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croyleje



Registered: 08/22/22
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Question about pairing haploids?
#27948546 - 09/13/22 06:27 PM (1 year, 4 months ago) |
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I have managed to isolate several haploids by using the serial dilution method and then streaking the solution on plates. This is my first venture into advanced mycology and I have selected the strongest isolates (Not sure if this is the correct term.) and moved those to a separate plate my question is when the two haploids grow together assuming they are compatible is it only the point after which the two have grown together that contains the mated genetics or is it like an embryo is there a chemical reaction or something that takes place making the whole mycelium plate genetically the same? The reason I ask is I sectioned the plate and failed to label things well and now am unsure which pieces came from where on the plate. Will I just have to grow them all out and see what fruits and what doesn't? I guess I maybe able to tell once they start growing on the grain I assume the single line genetics will grow slower then the mated line. Again sorry I am sure I am using the wrong terms in the wrong places. I don't expect much from this experiment but if I can consistently do this I am hoping to be able to make my own hybrid (Probably won't be any good but I want to try.).
EDIT: I may be using the wrong terms in wrong place but I believe I have the basic method down. And maybe my question is just me over thinking or maybe I am an idiot? I maybe asking the question wrong, my thought is only the growth after the mating point would be viable but this then raises the question of how to tell it has mated and not just grown over the other?
-------------------- Learn Linux and cut your ties to big tech, remember if a service is provided to you for free you and your data are the payment. https://www.fsf.org/
Edited by croyleje (09/14/22 02:44 PM)
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Farm3r
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Re: Question about pairing haploids? [Re: croyleje]
#27950796 - 09/15/22 06:26 AM (1 year, 4 months ago) |
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If you didn't use a hemocytometer and don't have a microscope you probably did not.
Sadly Gary at fresh farm fungi started this with his stupid breeding series....
If you want to isolate monokaryotes/haploid cultures, there's a single spore isolation Tek on my YouTube.
To mate haploid cultures you simply put them on the same plate. If they're the same species they will mate.
Check out my channel, reach out for help, suggest content. Thanks.
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PsychdelicSpore
Funginner


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Re: Question about pairing haploids? [Re: croyleje]
#27951182 - 09/15/22 11:21 AM (1 year, 4 months ago) |
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Wumbo goes over this on YT. He and a few others on YT that talk about it. Mycogeek is another he was on. Hope this helps. 👍
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Farm3r
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Does wumbo have a hemo and verify clamp connections? Wumbo also has a kit that suggest you can breed fungi like bacteria, and doesn't seem to have proof that it works. Much like Doma at magic Myco.
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croyleje



