|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
The Weirding Way - Advanced Bene Gesserit Techniques 11
#27958711 - 09/20/22 09:06 AM (1 year, 7 months ago) |
|
|
ʬʬ ₸he Ѿeirding Ѡay ʬʬ
ϾϿ
Advanced Tools and Techniques of the Bene Gesserit for Manipulations in the Breeding Programme

[Weird- from Old Norse wyrd] The Weirding is a seemingly mystical power of select groups of people in Frank Herbert's “Dune.” Dependent upon highly developed mental and physical discipline, and sometimes the spice melange, the abilities of the Bene Gesserit- and those they train- allow them to stealthily control and manipulate individuals as they need, without detection, to orchestrate their grand breeding programme, to bring about the Kwisatz Haderach. To the Bene Gesserit, these abilities are described as Prana Bindhu control, and range from teleportation to transmutation. This section of Breed Like the Bene Gesserit contains things that, at first glance, may sound and look weird or magical. I assure you, they are not. With exceptional discipline and rigorous application, you too can learn how to levitate spores, control conception, transmute cultures, and reduce them to their base components. While attempting these procedures, you will fail. If this is because of constant contamination, you are not ready for these procedures, and need to return to developing basic cultivation skills. If not, take some Sufi advice and rejoice that you get to experience the freshness of the nubile state once more. Where possible, I will try to offer theories of the mechanisms, both personal and with links to published studies, as well as my personal notes of my own learning curve with these methods. Some will be rather involved, and I understand someone (many people) not wishing to get so involved. That is okay. Breeding isn't for everyone, and this section isn't for all breeders. It's fine to throw spaghetti at the wall, just to see what sticks- and believe when I say that you can find strange and wonderful things that way- but for the select, spaghetti art isn't enough. For the select, we judge our success by a self-directed vision, and how closely we can make reality match that vision. This is our secular magic, our Godless religion. This is our Art. Fortuna Juvat Perseverantiam (Fortune Favors the Persistent)Teks and Explainations:* Spice Agony, 1: Symmetrical Dedikaryotization* Fremen Tools, 1 - Induction Sterilizers* Trial by Gom Jabbar, 1: Trich Farming* How to Swap Mitochondria
Edited by Muad.Dweeb (01/30/23 01:13 PM)
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb] 1
#27958714 - 09/20/22 09:07 AM (1 year, 7 months ago) |
|
|
Symmetrical Recovery of Neohaplonts from Dikaryon Fragmentation
Sources: A Patent
PDF below Abstract
A more recently published use of this technique
My Notes
A combination of previously outlined procedures and tools will be utilized. The primary benefits of this approach are recovery of both monokaryon types, with low mutation and damage potential. It can be thought of in two phases: the initial phase, in which the dikaryotic culture is minced, and the recovery phase, in which colonies are regenerated from the minced culture.
Quote:
Materials:
Dedikaryotizing Broth (“DB”); 2% Dextrose, 2% Peptone, sterilized 15 min. Osterizer Water agar Nutritional agar Inoculation loop Syringes or pipettes 15mL Centrifuge tubes with 9mL of sterile water, each
Set up:
1. First, assemble the Osterizer jar. If using a Waring blender, sterilize all parts that will contact the liquid and culture. With the Oster blender, I recommend grinding or filing down the blades so they are thinner, and using a polishing stone to smooth the blade edge before sterilizing. It's not necessary, but at least polishing the blade edge will reduce cell damage to the mycelium.*
* [ETA] ...actually, a fresh blade has proven ideal. A polishing stone to smooth out knicks is fine, but the best results have come from the newest, un-modified blade base I have.
2. Prepare the dedikaryotizing suspension broth in the Osterizer jar with 2% dextrose and 2% peptone. Sterilize 10-15 minutes. With the half pint jars, *100mL* is a good amount.
3. Generate clean liquid culture from agar. I recommend using the EzLC method, but any traditional method will suffice. The starting material should be dikaryotic. If blending agar cultures, they should either be soft agar (1.5%-1.75%) to ease blending, or stiff agar (2.5%-3%) to make the culture easy to scrape off the surface.
Procedure:1. Add a square of colonized agar approximately 30mm (or 10mL of LC) to the Osterizer. 2. Blend for 15-20 seconds, pulsed in 5 second increments (to reduce heating the culture). An emulsion will form, which will help to cushion and partition the cleaved hyphal compartments. The dextrose will slowly settle back into solution after a short while. 3. Incubate in situ 3-4 days at room temperature. Gently swirl the culture periodically, to prevent clumping. If new growth hasn't formed within this time frame, start over with a lower blending time, sharper blades, or both. 4. Blend for 5 seconds. This is only to fragment new growth, not completely macerate the culture. 5. After the emulsion settles, swirl well and add 1mL to a centrifuge tube with 9mL sterile water.  6. Agitate thoroughly for 10 minutes and streak on water agar or low nutrient agar (<1%). Alternatively, 1mL can be drawn up and a single drop can be added to several centrifuge tubes with ~9mL of sterile nutrient broth or water. The colonies in these tubes will grow into spheres of monokaryotic mycelium, which can be fished out and minced/streaked separately.  7. Incubate at room temperature until visible colonies form (~1mm). With an ultra fine marker, circle potential isolated colonies with at least a centimeter of isolation, and inspect the colonies under a microscope. Check each colony for absence of clamps, and absence of minute proximal colonies not casually visible. Young, isolated colonies should be considerably circular, with frequent branching of hyphae in a “peg” style. Note on the plate where there are any nearby colonies to avoid, and transfer them methodically, with great care to absolutely minimize sample size. [clamps highlighted with red]
   8. Transfer colonies into isolation on nutrient agar and label them in series (1, 2, 3, etc...) When they have grown enough to transfer from, set up a series of comparison plates with 1x2, 1x3, 1x4, etc...  9. The first recovered neohaplont will arbitrarily be labeled nuclear type A. Colonies that fuse with “1” and do not produce clamps are nuclear type A, while colonies that do form clamps after growing together are nuclear type B. For further personal confirmation, fruit the A/B cultures on a fruiting medium and observe the original fruit phenotype.    10. Label the corresponding nuclear type to the cultures, and save redundant axenic samples of both nuclear types on slants. Notes:This is a “One Pot Recipe,” if you will, designed to minimize the potential for contamination and maximize dedikaryotization. The most accessible materials possible were used to execute this procedure with repeated success (with P. cubensis) with high degree (approaching 100%) of dedikaryotization. Minor fine tuning as needed per specific strain is to be expected. Entirely different tolerances may be required for other species, but this technique should prove a good starting reference. Many published research papers using this or a similar technique call specifically for peptone type P “Oxoid” and glucose. However, standard bacteriological peptone from Hi-Media was used, and the very available dextrose is the d-glucose enantiomer. Potentially, 2% nutrient yeast could be substituted for peptone, as well. Additionally, great results have been obtained in many cases using only water in place of any nutrient broth, though colony growth is minimal. The key points to perfect are blending time, and a proper dilution after the second blending. This is a complex procedure. Always double check the work and the tek while working. Several attempts may be required for success.
Edited by Muad.Dweeb (10/04/22 11:47 AM)
|
Rotnpins
🤮 Rotten-Pins 🍄



