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SHJ
Second hand Jesus


Registered: 12/22/15
Posts: 433
Last seen: 8 years, 2 months
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What am I doing wrong?
#23191150 - 05/05/16 05:28 AM (8 years, 8 months ago) |
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Guys... I need your help because I'm getting pretty nervous.
My first grow went relatively well, considering that I inoculated grains straight from spore syringe. After that, and after having learned a few more things, I began to work with agar. Now I'm having a few problems with that and I really need your help to understand what am I doing wrong.
I began to try to isolate some good genetics, but I understood that this wasn't a good idea since I'm new to this and isolates are a bit challenging sometimes. When I understood this, I put a few more spores (cubes) on agar to grow some MS and clone. Tese little bastards refuse to germinate now, but this is another story.
Back to isolates... During the maybe 4th transfer I made my first bad mistake with the agar recipe. The first batch of pasty plates where too liquid, but I didn't recognized that and did the transfers anyway. I didn't trashed the donor plates and the day after I decided to do some more transfers. After a few more days, I noticed the bad consistence of the first plates and trashed them (I wish I didn't however ), thanking God for having done a few other transfers. But, of course, these where the second-choice transfers. I did two or three more transfers since then and then another accident happened: too much ME in the plates. Extra-slow growth. I did some early transfers as soon as I saw some decent growth. Extra-slow recover of course and here we are today.
Growth is not so fast. These are some of the best plates I have now. Today is 8 days since transfer (they come from the extra-ME plates).

And these are a few plates that I trashed today due to bad growth. They are approximately 1/3 of all plates.
Now a few questions.
1 Do you think that this bad growth is due to my bad sterile technique? 2 Do you think it's up to genetic? 3 Is this just normal while isolating? 4 Do you think my best plates are too slow or they are just fine?
Honestly, I would just start over, if only these spores decided to germinate. For now, my best plane is to test plates before I get isolates. My best bet is to clone the first nice cluster that comes out and start from there. I'm confused...
Plus, I'm hating these containers. I cant get rid of condensation. It doesn't matter how slow I go up and down with themperatures, they still have tons of condensation that finally flow my coltures
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SHJ
Second hand Jesus


Registered: 12/22/15
Posts: 433
Last seen: 8 years, 2 months
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Re: What am I doing wrong? [Re: SHJ]
#23191165 - 05/05/16 05:39 AM (8 years, 8 months ago) |
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Also: could it be that I don't change media recipe? Could only something like 8 transfers affect mycelium strongness?
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blindingleaf
blue collar underworld


Registered: 07/19/13
Posts: 22,008
Loc: sub-surface unseen
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Re: What am I doing wrong? [Re: SHJ]
#23191690 - 05/05/16 09:57 AM (8 years, 8 months ago) |
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if it was sterile technique, u'd be seeing satellite colonies of mold, so i think ur procedure is fine.
my guess is ur recipe. either way to stiff agar or too much nutrient like u mentioned.
here are cube plates at 6 days, slightly larger than urs. these are standard nutrient (10g nutrient, 11g agar).

MS at 5 transfers, clone at 4 transfers, and MS at 6 transfers. pretty similar size/speed despite the different genetics.
-------------------- A few thoughts on cultivation
MICROBIAL HUSBANDRY!!!!
The whole is greater than the sum of its parts
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SHJ
Second hand Jesus


Registered: 12/22/15
Posts: 433
Last seen: 8 years, 2 months
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Uhm... My recipe is 2% agar and 2% ME. I'm wondering if my particular ME is somehow too nutrient? today I'm gonna buy some dextrose and do some PDA or maybe PYDA. Let's see what happens. Thank you
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SHJ
Second hand Jesus


Registered: 12/22/15
Posts: 433
Last seen: 8 years, 2 months
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Re: What am I doing wrong? [Re: SHJ]
#23197782 - 05/07/16 02:55 AM (8 years, 8 months ago) |
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I finally discovered that I was using ME but not the light one. Now I'm sterilizing a batch of plates with 2% of LME and in the same time a few cotton swabs in a jar. Let's see if this way I'll get some germination from my plates.
Also, I forgot to mention that I was using ME for shiitake and pioppino too, and these two, especially shiitake, are doing great! This is strange because I always read shiitake is slow. I guess I've got a good genetic from the clone I took...
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