Puffin said:

Quote:

BeefSupremeJr said:fascinating.  do you have your own thread? id like to follow along.  im interested to see what youre up to.  also i cant have you going around unrated pardner

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My ambition is to create novel hybrids using protoplast fusion. For the challenge and adventure more than anything.

The first stage is to isolate monokaryotic cultures for as many Psilocybe species as possible. I started with subaeruginosa and natalensis today; creating spore solutions, counting spores under the microscope, doing serial dilutions to make a solution with ~25 CFUs per 0.1ml (single spores or clumps is how I'm defining that), and spreading it onto plates. I'll be watching these for single spore germinations and doing early transfers, then proving monokaryotic status with a microscope. I've got spores for hispánica, ovoideocystidiata, cyanescens, azurescens and cubensis up next.

The stage after this is to generate auxotrohpic strains for each monokaryotic culture using UV mutagenesis. Auxotrophs are mutants that can't create a certain nutrient so rely on it being in their environment to survive. The idea is that each parent has a different auxotrophism, then after protoplast fusion (forced mating essentially), when the resulting cultures are cultured on minimal media, the only ones that will survive and grow are hybrids, with the working gene from the other parent for to fix their auxotrophism. Self mated parents do not survive do not survive, so it's a way to find 'the needle in the haystack' when a hybridisation does occur.

I've started writing it up in my shromery journal but it's all very early days and most things are super draft. This will be the main thread at the moment:
Isolating Monokaryotic Cultures, but it is part of a bigger series.

I'll write up today's process in there at some point tomorrow and start using that as a log.

I can DM you any time anything notable happens, if you're interested?

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Puffin said:

The stage after this is to generate auxotrohpic strains for each monokaryotic culture using UV mutagenesis. Auxotrophs are mutants that can't create a certain nutrient so rely on it being in their environment to survive. The idea is that each parent has a different auxotrophism, then after protoplast fusion (forced mating essentially), when the resulting cultures are cultured on minimal media, the only ones that will survive and grow are hybrids, with the working gene from the other parent for to fix their auxotrophism. Self mated parents do not survive do not survive, so it's a way to find 'the needle in the haystack' when a hybridisation does occur.

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Synack said:

Most of my otherwise healthy-seeming plates have this ring of shiny growth on the leading edge. Is this a bacterial "halo" that you guys talk about or is it just what happens on the leading edge of growth? 




This is a transfer from a vendor LC test plate which has been going for a few weeks and still shows no obvious sign of contam (bacterial or otherwise). The transfer was made in front of a LFH with an alcohol-soaked and then freshly torch-flamed agar punch and scalpel while wearing gloves and mask. I have a bunch of other similar test plates and T1/T2 transfers that are from different LC but still look like this. If I'm still picking up bacteria I'm struggling to figure out where, so I'm really hoping this is just paranoia....

Tell me it's all going to be ok!

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misterswisher said:

Synack said:

Tell me it's all going to be ok!

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Looks healthy to me. That “halo” seems like temp change imo. It also does grow out thinner and get thicker. Bacteria is usually on top of the agar. It doesn’t burry itself in so if you look at the plate at an angle you can usually see bacteria colonies bulging out of the agar.

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Puffin said:

My ambition is to create novel hybrids using protoplast fusion.

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misterswisher said:

Looks healthy to me. That “halo” seems like temp change imo. It also does grow out thinner and get thicker. Bacteria is usually on top of the agar. It doesn’t burry itself in so if you look at the plate at an angle you can usually see bacteria colonies bulging out of the agar.

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Synack said:

Most of my otherwise healthy-seeming plates have this ring of shiny growth on the leading edge. Is this a bacterial "halo" that you guys talk about or is it just what happens on the leading edge of growth? 




This is a transfer from a vendor LC test plate which has been going for a few weeks and still shows no obvious sign of contam (bacterial or otherwise). The transfer was made in front of a LFH with an alcohol-soaked and then freshly torch-flamed agar punch and scalpel while wearing gloves and mask. I have a bunch of other similar test plates and T1/T2 transfers that are from different LC but still look like this. If I'm still picking up bacteria I'm struggling to figure out where, so I'm really hoping this is just paranoia....

Tell me it's all going to be ok!

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cronicr said:

...after you're next transfer taste the old plate it should taste like mushrooms and not a bitter sweet lol

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cronicr said:

But I'm half buzzed , am I tripping or does the growth itself look suspect?

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cronicr said:

The halo is fine , the powdery unorganized look seems a bit off but Nats can be strange , after you're next transfer taste the old plate it should taste like mushrooms and not a bitter sweet lol

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This is definitely trolling, right?

paff said:

cronicr said:

But I'm half buzzed , am I tripping or does the growth itself look suspect?

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It's looking a little like how Trich would grow out.

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cronicr said:

Naw not even a bit lol, clean plates taste great.

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cronicr said:

I agree it looks powdery compared to my other cultures. I assumed this was a "feature" of Ochras/Nats, or at least of this lineage of them?

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cronicr said:

Naw not even a bit lol, clean plates taste great.

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MEA plates are just sweetened agar, so I expect it would taste a little like shitty jello. However, I am less optimistic about the taste of the myc. The fruits taste like shit, why would the myc be any better?