Registered: 01/11/22
Posts: 4,738
Loc: in (front of) the hood
Last seen: 1 year, 4 months
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb]
#27958730 - 09/20/22 09:23 AM (1 year, 7 months ago) |
|
|
I've read through your other thread a couple times, good stuff thanks for sharing
|
DERRAYLD
Constructus


Registered: 05/13/02
Posts: 10,170
Loc: South Africa
Last seen: 12 hours, 11 minutes
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Rotnpins]
#27958736 - 09/20/22 09:27 AM (1 year, 7 months ago) |
|
|
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: DERRAYLD]
#27958755 - 09/20/22 09:51 AM (1 year, 7 months ago) |
|
|
"approaching 100%" I should qualify...
Read my notes (linked), and the "recovery rate" was 12/23, or 52.17%, which is in the range of other published results. Of those 12, identification as a mon was possible before pairing 100% of the time.
It's always a good idea to keep any old plates around while doing this for a few weeks at least. However, with the results I have been obtaining, the timeline and response of paired mycelium is pretty straight forward. When simultaneously, some plates start forming clamps, while their controls and other plates do not, you can confidently fill in that sudoku box.
-------------------- 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
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb] 4
#27959002 - 09/20/22 01:40 PM (1 year, 7 months ago) |
|
|
Induction SterilizersA couple years ago, I did a write up in the Facebook Shroomery group on Induction Sterilizers. I wasn't exactly the first person to come up with the idea, but after I made the post, a bunch of people were suddenly making the cheap, plug-and-play version I offered to people who lack solder skills and just want one as cheap as possible. And you wouldn't believe the outrageous prices people are charging/paying for a handful of chinese parts, a box and a foot pedal. So here is how you make your own. You'll Need:A ZVS Module: https://www.amazon.com/s?k=ZVS+module&crid=B9UO1MP6O3D6&sprefix=zvs+module%2Caps%2C223&ref=nb_sb_noss_1A Beefy Power Supply (12v to 24v, 10 to 20 amps) I'm partial to the Alitove brand (not affiliated): https://www.amazon.com/s?k=12v+power+supply+alitove+10A+20A&crid=3IL88KFC5CP06&sprefix=12v+power+supply+alitove+10a+20a%2Caps%2C167&ref=nb_sb_nossA Beefy Foot Pedal like this: https://www.amazon.com/Twidec-Momentary-Nonslip-Handsfree-Industrial/dp/B07XXWN331/ref=sr_1_17_sspa?keywords=foot+switches&qid=1663696911&s=industrial&sr=1-17-spons&psc=1Some connector crimps (optional, if you're adding extra wires): (you can get these from any hardware store really...) https://www.amazon.com/Connectors-Electrical-Connector-Insulated-Terminals/dp/B09FG49BZX/ref=sr_1_27?crid=24X3TXTF3DCTC&keywords=crimp+connectors&qid=1663697047&s=industrial&sprefix=crimp+%2Cindustrial%2C188&sr=1-27Optionally, you can put this in a project box to tie it all together neatly: https://www.amazon.com/s?k=project+box&i=industrial&crid=CA5XDGWX8VDJ&sprefix=project+box%2Cindustrial%2C190&ref=nb_sb_noss_1*** For the ZVS module, the 5-12v is plenty and you'll be able to sterilize your scalpels in 5-10 seconds without generating a whole lot of extra heat. Bonus, they always come with a pre-made coil, so that's one less thing to worry about. If you want the instant sterilizing of the 1000W ZVS modules, make sure you get one that comes with the coil, as a lot of them don't. Without the coil, you'll also have to get some 12g or 14g magnet wire, or thin copper tubing to make your coil.  If you can solder, soldering the coil directly to the large contact pads is preferable to the plastic screw-down terminals installed for the coil. Over time and with extended use, the terminals themselves will warp and fail. The screw-down terminals for the power supply are perfectly fine to use, though, as they do not conduct much heat.  For the power supply, the minimum/maximum requirements will usually be printed on the module PCB, or even in the description of the listing. The bare minimum you'll need is 12v that can deliver 10 amps (12v 10a), and that will drive the 5-12v ZVS just fine. The maximum for the 5-12v ZVS is 20 amps, but at 12v, that will probably burn out pretty fast. I would cap it at 15 amps for the small guy. For the 1000W module, 12v 10a is going to be the lower end of effective power, and up to 24v 30a could be used, but again it's likely to burn out sooner if you are pushing the higher values.  I like the Alitove (A Lite of Love) power supplies, personally, and they come with a screw-down adapter that makes this whole thing very seemless without buying extra parts. You need two small pieces of wire that connect + to + and – to –, then the power supply just plugs in. To add the button or foot switch, we just put it place of the wire between the + of the connector tie downs. Now, power only comes in when the switch is closed. *DO NOT POWER UP THE MODULE WITHOUT THE COIL ATTACHED* That is an excellent way to burn up the mosfets.  If you want to add a project box, some additional layout planning and drilling/cutting is required, maybe some epoxy gluing too. But that's really all there is to it.  So let's add all that up. Under 10 for a project box 10-40 dollars for the ZVS module 20-40 dollars for the power supply 5-6 bucks for crimps (maybe less) 5-10 clams for a switch That's 50-106 USD at the time of writing this in parts to have something that sterilizes blades on the low end, all the way to melting them on the high end. There are people out there using the 50 dollar option to sell for 150+. Don't be a sucker. Now if you're paying someone for one purely for the convenience or their craftsmanship, then fine, pay what you feel is fair. But know what you're getting. And how much more personal making one yourself can be.    [custom inductor skulls I have made. Do not message, I don't sell these.]
-------------------- 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
Edited by Muad.Dweeb (09/20/22 01:48 PM)
|
smalltalk_canceled
Babnik



