the trick. The only problem is that it superheats and rises up the container, then collapses in on itself, leaving a large, gooey dent. Here's a pic of the gel after I smoothed it. This is not the actual nutrient solution, I plan on practicing with how it sets in a pressure cooker before using my actual concoction.

<a href="http://www.flickr.com/photos/84442298@N03/8547536126/" title="DSC02197 by Sundrew, on Flickr"><img src="http://farm9.staticflickr.com/8391/8547536126_3f4b2748f6_z.jpg" width="640" height="480" alt="DSC02197"></a>


In other news, I put together the pressure cooker! It looks very nice.

<a href="http://www.flickr.com/photos/84442298@N03/8547536032/" title="DSC02191 by Sundrew, on Flickr"><img src="http://farm9.staticflickr.com/8238/8547536032_92737df66c_z.jpg" width="640" height="480" alt="DSC02191"></a>

 

[BigBella]

One of the primary issues with using corn starch as a gelling agent, is that it becomes difficult to tell whether cultures become contaminated -- since it already appears compromised . . .

 

[pokie22]

Chinese food utilizes corn starch as a sauce thickener. And yes, I have to agree, most of the time the take out has been compromised.

 

[richjam1986]

Agar powder is also frequently used in Asian cooking, and is available at Asian markets (if you're lucky enough to live close to an Asian market). While corn starch may work, it seems like there may be some issues in dealing with it (like the fact that it bubbles up and makes a mess, like you said).

It may certainly be worth experimenting with cornstarch as a medium if agar isn't easily available, but I got a packet of culinary agar power for like $2 at a nearby Asian market.