FlytrapCare Carnivorous Plant Forums

Hello! A friend and I are working on tissue culture. Are our steps adequate? We are going to grow cephalotus, sundews and venus fly traps.


Our Steps.
Prepare The Media

1. Measure 1 quart of distilled water in a clean measuring cup.
2. On an electronic scale measure 36 grams of MS, with gelzan and vitamins.
3.Mix the two thoroughly.
4.Test the ph 5.6-5.8 (if ph is to low add sodium bicarbonate, if it's to high add white vinegar)
5.Once the desired ph is reached, place 30 ml of the mixture into each baby food jar.
6.Place in Cooker (refer to “Sterilize the Media”).

Sterilize the Media
7.Place in pressure cooker for 22 minutes
8.Wait for it to cool down

Sterilizing the seeds
9.Put seeds in coffee filter .
10.Dip the coffee filter in rubbing alcohol briefly and take it out.
11.Soak in distilled water for 24 hours
12.Soak in 3-4% Hydrogen Peroxide for (specify later).
13.Rinse seeds

Putting it in the Media
14.Use forceps to put it into media 3-5 seeds per jar
Transport
15.Bring the seeds from Davids House to my house and put all the jars in a bin. Keep it on brightest window sill.


Thanks!

I'm no expert on TC but I have a few recommendations. The forceps used would need be sterile, and make sure the jars don't overheat on a window

Leathal_Traps wrote:I'm no expert on TC but I have a few recommendations. The forceps used would need be sterile, and make sure the jars don't overheat on a window

Thanks! I will put it in a cooled terrarium. We will also clean the forceps with rubbing alcohol

You're also going to need to flame sterilize your tools -- forceps, scalpels, etc; don't rely only upon an alcohol soak. Even a small amount of residue, can kill seeds and explants. Add, at the very least, an inexpensive alcohol lamp to your list -- and only use denatured alcohol as a fuel.

Also, buy a large roll of Parafilm to reliably seal the vials after explants, etc. are introduced.

Always have a fire extinguisher on hand . . .

BigBella wrote:You're also going to need to flame sterilize your tools -- forceps, scalpels, etc; don't rely only upon an alcohol soak. Even a small amount of residue, can kill seeds and explants. Add, at the very least, an inexpensive alcohol lamp to your list -- and only use denatured alcohol as a fuel.

Also, buy a large roll of Parafilm to reliably seal the vials after explants, etc. are introduced.

Always have a fire extinguisher on hand . . .

Thanks! Flaming sounds a little extreme. Could I light a candle and just flame that way?

blucksy wrote:Thanks! Flaming sounds a little extreme. Could I light a candle and just flame that way?

Even the best of flames don't get hot enough to kill all bacteria. A combination of alcohol and flame does a decent job, but bacteria containation is still common.

Best to run the tools through a pressure cooker and then alcohol dip and flame them between touching anything with them.

Matt wrote:

blucksy wrote:Thanks! Flaming sounds a little extreme. Could I light a candle and just flame that way?

Even the best of flames don't get hot enough to kill all bacteria. A combination of alcohol and flame does a decent job, but bacteria containation is still common.

Best to run the tools through a pressure cooker and then alcohol dip and flame them between touching anything with them.

I should have mentioned that I store my tools, while working, in a small beaker of 99% isopropyl alcohol and flame them between each use; either that, it use a glass bead sterilizer, but those are generally not used by those just outfitting their labs . . .

blucksy wrote:

BigBella wrote:You're also going to need to flame sterilize your tools -- forceps, scalpels, etc; don't rely only upon an alcohol soak. Even a small amount of residue, can kill seeds and explants. Add, at the very least, an inexpensive alcohol lamp to your list -- and only use denatured alcohol as a fuel.

Also, buy a large roll of Parafilm to reliably seal the vials after explants, etc. are introduced.

Always have a fire extinguisher on hand . . .

Thanks! Flaming sounds a little extreme. Could I light a candle and just flame that way?

Safety first my friend!!! You don't want to earn few bucks and see "Jully 4th" in your house. It costs you hundred thousand dollars. If you want to get rid of "small amount of residue" then sterilize paper towel sheets in a jar and wipe your tools. It costs nothing and no need to buy "alcohol lamp and denatured alcohol"

BigBella wrote:

Matt wrote:

blucksy wrote:Thanks! Flaming sounds a little extreme. Could I light a candle and just flame that way?

Even the best of flames don't get hot enough to kill all bacteria. A combination of alcohol and flame does a decent job, but bacteria containation is still common.

Best to run the tools through a pressure cooker and then alcohol dip and flame them between touching anything with them.

I should have mentioned that I store my tools, while working, in a small beaker of 99% isopropyl alcohol and flame them between each use; either that, it use a glass bead sterilizer, but those are generally not used by those just outfitting their labs . . .

Okay!