Registered: 07/13/20
Posts: 2,906
Last seen: 7 hours, 10 minutes
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb]
#27959115 - 09/20/22 03:13 PM (1 year, 7 months ago) |
|
|
-------------------- Willpower is the one true virtue
  
|
The Blind Ass
Bodhi


Registered: 08/16/16
Posts: 27,994
Loc: The Primordial Mind
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb]
#27959416 - 09/20/22 06:17 PM (1 year, 7 months ago) |
|
|
Some things are so good they hurt because we may lack the proficiency to express our feelings regarding them. Almost like the life of a chair dog but not quite as horrific and minus all the good.
Anyways. This thread is one of those things, but with all the good and no chair dog... 
-------------------- Give me Liberty caps -or- give me Death caps
Edited by The Blind Ass (09/20/22 06:26 PM)
|
DERRAYLD
Constructus


Registered: 05/13/02
Posts: 10,170
Loc: South Africa
Last seen: 12 hours, 11 minutes
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: The Blind Ass]
#27959970 - 09/21/22 12:19 AM (1 year, 7 months ago) |
|
|
Damn those skull induction coils are ridiculous dude,
|
hummingbird

Registered: 06/29/14
Posts: 2,169
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: DERRAYLD]
#27960044 - 09/21/22 02:20 AM (1 year, 7 months ago) |
|
|
, cool thread!
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: hummingbird]
#27960332 - 09/21/22 08:42 AM (1 year, 7 months ago) |
|
|
Thank you, everyone! I wish I could claim credit for any of this, but really I'm just pulling bits and pieces out of humanity's collective knowledge, and then doing work. I'm still working on both of these things, I'd like to make a much better inductor TBH, but I'll have to put research and testing time into the circuit and I'm not ready to commit to it atm.
The Dedi procedure I'm trying a lot of variants of still, and I'm moving out to other strains and species. I'm also preparing for an attempt at protoplast prep that would be reasonable for use in a hobby lab setting. So, exciting things coming up!
These things should be standard tools we all have the ability to use as needed.
-------------------- 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
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb]
#27964133 - 09/23/22 11:00 AM (1 year, 7 months ago) |
|
|
Update on the Dedi test plates:
As they continue growing, more macro-morphological differences are emerging. The A type has a partial tendency to grow asymmetrically, but regardless, it has a higher sensitivity to temperature changes (indicated by the concentric banding as room temp changes throughout the day). But the asymmetry is also in the local mycelial density, as illustrated by the third picture. Out of the 5 recovered A type, 2 display this asymmetrical, floccose behavior, plus the original A type recovered from a previous attempt.
  
The B type is very consistent, with pseudorhizomorphic striation in the colony pattern.

When the A/B plates are compared to either type, you can see the morphological distinctions. The pairing seems to consistently prefer increased development on the B side, with the A side not developing increased rhizomorphs or often size, significantly.
 
Compared to the A/A plates, B type again stands out, with the A/A colony preference falling to the more vigorous mycelium. The floccose A type gives way to the tomentose, circular A type. In some cases, it stops growing altogether, perhaps to allow growth from the more rigorous subculture to colonize the resources and support the weaker subculture, and increase the likelihood of an optimized pairing.
 
Looking now at the A/A and A/B plates compared to a control plate I allowed all the cultures to fuse on, we can see the A/B starting to resemble the rhizomorphs of the control plate.
 
Thanks for Watching!
I'll update with fruits of the re-paired culture and the original culture later when they fruit.
-------------------- 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
|
ReverendMyc
succinct is not my forte

Registered: 03/29/19
Posts: 1,849
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb] 1
#27972389 - 09/28/22 02:34 PM (1 year, 7 months ago) |
|
|
-------------------- Stoned Gummys tek (Gummies from sclerotia or mushrooms) *Not just for stones any more"Psychedelics are powerful substances. Nothing that powerful is completely safe... and nothing completely safe is that powerful!" - Abigail Calder at ALPS 2023 Don't Panic   
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: ReverendMyc]
#27972615 - 09/28/22 05:09 PM (1 year, 7 months ago) |
|
|
Pics were acting wonky yesterday, so I didn't post this update:
Three new cultures were selected for variations of the maceration technique- Gan.CL1 (a Gandalf clone), KS.CL1 (my favorite squat clone), and BLM.F3.CL2 (a lion's mane culture inbred from a wild N. China clone).
0.1mL LC of each were added to the same dex/pep/oster set up, and each blended 10 seconds. Incubation was for a total of 6 days. At 3 days, spheroid colonies were developing in the KS broth. Even at 6 days, Gan and BLM failed to culture. A 1cm sphere was retrieved from the KS broth and viewed at 1000x and determined to be fully monokaryotic. The remaining colonies were blended for 5 then another 5 seconds, with 101 dilutions made of each blend time [BT].

