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OfflineHiroller
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Registered: 09/23/00
Posts: 29
Last seen: 14 years, 7 months
Live Tissue Culture
    #406899 - 09/27/01 11:30 PM (15 years, 2 months ago)

I recently decided to to start a live tissue culture from a mature mushroom. I split open a mushroom and cut out the tissue from the inside an dplaced it on a culture plate. Unfortunately, it's haas been two weeks not and the growth is little. I'm not sure if the strain is old or if I'm just being impatient. The mushroom was a third flush mushroom but the jars were spore incoulated. Any ideas or suggestions?



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OfflineAbudiwa
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Registered: 08/17/01
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Re: Live Tissue Culture [Re: Hiroller]
    #406942 - 09/28/01 12:01 AM (15 years, 2 months ago)

HaHaHa

I thought you were going to post something about injecting spores into your bloodstream or something. funny.

Ok, to your post:
1) What medium are you using as culture
2) How old is the mushroom? and Is it fresh or dry?
3) How firmly did you push the piece of cap into the medium?
4) How clean were you? (That sounds dumb, but think about it...) It requires as much if not more sterilization as innoculating jars, because the medium is more nutritious than a substrate in a jar. This begs for contamination.
5) Average temperature of dishes?

That's it... Although I havn't utilized this method before, I'm starting some PDY broth jars either sat or sun using some mycelium from my colonized jars that are going to be cased this weekend too. I have read the tek i'm pretty sure you are using so many times I could recite it from memory... So I know it very well.

cheers,

abudiwa

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OfflineMattyB
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Registered: 10/13/00
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Re: Live Tissue Culture [Re: Hiroller]
    #406996 - 09/28/01 12:41 AM (15 years, 2 months ago)

Two weeks on a culture plate without growth means you need to try again. Are you using agar? You didn't mention what you were using to grow the live tissue on. Also, did you only prepare one plate? If so, you should always do three or four since not all of them will grow. If you fail with agar, you can grow out live tissue on regular substrate as well. Just prepare the substrate, pack it into a jar (or even a petri dish) and using a scalpel, press tissue from the cap into the substrate. Cover it and watch it grow...



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OfflineHiroller
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Registered: 09/23/00
Posts: 29
Last seen: 14 years, 7 months
Re: Live Tissue Culture [Re: Abudiwa]
    #407807 - 09/28/01 08:34 PM (15 years, 2 months ago)

I used Bob's Malt Agar from the Mushroompeople. It's made from malt(never could have guessed huh? =) ), cornsteep, Calcium Hydroxide, and Potassium Phosphate. The mushroom had just been picked when I placed it on the original agar but then I transfered it to isolate the strain from contaminates that began to appear on the plate. As for pushing it into the agar, I never knew I had to something as such. I usually just place the agar wedge or tissue on top of te agar. I typically leave it incubated right around 84 to 86 degrees, never going above 86 and not below a low of 82.
TO give you a better idea of what has happened, I transfered the 3 wedges from the orginal plate to 3 different plates. I kinda left them alone for two weeks and when I checked them the mycelium had barely grown off the transfered wedges and onto the new agar.
Does pushing the wedges/tissue really matter that much? I mean, it could be that or maybe the agar isn't good for cubes. When I tried to do strain selection with spores, the spores grew into mycelium, but the mycelium was rather weak looking and again didn't grow that far.
PLUR



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OfflineHiroller
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Registered: 09/23/00
Posts: 29
Last seen: 14 years, 7 months
Re: Live Tissue Culture [Re: Abudiwa]
    #407808 - 09/28/01 08:36 PM (15 years, 2 months ago)

I used Bob's Malt Agar from the Mushroompeople. It's made from malt(never could have guessed huh? =) ), cornsteep, Calcium Hydroxide, and Potassium Phosphate. The mushroom had just been picked when I placed it on the original agar but then I transfered it to isolate the strain from contaminates that began to appear on the plate. As for pushing it into the agar, I never knew I had to something as such. I usually just place the agar wedge or tissue on top of te agar. I typically leave it incubated right around 84 to 86 degrees, never going above 86 and not below a low of 82.
TO give you a better idea of what has happened, I transfered the 3 wedges from the orginal plate to 3 different plates. I kinda left them alone for two weeks and when I checked them the mycelium had barely grown off the transfered wedges and onto the new agar.
Does pushing the wedges/tissue really matter that much? I mean, it could be that or maybe the agar isn't good for cubes. When I tried to do strain selection with spores, the spores grew into mycelium, but the mycelium was rather weak looking and again didn't grow that far.
PLUR

P.S. I was going to try to inject myself with some spores but I caught a glimpse of a picture on this website of a shaman with mushrooms with fingers. I thought that would be cool until I realized I would have family and friends gnawing on my hands, Heh



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OfflineMattyB
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Registered: 10/13/00
Posts: 238
Last seen: 3 years, 11 months
Re: Live Tissue Culture [Re: Hiroller]
    #408006 - 09/29/01 03:18 AM (15 years, 2 months ago)

Did you let the agar cool completely before placing the tissue on it? You may need to experiment with different incubation temps. I've successfully grown out tissue on agar at room temperature as well as 84F. Try it again and see what happens. If at first you don't succeed...



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