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TCMaster wrote:

blucksy wrote:

BigBella wrote:You're also going to need to flame sterilize your tools -- forceps, scalpels, etc; don't rely only upon an alcohol soak. Even a small amount of residue, can kill seeds and explants. Add, at the very least, an inexpensive alcohol lamp to your list -- and only use denatured alcohol as a fuel.

Also, buy a large roll of Parafilm to reliably seal the vials after explants, etc. are introduced.

Always have a fire extinguisher on hand . . .

Thanks! Flaming sounds a little extreme. Could I light a candle and just flame that way?

Safety first my friend!!! You don't want to earn few bucks and see "Jully 4th" in your house. It costs you hundred thousand dollars. If you want to get rid of "small amount of residue" then sterilize paper towel sheets in a jar and wipe your tools. It costs nothing and no need to buy "alcohol lamp and denatured alcohol"

Thanks also! I will probably do this.

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Wiping tools with sterilized paper towel in an some vain effort to avoid contamination is an exercise in absurdity. The primary reason that people get discouraged from TC, is that the advice given is always in half measures and in efforts at jerry-rigging everything. Failures in this hobby are legion; and I have either purchased outright or have inherited lots of equipment of those who have simply given up.

I have either flamed tools or used bead sterilizers for years without the slightest issue; and my contamination rate remains below two percent.

What's the big mystery? Who hasn't used bunsen burners in school and have lived to tell about it?

BigBella wrote:Wiping tools with sterilized paper towel in an some vain effort to avoid contamination is an exercise in absurdity. The primary reason that people get discouraged from TC, is that the advice given is always in half measures and in efforts at jerry-rigging everything. Failures in this hobby are legion; and I have either purchased outright or have inherited lots of equipment of those who have simply given up.

I have either flamed tools or used bead sterilizers for years without the slightest issue; and my contamination rate remains below two percent.

What's the big mystery? Who hasn't used bunsen burners in school and have lived to tell about it?

In my chemistry class I tried using a bunsen burner and I almost died! (sarcasm)

There is no doubt that your sugestion works well; however, the discussion is about safety.
You know that it's not quite safe and it may catch fire so you wrote " Always have a fire extinguisher on hand . . .". Sterilize equipments, soak in alcohol (Matt suggests) then wipe with sterilized paper to get rid of residue is my suggestion. Working in school/lab is quite different than at home: space, safety equipments, instructors/teachers watching/helping... You don't have that luxury at home. Many houses don't have extinguisher. This is the reality: one guy in THIS FORUM blows his glow box by burning alcohol. We don't want to see people get hurt.

We help people in TC . We may have different opinions and we come here for fun.

TCMaster wrote:There is no doubt that your sugestion works well; however, the discussion is about safety.
You know that it's not quite safe and it may catch fire so you wrote " Always have a fire extinguisher on hand . . .". Sterilize equipments, soak in alcohol (Matt suggests) then wipe with sterilized paper to get rid of residue is my suggestion. Working in school/lab is quite different than at home: space, safety equipments, instructors/teachers watching/helping... You don't have that luxury at home. Many houses don't have extinguisher. This is the reality: one guy in THIS FORUM blows his glow box by burning alcohol. We don't want to see people get hurt.

We help people in TC . We may have different opinions and we come here for fun.

Oooh, big text; big text. Being new to the interwebs, that means real knowledge and sincerity, yes?

The thread, had you forgotten or stroked out in the interim, was whether their steps were "adequate for tissue culture" and they were clearly not. In one private communication, their protocol was to sterilize seed in 100 degree Celsius water for twenty minutes or more. Yeah, that'll work . . .

Everyone should have a fire extinguisher; it's common sense safety and they're cheap as hell. I have one in my kitchen as well and have never had to use it -- not that I somehow knew that cooking over an open flame is inherently dangerous. Common sense.

Since you've appointed yourself safety officer, complete with a colorful sash, has anyone here mentioned the real risks associated with pressure cookers? I was once at a UC lab when a steam sterilizer (basically, just a high end cooker) blew and sent playing card sized pieces of shrapnel into brick and flat-wall. Thankfully, everyone was out of the room.

Should we all just wring our hands; microwave and hope for the best? Yeah, a half inch flame is the real issue here.

Sure, I am a madman for suggesting an alcohol lamp, which I have had and used, for one thing or another, since my middle school chemistry kit. Never burned myself or anyone else.

I have trained more people -- successfully -- in tissue culture within the last fifteen years or so, than a bunch of people pulling their puds, and offering "safe" failure-prone half measures.

Dollars to doughnuts, after a few months of green muck in vials and hard-earned monies spent, he'll sell his gear . . .

Different people have different opinions. There's nothing wrong with your opinion and your performance. It's excellent and you got beautiful and rare Heliphora, Cephalotus, Nepenthes. I love them and wish I get them too. It's better to modify the standard procedure to reduce dangerous steps for newbies as much as possible because they don't have much practice and experience like we do. It's another opinion. You don't like my solution: no big deal, no angry. This is the forum. Smile.