After thoroughly agitating, each BT was drawn into a 1mL syringe each, and 0.1mL was added to five water agar plates for both BT's, and each cross streaked for recovery of colonies. After retrieval, subcultures of each recovered neohaplont will be paired as before to determine nuclear type.
1 day after streaking, all 10 plates are showing growth, and 7/10 have colonies spatially isolated enough to transfer. I'll be circling them, inspecting them under the scope and transferring them to MEA later. Confirmed monos will then be paired, and the best representatives of nA and nB will be slanted. I'll try to remember to take pics.
Also, new broth will be sterilized and Gan/BLM will be attempted again with more care to ensure inoculum is introduced, and it is not over blended. Currently, I only have 3 blade bases, so I'm limited to 3 trials with this procedure at a given time.
-------------------- 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
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb] 1
#27981032 - 10/04/22 11:28 AM (1 year, 7 months ago) |
|
|
Trich Farming
intro to protoplast prepI have recently acquired the last pieces I think I'll need for conducting Protoplast Preparation. The "affordable, home lab" version necessitates more footwork to accomplish, but even with some lacking, I have already achieved spheroplasts (some cell wall remains, creating a "spheroid" with the protoplast inside) using both a Metarrhizum culture and a Penicillium culture (neither are ideal for full cell wall removal, but could have application similar to snake venom, since they are mainly proteolytic as I understand it...) However, some select extracts and a source of the preferred Trichodermas- harzianum and viride- are now in my possession, so I will be updating as I cultivate the trich, extract the enzymes (maybe show the precipitation of the enzymes, too, though it isn't necessary), lyse the cells, collect the protoplasts, and regenerate the protoplasts on agar. I'll use the premade extracts (used in brewing as Beta Glucanase and clarifying agents like "Clarity Ferm," respectively) in a side-by-side to see their efficacy as an alternative to having to cultivate mold. Up front, I expect the trichs to be far more effective, as between the two there is a full cell wall degrading complex that covers the glucans, the lipoproteins, and the chitin we need to get rid of. The specific cultures/enzymes used will be: - Bioglucanase TX (from Trichoderma reesei)
- Clarity Ferm protease (from Aspergillus niger; White Labs)
- Mykro-Root (T. harzianum and T. viride, 2x107 CFU/g; Microbial Applications)
* * *Simply to test the viability of the trich, I dabbed a swab into the package (in still air) and agitated the swab in a centrifuge tube with ~10mL of water. After mixing well, I added 0.1mL to 10mL of 1% LME broth and 0.1mL to an MEA plate, cross streaked. After 12 hours in distilled water, flecks of translucent white mycelia are visible when held to the light. There is no growth on the plate yet, and the LME clouds the broth too much to assess just yet. In order to ensure ease of cultivation, I will be trying three methods that are supposed to prevent the production of conidia (exponentially potentiating possibility of contam outbreaks). The first two I will relate here, since they are well known, and the third I will elaborate on later once I have another ingredient and have tested it a few times (it's experimental at the moment, and only theoretically applicable to trich). The two main methods of controlling sporulation of the molds are temperature control (higher temp incubation), and submerged cultivation (trich LC). Incubation Phase:Using LC is the easiest means of preventing mold spores. The addition of a stir bar/stir plate helps to ensure that the moving liquid keeps the culture submerged. While they can still easily produce oidia (shorter cell compartments that fragment off easily to increase asexual propagation), the nuisance conidiospores that go airborne don't form as readily. Heated incubation is typically not something we do in cultivation, but in this instance a reptile heat mat could potentially be used for constant temp heated incubation, provided it doesn't exceed the thermal limits of the fungi. Trichodermas can usually withstand 130-160 F (54-71 C), depending on species and strain. While 115 F (46 C) will be sufficient for mold cultivation, other, higher fungi will usually die around 110-115 F (43-46 C), so the preferred experimental range while generating the mold culture and for generating the enzymes is 90-100 F (32-37 C). Fermentation Phase:Now, for actually generating the enzymes, we have to stimulate the mold to produce them by feeding them what we want to break down later. In this case, liquid or solid medium with fungal tissue as the only food source, i.e. a mushroom powder broth or mushroom powder agar, or if you are feeling particularly sadistic, you can add the trich inoculum directly to colonized agar or LC with the target species of mushroom we will protoplast later. Allow 14 days to "ferment" the mycelium. If using LC, keep in mind that the final liquid volume should be minimal, as we cannot heat these to reduce the liquid without denaturing the enzymes (they begin degrading below 100 C, so boiling isn't an option). Collection and Purification:First, we need to separate all the fungal mass from the serum of water and enzymes. To do this, we will blend up the ferment (LC + Oster; or Agar + Water + Oster... remember to minimize the liquid), then do a coarse filter and a fine filter. So load up the oster jar (really, you can use any blender for this, as we purify it later) and blend it for at least 60 seconds. This is to kill any remaining fungi. It won't be 100% lethal, but enough to eliminate what minor risk remains in opening the jar. For the coarse filter, we need a funnel and some cotton, cheese cloth, polyfill, or similar to pack into the spout of the funnel. Filter through this, change the filter, then filter it at least once more. There will still be some solid particulate in this filtrate, and as an option, it can be refrigerated to help settle those particulates overnight, then decanted through the coarse filter again, leaving the sediment in the jar. The fine filtration will be done with a 0.22μm syringe filter (PES or acetate, NOT PTFE... hydrophobic filters don't like water), and be fairly slow. Don't apply too much pressure to the syringe as you filter it into a sterile container. Forcing it through can distort the membrane and allow impurities and contaminants to pass through. When the flow through the membrane decreases and the syringe plunger gets stiff, switch to a new filter and continue. The course filter (and overnight sedimentation) help immensely in reducing clogging of the filter, allowing more serum to be filtered before requiring changing the syringe filter. Light and heat are the main degrading components, so store in the fridge or freezer, and if desired, in amber glass, opaque bottles, or wrap in foil to block light. Label and date it as well. There is an additional step for refinement that can be taken that I haven't tried yet, as the chemicals aren't as easy as visiting a big box store to acquire. However, a precipitation step can be taken after fine filtration by adding potassium sulfate or magnesium sulfate to the serum, and chilling the serum. The precipitate can be collected on filter paper (coffee filter is fine), dried, and stored in glass in a refrigerator. This would be the closest to a quantitative recovery that would be measured and used around the same percentages of most published procedures for protoplast prep (typically 4-6% lysing agent). For use of serum, there will be more art and trial involved for getting the correct balance, as concentrations will be much lower. I have no idea what the shelf life of serum actually is, but I would hazard a guess that it is best used within 6 months, when stored in the proper conditions. Precipitated enzyme stored in the same conditions, perhaps a year.
-------------------- 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
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb] 2
#27983235 - 10/05/22 05:43 PM (1 year, 7 months ago) |
|
|
I can't upload this because of data limits, but I spent the day watching trich grow:
https://imgflip.com/gif/6vw0jg[animated .GIF]
I find it especially interesting how the old growth forms the cross-bar, fat hyphae to support aerial growth. I expect there will be condia to help me ID which is which soon.
Isn't she beautiful?
-------------------- 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
|
smalltalk_canceled
Babnik


Registered: 07/13/20
Posts: 2,906
Last seen: 7 hours, 10 minutes
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb]
#27983301 - 10/05/22 06:16 PM (1 year, 7 months ago) |
|
|
Intimidating and beautiful at the same time.
-------------------- Willpower is the one true virtue
  
|
DERRAYLD
Constructus


Registered: 05/13/02
Posts: 10,170
Loc: South Africa
Last seen: 12 hours, 11 minutes
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb]
#27983966 - 10/06/22 02:30 AM (1 year, 7 months ago) |
|
|
Some beautiful work
|
mind.at.large
Myconerd


Registered: 12/13/16
Posts: 1,230
Loc: Floating in liquid gardens
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: DERRAYLD]
#27984261 - 10/06/22 09:13 AM (1 year, 7 months ago) |
|
|
-------------------- Mind's Easy Bag 2 Bag Grain Transfers Endless Sub Tek ...the doll's trying to kill me and the toaster's been laughing at me...
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: mind.at.large]
#27988643 - 10/08/22 04:23 PM (1 year, 7 months ago) |
|
|
Cultivating trich is going to be... still harder than I thought.
I did not account for the fact that this Mykro-Root is a horticultural supply, and gets mixed into dirt, so no consideration for sterile manufacture or packaging was made, and that beautiful trich from the .gif is overrun with a pinmold now. The six plates I made from the streak plate are pinmold (maybe with trich hiding at the center, but effectively it is just pinmold, given how fast they grow).
But God doesn't spit in your face without cupping your balls, or something, and an old spent tub that I didn't toss finally seems to have attracted trich. So I'm trying to propagate that, and I'll return to serial diluting the Mykro-Root and seeing if I can get clean isolations. At the least, it doesn't appear that this is a germ cocktail, just an unclean product.
Wish me luck! I might burn through a few hundred plates before I update this particular topic.
-------------------- 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
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb]
#27993648 - 10/11/22 12:10 PM (1 year, 7 months ago) |
|
|
The mold in the tub was, in fact, trichoderma!
I cultured it on water agar and eventually it formed conidia:

