(Stranger)

05/11/08 08:56 AM

Tissue culture

Hi everyone,

I am new member here.Hopefully to learn a little about tissue culture of Cordyceps Sinensis and its cultivation.I see so many expects here, hope someone will share some knowledge with me.

mak

(Crazyness)

05/11/08 09:07 AM

Re: Tissue culture

Welcome to the shroomery

im no expert in tissue culture, but I think it involves sterilizing the tissue with something mild like peroxide, but other then that I know nothing.

legallyhomeless

(mooch)

05/11/08 09:45 AM

Re: Tissue culture

To clone from a piece of tissue you will need some agar work done. You would clean the piece of tissue in h2o2. Then give it a couple h2o baths and then place it on some agar. You would have to transfer it a few times because of contamination.

(MysticalPhysician)

05/11/08 10:32 AM

Re: Tissue culture

h2o2 will hurt your sample. It will slow the growth of the mycelium. It is used to treat the casing layer in dire cases of contamination but it ALSO hurts the mycelium.

If a contaminate is present it will be evident on the Agar, so cloning to agar gives you the opportunity to isolate mycelium growth AWAY from a contaminate. Using H2O2 will slow the growth of the mycelium because the mycelium needs time to recover from the H2O2 bath and ideally we would want the mycelium to out pace any contaminate in an agar dish.

If an H2O2 bath were a guarantee of sterility than we would probably clone by just doing an H2O2 bath and then go directly to LC . . . of course doing that would just get you a dirty LC so instead Common Practice is Agar Work to guarantee clean clones.

I'm sure a lot of people do okay using H2O2 . . . but it just slows things down and if too much is used you just killed your grow. Read up on Agar and do some isolating.

OR you could attempt a LC in a glove box. Don't cut your sample out of the fruit body outside the glove box and be as sterile as possible.

(Mycologist/Fisherman)

05/11/08 10:37 AM

Re: Tissue culture (moved)

This thread was moved from Mushroom Cultivation.

Reason:
this is better suited for the Gourmet and Medicinal Mushroom forum.

legallyhomeless

(mooch)

05/11/08 06:53 PM

Re: Tissue culture (moved)

I never said it would make the sample steril. It will CLEAN the sample. The h20 baths after wards wash away the h202. It slightly hurts your mycelium but I dont think RR is wrong.

(Stranger)

05/13/08 08:30 AM

Re: Tissue culture (moved)

Hi all,

I am a bit mixed up with sending question to this forum as I just a newbie here but disinfect mushroom's explant and then put into a right medium for growing is not consider tissue culture anyway ?

Thanks,
Mak

()

05/15/08 08:54 AM

Re: Tissue culture (moved)

Hi Mak

Not sure if you want help on tissue culture or cloning. Mostly cloning involves taking a clean (generally use the inside of a mushroom near to where it forms the spores, as this is the most genetically diverse part), piece of tissue from a mushroom that is already growing and then allowing it to grow on agar medium. The simplest way to get it clean is to clean the outside and then rip it open being sure not to touch inside. With a sterilized needle take/cut a small piece out and place on agar medium. If you see contamination (generally any green or other colored growth ) you will need to do further isolations until you end up with a clean culture. Once it has grown out and is only mushroom mycelia. After this you can start to reproduce it either in grain masters/suitable spawn material and then grow it on a fruiting substrate.

Also remember that the more clones you make the weaker the mushroom strain becomes. To avoid this you can make a large quantity of isolations from your first mushroom and store these with longterm storage like sterile water or oil slants. Then when your strain starts becoming "weak" you simply start the strain again with one of your cultures in storage.

I am not too sure of Cordyceps but try Stamets "growing gourmet and medicinal mushrooms" probably the best book to start with.

The above procedure assumes that you have access to some lab equipment, if not you will have to improvise and see what works best for you.
ciao

(Doctor of FoolyC<wbr>ooly)

05/15/08 06:47 PM

Re: Tissue culture (moved)

Quote:

solumvita said:
Hi Mak

Not sure if you want help on tissue culture or cloning. Mostly cloning involves taking a clean (generally use the inside of a mushroom near to where it forms the spores, as this is the most genetically diverse part), piece of tissue from a mushroom that is already growing and then allowing it to grow on agar medium.

When cloning you usually DON'T want genetic diversity. A clone usually is a exact copy of the genetics of the mushroom being used. Any part of the mushroom would work for cloning since all the tissue that makes up the mushroom is mycelium in a different form. Any piece will go back into vegetative form once placed on agar.

If you want genetic diversity start from spores.

()

05/18/08 03:09 PM

Re: Tissue culture (moved)

Absolutely, a clone is a clone. The point I am trying to make is that the best place to use (in cloning) and which may help in strain degradation is to use parts from the reproductive tissue. This is the region with the greatest diversity in the mushroom. Hopefully that makes what I am trying to say clearer? If not let me know.

(Bans forPleasure)

05/18/08 06:41 PM

Re: Tissue culture (moved)

What are you using to get the sample? If it's a dried C sinesis, you'll have a very hard time getting live tissue, but you can grow from spores.

Cordyceps spores can withstand what no other fungi spore I know of can. If using a dried fruit, wash it well with clean water, and then wash well with 70% isopropyl alcohol. Rinse again with clean water, and then drop the fruit into a 10% bleach solution for fifteen minutes. After that, slice into 1mm sections and place on insect agar using sterile technique. Simply blend up 20 or so crickets in 1 liter of water and add 20 grams of agar agar prior to sterilization.
RR

(Fruiting)

05/19/08 02:46 AM

Re: Tissue culture (moved)

Quote:

RogerRabbit said:
Simply blend up 20 or so crickets in 1 liter of water and add 20 grams of agar agar prior to sterilization.
RR

That Sounds NASTY

cricket shake, ahhh

(doofing biologist)

05/19/08 09:31 AM

Re: Tissue culture (moved)

Ever been to South-East Asia? They love deep fried crickets there!