Registered: 08/22/22
Posts: 122
Last seen: 1 year, 13 days
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Re: Question about pairing haploids? [Re: Farm3r]
#27951715 - 09/15/22 04:52 PM (1 year, 4 months ago) |
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Thank you for pointers and a place to start and your correct about where I saw the original process I will check out your channel and see if I can work it out from there and will definitely reach out after I take it all in. I am fairly certain the spore isolation worked but a few of the terms you used I am not familiar with but will watch your videos and and see what I can learn from there.
I have no issue putting out the money to purchase the needed equipment but didn't want to until I knew exactly what else I would need and thank you for the information I look forward to seeing the videos.
-------------------- Learn Linux and cut your ties to big tech, remember if a service is provided to you for free you and your data are the payment. https://www.fsf.org/
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Muad.Dweeb
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Re: Question about pairing haploids? [Re: croyleje]
#27952468 - 09/16/22 07:37 AM (1 year, 4 months ago) |
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you can't be absolutely certain that you have a monokaryon without a microscope.
However, you don't need a hemocytometer to do serial dilution. It's a great tool for knowing what your starting population is, roughly, so that you can project the expected population in successive dilutions, but without that you can just streak each dilution factor to calibrate.
Spore clumping is a real issue, however, and the main one to overcome with or without the microscope and hemocytometer. The spores are coated in fungal sugar to help get them started after germination, which makes a lot of them stick together. Some people have used TWEEN, or surfactants, or even a small amount of ethyl in the initial suspension to break up the spores. But the single most effective thing to do is shake the holy living shit out of them. 10 minutes minimum. A shaker made from a back massager is a good option, too.
Without a scope, the closest you can get to a confirmation of a mono/haplont is allowing a colonized plate to consolidate and wait for pinning (pinning means not monokaryon), and pair it in a plate with a clone culture. Pinning from the mono side of the paired plate, with no pinning on the mono plate is a very strong indicator that the monokaryon is in fact monokaryotic.
Now, when two compatible haploid cultures meet and undergo plasmogamy, each half copies the new nucleus that came in at a very rapid rate. The new nuclei then migrate through the septa in the hyphae to populate all the previously monokaryotic cell compartments. When this process is complete, the new growth on both sides will be dikaryotic, forming clamps between cells. When this happens, there is a marked change in the morphology of the new growth. It will get more dense, likely colonize faster, grow in a more uniformed pattern...
An entry level microscope in the range of 200-300 dollars would be acceptable for viewing cultures to see clamping, etc...
-------------------- A beginning is the time for taking the most delicate care that the balances are correct. This every sister of the Bene Gesserit knows.
How to Breed like the Bene Gesserit The Weirding Way - Advanced Bene Gesserit Techniques
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Farm3r
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Re: Question about pairing haploids? [Re: Muad.Dweeb]
#27952478 - 09/16/22 07:48 AM (1 year, 4 months ago) |
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You can use tween 20 or polysorbate 20 I think it is as a detergent to keep the spores from sticking together. I think I used it at about 1% maybe less.
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croyleje



Registered: 08/22/22
Posts: 122
Last seen: 1 year, 13 days
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Re: Question about pairing haploids? [Re: Farm3r]
#27953154 - 09/16/22 04:42 PM (1 year, 4 months ago) |
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I saw the polysorbate 20 mentioned in one of your youtube videos also, what ratio do you normally use. Is it like using Jet Dry where you only need a drop of two per 100ml? The only thing I could find online was for stabilizing homemade cosmetics and it was suggested at a 1:1 ratio which is way to high for what we are using it for.
I will have to look into Tween not familiar with that one.
-------------------- Learn Linux and cut your ties to big tech, remember if a service is provided to you for free you and your data are the payment. https://www.fsf.org/
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croyleje



Registered: 08/22/22
Posts: 122
Last seen: 1 year, 13 days
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Re: Question about pairing haploids? [Re: Muad.Dweeb]
#27953173 - 09/16/22 04:50 PM (1 year, 4 months ago) |
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Thank you for the information I appreciate it and am hopeful of future success. I started my search for a new microscope and am getting ready to upgrade my lab area to be a little more professional. I already have a FFU but have ordered a new larger setup to allow coverage of my whole bench and use my older 2x2 unit for a microscope station.
-------------------- Learn Linux and cut your ties to big tech, remember if a service is provided to you for free you and your data are the payment. https://www.fsf.org/
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Farm3r
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Re: Question about pairing haploids? [Re: croyleje]
#27953249 - 09/16/22 05:36 PM (1 year, 4 months ago) |
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"The M38-A document recommends the use of a drop of Tween 20 per ml (approximately 0.01 to 0.02 ml, 0.5 to 1%) (17) for inoculum..."
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1081276/
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croyleje



Registered: 08/22/22
Posts: 122
Last seen: 1 year, 13 days
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Re: Question about pairing haploids? [Re: Farm3r]
#27954697 - 09/17/22 02:34 PM (1 year, 4 months ago) |
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I had not thought about using research papers and doctoral thesis as source but these are full of excellent information thank you for the link. There are complete discussion and papers written about preparing spore suspensions, calculating CFU and spore count. Thank you.
-------------------- Learn Linux and cut your ties to big tech, remember if a service is provided to you for free you and your data are the payment. https://www.fsf.org/
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Farm3r
Human



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Re: Question about pairing haploids? [Re: croyleje]
#27955583 - 09/18/22 04:54 AM (1 year, 4 months ago) |
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When doing research, after entering you question into Google, add "Shroomery" or ".edu .org.
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