I'm not sure on the species, but my initial thoughts are harzianum or viride/aureoviride. My next move is to try and cultivate it without it producing conidia, maybe even conidiaphores.
-------------------- 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
|
Stipe-n Cap


Registered: 08/04/12
Posts: 7,843
Loc: Canada
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb]
#27998743 - 10/14/22 02:04 PM (1 year, 7 months ago) |
|
|
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb]
#27998943 - 10/14/22 03:54 PM (1 year, 7 months ago) |
|
|
Regarding the individual characters of monokaryotic mycelia contributing to the character of the resulting dikaryotic mycelia (P. ostreatus, but relevant):
https://scialert.net/fulltext/?doi=pjbs.2007.2334.2340
Scroll down for the pairings.
-------------------- 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
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Stipe-n Cap] 1
#27998944 - 10/14/22 03:55 PM (1 year, 7 months ago) |
|
|
Hey you!
-------------------- 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
|
Stipe-n Cap


Registered: 08/04/12
Posts: 7,843
Loc: Canada
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb]
#27998959 - 10/14/22 04:08 PM (1 year, 7 months ago) |
|
|
Nice thread, not often I find something interesting to read here
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Stipe-n Cap]
#28006121 - 10/19/22 09:31 AM (1 year, 6 months ago) |
|
|
So, something I'm experimenting with right now has some interesting first results.
What you see here is mycelium (trich atm) who's cell walls have become porous on a supplemented MEA. I'm still very into the experimental/investigatory stage, so details are going to be absent for now. Suffice to say, I was trying a method of controlling conidia production, and while it doesn't appear to work, this other effect could be very useful.

[photos on agar @ 1000x and 250x]
While the pores are not large enough to liberate a protoplast, they are large enough to potentially pass nuclei and plasmids (and molecules...) through the cell wall. And the effects are reversible (this isn't venom or a lysing enzyme) by removing the mycelium and transferring to standard media (there are effects besides making the cell wall porous related to up and down regulation of gene expressions, according to some papers. One effect has been lower to no color expression in the conidia.)
I'll be transferring some cubes to the media later and we'll see if it translates.
-------------------- 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
|
Stipe-n Cap


Registered: 08/04/12
Posts: 7,843
Loc: Canada
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb]
#28006187 - 10/19/22 10:14 AM (1 year, 6 months ago) |
|
|
Are you saying that the structures within the hypha are pores? It appears as though trich hyphae are non septate, so perhaps the photo is showing free movement within the cytoplasm, Cytoplasmic inclusions?
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Stipe-n Cap]
#28007759 - 10/20/22 11:41 AM (1 year, 6 months ago) |
|
|
1, the aerial mycelia and conidiophore don't have this pattern on the hyphae; it's only on the surface and submerged mycelia.
2, it definitely is septate, but the compartmentalization behavior is (I should think "obviously") different from basidiomycota.

Also, I have been able to observe nuclear or vacuole movement within other cultures' hyphae. These have been stationary.
-------------------- 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
|
Stipe-n Cap


Registered: 08/04/12
Posts: 7,843
Loc: Canada
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb]
#28007773 - 10/20/22 11:59 AM (1 year, 6 months ago) |
|
|
Ah, very nice. Nice snap of the septa.
|
smalltalk_canceled
Babnik


Registered: 07/13/20
Posts: 2,906
Last seen: 7 hours, 10 minutes
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Stipe-n Cap]
#28007877 - 10/20/22 01:47 PM (1 year, 6 months ago) |
|
|
"Also, I have been able to observe nuclear or vacuole movement within other cultures' hyphae. These have been stationary."
What is the general meaning of this? What does it suggest?
-------------------- Willpower is the one true virtue
  
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
|
Quote:
smalltalk_canceled said: "Also, I have been able to observe nuclear or vacuole movement within other cultures' hyphae. These have been stationary."
What is the general meaning of this? What does it suggest?
Just what it says. The nuclei and other cellular components move around inside the cell, sometimes they even hop between cells through the septa. They get pulled around by things like actin and dynein, and if you have the proper refraction through the hypha, you can see them move about in live mycelium.
By contrast, I'm calling the apparent pitting on the trich mycelium I'm viewing "pores opening in the cell wall," both because they are stationary and because it is a possible side effect of my agar supplement.
-------------------- 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
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb] 1
#28162841 - 01/29/23 12:41 PM (1 year, 3 months ago) |
|
|
From a convo with Rhizy:
Quote:
it would seem like the mating type genes would be one of the things regulating fruitbody development
Mat1 and Mat2 are the mating loci names, and Mat1 is a simple allele, whereas Mat2 is a complex of various traits that generally seem to inherit as a unit. These traits relate to pheromone production, clamp coordination and often fruit body cellular orchestration, from what I understand.
Quote:
I’ve heard some really strange stuff recently as well like traits flip flopping between generations (apparently not due to heterozygotes), and lines that blob from spores and revert from cloning. I also think that environment seems to have a big impact on expression, and it seems that some varieties are a lot less stable than is assumed especially when grown in a multi-dik environment.
I have a few ideas regarding this. I have been assured by a couple vets that it isn't a real problem, but Same Species Cross Contamination is one possibility. Especially when it comes to any degree of open air fruiting and heavy target species spore load. One great example of this is when CronicR was fruiting John Allen Strain and CRS at the same time. Spores from the CRS produced fruit with purple spore (F1) that in turn produced mixed purple spore and red spore (F2), with similar happening in the descendants from JAS. Pasty assured me that CronicR has a massive sporeload in his grow room (visible on walls even), but I think it would still be quite possible with typical circumstances. It's one experiment design I return to repeatedly, I haven't quite figured how to simulate it effectively.
Another is partial expression, which could tie in to mitochondrial preference.
Quote:
how many prints you need to run from F1 to catch the offspring of different nuclei/diks?
Any time a cross happens, regardless of the sexual mode, the offspring present in typical Mendelian fashion. But, "how many prints" is a bit of a statistical variable. If you are only looking for one recessive trait, it likely will pop up within a grow or two. The more non-linked recessive traits you are looking for, the less likely a given dikaryon will match the criteria. there are also undefined variables in play, like how likely two adjacent alleles are to inherit as a unit, or separately. Don't quote me- math is not my strong suit- but the probability of stacking n recessive traits is an inverse exponential. Another variable is how diverse the grow is. I try to make my MS grows fairly limited, but I'm probably still getting around 1-3k spores in there. Others, like Fahtster, are using substantially more and getting far more varied results early on.
I'm sure as a Maths lecturer you've put together that more iterations in a limited set increases the odds of a hit.
Quote:
about di-mon. Do you look for new dik myc growing away from the interaction zone (maybe using morphology and heterosis), subculture the interaction zone, or would you wait until nuclear migration has happened and subculture from the area that was originally the mono?
Literally, I wait until the plate pins. There are details that indicate the Buller Phenomenon has taken place, like abrupt change in morphology (thickening, increased colonization rate, formation of clamps in new growth...), but those can be variable. Clamps normally form in new growth after 48 hours with cubes, for example, but some pairings don't develop clamps for a week or so. But there are only a few examples of basidiomycete monokaryons that can produce asexual fruit bodies (not oidia, but full on fruits), so I use that as a definitive marker most times. I have never seen a "third culture" grow out of the interaction zone using Di-Mon pairings. Mostly, that seems to be a response some dikaryons have in the presence of other dikaryons/competitors.
Quote:
and then there’s also mitochondria!
Quote:
The stress of changing between vegetable and reproductive states, for example relating to cloning, must be greater than subculturing/asexual expansion, and is also really interesting. I was also thinking about what you said about enigma and cleaning up a culture, makes me wonder what was involved in cleaning it…
Yes, there certainly is. I asked my friend Sidnee about this a couple years ago when I first started trying to wrap my head around Enigma. I was pretty familiar with Boomer Smith and Moe Matteo, who brought Enigma into circulation. I can say pretty confidently that Enigma was isolated into a monokaryotic pure culture by Boomer before it was sent back to Moe, who fruited it, so repeat cloning wasn't a factor. Boomer also doesn't use any antibiotics or anything, so it was cleaned with serial transfers, so not a factor. Further, I know a lot of back story of that particular drama, and I'm pretty sure that Magic Myco (Doma, Tidalwave breeder) probably found another from spore, and they were arguing over 6 vs a half dozen. My own ability to track down blob-only cultures from spore certainly supports that.
Back to Sidnee, I asked if the mitochondria of that particular selection could play a part. His response was that while cytoplasm isn't exchanged in plasmogamy and you end up with two cultures with the same nuclear DNA but different mtDNA, it doesn't really matter. I didn't accept that answer, though, and have been designing an experiment where I can investigate this without access to DNA scanning. I have a good idea on how to go about it, but it requires competency in several lab techniques like dedikaryotization. I'm close to attempting it, I just need to carve the time out for it and select ideal candidates.
Quote:
It’s interesting that you mentioned about reversion as a type of mutation, as I was going to ask what you thought about it.
You can find more information on it with the term "synthetic recovery".
Quote:
about auxotrophs, or mutants that require some specific nutrient in the medium? I guess it’s hard to know without specifically trying different media, but these mutants seem to be useful in experiments as identifiers. I also wonder if any of the cube mutant iso/cultigens would fruit more normally with supplements or different environmental conditions.
Auxotrophs are interesting, but in typical cultivation practices we usually surreptitiously breed them out- at least, by my vague understanding of artificial and natural selection. One interesting example mentioned in (I think) A.C. Chang's "Genetics and Breeding of Edible Mushrooms" was an auxotrophic strain that required a nicotinic compound for normal metabolic health. Without that supplement, it doesn't fruit or even colonize effectively. I imagine that depending on the exact metabolic mutation, this could be survivable and present deformed fruits, but I don't know.
Quote:
it also interestingly mentioned that the mutated low spore trait in the oyster was stable for more than five gens. You could add the photoreactivation stuff to your UV tek, but from the papers it isn’t really clear when the mutant culture can be exposed to the ‘normal’ light (definitely by fruiting it seems).
Photoreactivation triggers DNA repair. From what I've read in a few AG science papers, it occurs mostly in the blue spectrum, so UVC treated fruits and veg should be kept in darkness or red spectrum light after sterilization. When I tried this with live cultures, it outright killed them, reddening the mycelium often and halting all growth. Samples would not culture on fresh agar. Exposure to light seems to be required to avoid a terminal outcome. And while this is a very accessible route to mutations, it will probably always require tinkering to get ideal results, and then a lot of fruiting to find beneficial mutations. This technique has been used with a good clutch of species already.
Quote:
It seems that for cubes and more complex fungi there are enough mating alleles that there are thousands of sexes, and incompatibility would be hard to find even if you wanted to.
True, until you start inbreeding. Then, Mendelian inheritance applies to the predictability of Mat1 and Mat2. In a wild setting, inbreeding happens on much lower scale, and homozygous recessive strains present less frequently. At least, that's my assessment. Ecological studies of anomalous wild specimens could prove me wrong. It's proving wrong with several species like Boletus edulis, which is showing that the environment has more to do with their phenotypes than the genotype does. Even transplanting European varieties in the USA, the European variety takes on the characteristics of local strains, even though the genome stays intact. [ https://attheu.utah.edu/facultystaff/a-tale-of-terroir-porcinis-evolved-to-the-local-environment/ ]
Quote:
You mentioned something about a sort of di-di mating through exposure to chemicals, could any mutagen/shock treatment be used, like heat, UV, electroporation? What is the mechanism for this, I’m guessing it would need to prevent or delay somatic incompatibility in the interaction zone.
To be clear, Di-Di mating isn't a thing, in my observation and opinion. But certain events could appear to be Di-Di mating. Hypothetically, this would involve some sort of dedikaryotization (dedi) event to happen when two strains are in close proximity. There are natural events that can cause this, some of which I'm investigating (see me Swinger Tek post in Bene Gesserit). IIRC, chemical dedi can happen in the presence of things like cholates (biles), with a fair chance of inducing damage mutations to the recovered mono. Using high enough concentration to do this prevents one nucleus from replicating and the new growth becomes mono. On a plate with cholates, or even theoretically in an animal's GI, this can cause one or two dikaryons in close proximity to become available for a new di-mon or mon-mon pairing. And there are a host of events that can do this. It can even happen randomly for undefined reasons, particularly in new growth. My contention is that people who have successfully "crossed two dikaryons" have actually exploited a form of dedikaryotization.
Quote:
I saw a ‘gold member’ variety for sale online, is this your cross or just a coincidence? I guess the later as it says PE x GT.
Nah, not mine. That is a "variety" (unstable from what I've seen). Mine is a clone name, just because my labeling system isn't verbally friendly. But, I do know the person who made it (they are pretty active on facebook) and they named it Gold Member as an attempt to slight me. That is actually something I anticipated though, which is why I haven't named the variety beyond the project name (GOGH, just initials that mark the original parents)
-------------------- 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
|
HFM
hairy fecal matter


Registered: 06/25/21
Posts: 372
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb]
#28163176 - 01/29/23 04:22 PM (1 year, 3 months ago) |
|
|
.
-------------------- Trees lay chipped across the paths, we hunt their souls to eat the wrath-HFM
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: HFM] 2
#28164374 - 01/30/23 01:12 PM (1 year, 3 months ago) |
|
|
Mitochondrial InvestigationsTo investigate the influence of mitochondrial DNA (mtDNA) in abnormal fungal behaviors, in particular the formation of fruit bodies, the following procedures have been designed to generate cultures with identical nuclear DNA (DNA) but disparate mtDNA, for conducting experiments in linear growth rate (LGR), biological effeciency (BE), carpophore formation (CF), and any other metric that could potentially be affected by mitochondrial activity. Introduction During plasmogamy, when two monokaryotic cultures exchange nuclei, only a small area where the anastomatic connection is formed contains the cytoplasm of both monokaryotic cultures. In this small area, copies of respective nuclei are transferred to the other cell, and rapid nuclear division of the new DNA takes place, with the donated nuclei traveling through the culture via nuclear migration through the cell septa. The new DNA now operates in tandem with the host DNA and the native mitochondria. In vivo, this results in two cultures with the same binucleate DNA being utilized by separate mtDNA genomes. Using the simple tools of serial dilution and dedikaryotization by mincing cultures, it is possible to replace the mtDNA of a given monokaryon or dikaryon to study the specific effects that mitochondria produce while utilizing DNA. Moreover, the procedure can be used starting with a wild or domestic clone, or starting with spores to produce monokaryotic cultures. Competency with agar, serial dilution and dedikaryotization is required to execute this procedure. Quote:
Materials
An Oster/Waring/Eberbach Graduated centrifuge tubes Syringes/pipettes plenty of agar/LC
Before anything, monokaryons need to be generated with serial dilution (or a suitable method that accomplishes the same). These monokaryons cannot have the same mtDNA as the target culture for the procedure to produce viable data. In domestic cultivation, this means selecting varieties with disparate pedigree- which will require some degree of knowledge regarding such. This is a simple matter when investigating specimens that have particularly unusual morphology or metabolism, as wild-type mtDNA is (theoretically) plentiful in the population. In such case, generating monokaryons from wild-type leaning varieties is important. These will become the “normalcy” benchmarks for later experiments.
Tandem Dikaryotic Cultures (TDC)
After isolating monokaryons from disparate lineages, pairing monokaryons from those respective parents results in two dikaryotic colonies, each colony retaining it's original mtDNA. After confirmation of plasmogamy, each colony should be subcultured and maintained separately. Agar is the preferred method for tracking LGR, and can easily be tracked by marking a graduated grid on the bottom of the plate. Expanding and fruiting each maintained culture to observe differences in CF and BE is possible now. Dedikaryotizing the cultures will produce both DNA types times both mtDNA types.
Replacing mtDNA in a Dikaryon With cloned, dikaryotic cultures, attempting to utilize the Buller Phenomenon (di-mon mating) will result in a novel dikaryon with the host monokaryon's native mtDNA intact. To achieve the desired replacement of mtDNA, the dikaryon must be separated into its component monokaryons (neohaplonts). Both neohaplonts will have the same mtDNA, but pairing them with other monokaryons (generated from spore or an unrelated dikaryon) as when starting from spore, the respective pairings produce a TDC. Subculturing the TDC from the non-neohaplont side will select the novel dikaryon with different cytoplasm and mtDNA than the starting dikaryon. Further dedikaryotization of the novel dikaryons will produce the monokaryotic components of the original clone with new mtDNA.
-------------------- 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
|
Rhizy
Stranger


Registered: 11/13/22
Posts: 2
Last seen: 3 months, 22 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb]
#28212311 - 03/03/23 10:37 AM (1 year, 2 months ago) |
|
|
First post, woohoo! Great to see some of our chat in the thread! (Looks like a typo about Enigma being isolated to a mono). Have to say, as usual, loads of great original content! Really impressed to see the new mito-swapping experiment design, a lot of work though lol!
I’ve only been using a microscope for a few weeks, but I’ve seen movement of small black organelles, even moving through the septa as you said, and what seem to be various types of vacuoles in the hyphae at 1000x (see pictures below and clip of movement is on discord microscope channel). I had hoped that the small black dots were nuclei, but after speaking to someone more experienced, looking some more, and looking at micrographs in the literature, then I think seeing nuclei with a standard light microscope without staining is very rare, and these are more likely vesicles or some other type of vacuole moving around.
The following pictures are from a standard light microscope at 1000x, with a hand held phone camera using some zoom. Some possible vesicles circled in the first photo.

|
Rhizy
Stranger


Registered: 11/13/22
Posts: 2
Last seen: 3 months, 22 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Rhizy]
#28216210 - 03/05/23 05:08 PM (1 year, 2 months ago) |
|
|
P.S. I went back to Chang and Miles (Mushrooms: Cultivation, Nutrition Value, …) to see what they say about UV mutagenic treatment. It seems that UV mutation works by forming dimers that prevent correct DNA synthesis, and they seem to suggest that spores (or conidia), or mycelial fragments are treated with mutagens.
So, I’m guessing photoreactivation needs to be prevented until after DNA has been synthesised in the treated sample, like the spores germinate or the fragments start to extend/grow. They also say that yellow light can be used to illuminate the work area for UV without causing photoreactivation.
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Rhizy]
#28225059 - 03/11/23 12:11 PM (1 year, 2 months ago) |
|
|
yeah nuclei are pretty tiny, not nearly the scale they are usually shown in illustrated diagrams.
Right now, I'm doubling back to do a deeper investigation into "swinger tek" using my Gold Member (GM) culture and Enigma (EN). I figure the clear visual difference should highlight patterns of inheritance pretty well.
There are actually 2 possible outcomes if the procedure does produce novel dikarya, dependent upon if the mtDNA is significantly different. The first is 4 novel combinations, plus the two original dikaryons, with no significant variation of mtDNA. If variant mutation(s) exist in at least one of the mtDNA lines, phenotypic expression can be between 5-8, plus the 2 original. Concurrently, I'm dedikaryotizing Enigma to do controlled matings with GM and EN (GMEN), to produce 8 total subcultures (transferring from both sides of each TAD to subcuture) as reference/control.
Presently, I have completed a battery of grows using the swinger tek for pheno hunting. I have identified 4 general phenotypes from the fruit, with possible mitochondrial variations of each.
The base phentypes are Wild Type (WT), Hat Type (HT - wild type with a party hat), Variegated Type (VT - having varied pigment on one cap from none to tan), and Conical Type (CT - cap starts conical and retains a nipple upon maturity); the possible variant phenotypes are the base types, plus wrinkled caps and stipes, leaning towards thicker stipes, but generally still wild type with modifications. Both leucistic and carpophoroid/pseudocarp phenos (GM and EN) appeared in most of the grows at a low rate.
Given the dominant alleles of each- GM produces generally agaricoid fruits, while enigma has "cap pigment" alleles- the predicted permutations of their nuclei should produce WT leaning phenos, including pigmentation of flesh and spores, which is what is showing up in the swinger tek grows. The 4 base phenotypes are already on grains, and a MS of each is also on grain, while I finish dedikaryotizing Enigma and make my reference pairings. I cloned the wild type and variegated wrinkle-phenotypes for comparison, as HT and CT didn't present wrinkle-phenotypes. I figure if there is a noticeable difference it will play out with the controlled pairings anyway.
Another possible cause of the variation is overall sub volume and spawn ratio. Notably, and perhaps obviously, the half pint cake/mono tube produced the smallest fruit, while the large filter patch bag was about 5 qt volume with a 1:4 ratio and the largest fruit. Wrinkled variants did occur in all but the half pint grow, though. To eliminate this potential, the clones will be standardized 1:3 in large grow bags sealed after spawn, as will the controls.
The prediction for the MS is that each of the novel phenotypes will produce a diverse fruiting population with more individualized recessive traits of either parent presenting in 3:1 or 1:2:1 ratios, perhaps some looking strikingly like either parent; this diversity will be drastically higher than the initial swinger tek grows and not lean so heavily towards wild types. Increased homozygosity respective to selection(s) is also predicted in the F3.
Phenos:

Wild Type - there appears to be a nipple, but it smoothed out in maturity and wasn't conical as a pin.

Hat Type

Conical Type - retains cone shaped cap until maturity, retains a nipple once the cap is (near) planar.

Variegated Type - while all caps with pigment will have a hygrophanus color gradient, I am only considering it variegation if it is present as a pin.
Swinger Grows in situ:

Most of the grows were 1:3 rye to CV in domed pint cups (dub tubes)

One cup (half pint) of mixed 1:3 spawn/sub was fruited in a single unmodified quart cup (mono tube).

In a bag, at 1:4 with CV. Note the distinction between conical and wild type here.
-------------------- 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
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb]
#28225068 - 03/11/23 12:19 PM (1 year, 2 months ago) |
|
|
...as such, I haven't circled back to UV experiments yet.
-------------------- 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
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb]
#28299066 - 04/28/23 03:53 PM (1 year, 21 days ago) |
|
|
I have some time, so I thought I'd update a couple things.
Apr. 1
Stage 2 of my Swinger Tek tests is underway. Here are the neohaplonts from dedikaryotizing Enigma and Gold Member, clone cultures I used in the initial GMEN blender experiment. They are much more flat and translucent than either dikaryotic culture. I'm waiting for the controlled pairings of GM×EN from their respective monokaryotic cultures to compare to phenotypes from the blender (and other) experiments.
GMEN.F1 [Control Set]
 The respective nuclear types were paired on agar and I waited for clamps to show. Clamps were confirmed with visual alterations of macromorphology and in situ microscopy @100x. Here is the most dramatic change in morphology from the GMEN.AA plate:  Apr. 14-28 Proceeding, the "TADs" were subcultured from respective colony sides and labeled according to cytoplasm type (mtGM or mtEN). Comparatively, we can see significant differences in mycelium character:  
 
 
  The result about to go to grain is 8 unique cultures that should show similar results to the GMEN swinger experiment. These were subcultured again and will go through several serial transfers to continue observing mycelium habit. In the meantime, I only ran 2 of the GMEN.F1 clones (Wrinkle and Hat), which showed opposite/swapped phenotypes (not a mix up, they were cloned on different days). They were 1:3 in sealed grow bags. Otherwise, same general (wild type) phenos.  Finally, I found 2 gold spore squat specimens in the GCKS.F2 (plus a couple other phenos I'll be pursuing), named Hotei and Goku. Forgot to grab pics of Goku, but they are both being cleaned and headed to grain soon. GCKS, if you've been following along, was also started with Swinger Tek. These two finds (after approx. 10 F2 multispore grows) completes the first demonstration/trial of Swinger Tek, combining the gold spore descended from Golden Halo (Golden Child F2 clone from my GOGH breeding project) and Koh Samui Squat's rotund, short stature. Proof of concept. Now with GMEN, we will have a deeper look at what's happening, hopefully.
-------------------- 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
|
YoshiTrainer
Onion tied to belt



Registered: 04/30/22
Posts: 1,382
Loc: Castles made of sand
Last seen: 56 minutes, 48 seconds
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb]
#28322295 - 05/16/23 06:32 PM (1 year, 3 days ago) |
|
|
Some great reading, happy to be following along!
|
Camera93
We got dicks like Jesus



Registered: 08/15/18
Posts: 3,346
Last seen: 1 day, 2 hours
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: YoshiTrainer]
#28322306 - 05/16/23 06:44 PM (1 year, 3 days ago) |
|
|
-------------------- All I need are some tasty waves, a cool buzz, and I’m fine. Whatever you decide won’t really impact our survival Close your eyes, and do the best that you can
|
Haywire
Wetspot Wizard



Registered: 12/29/13
Posts: 1,620
Last seen: 1 day, 19 hours
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Camera93] 3
#28344069 - 06/02/23 06:49 AM (11 months, 10 days ago) |
|
|
Hi
Wanted to let you know I made this because of you. Absolutely love it!
-------------------- Ciao mamma, guarda come mi diverto My grows Outdoor patches
|
Mandrake Erpelmann
St. Ranger


Registered: 12/30/22
Posts: 203
Loc: Germany
Last seen: 7 days, 17 hours
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Haywire] 1
#28347777 - 06/05/23 02:24 AM (11 months, 7 days ago) |
|
|
Quote:
Haywire said: Hi
Wanted to let you know I made this because of you. Absolutely love it!

Same goes actually for me ^^ Scalpel glows red hot in less than a second! <3
|
michaelmichael
sus

Registered: 06/28/23
Posts: 13
Last seen: 6 months, 7 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb]
#28385884 - 07/05/23 12:00 PM (10 months, 8 days ago) |
|
|
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Mandrake Erpelmann]
#28386849 - 07/06/23 08:23 AM (10 months, 7 days ago) |
|
|
Quote:
Mandrake Erpelmann said:
Quote:
Haywire said: Hi
Wanted to let you know I made this because of you. Absolutely love it!

Same goes actually for me ^^ Scalpel glows red hot in less than a second! <3

Hey, that's dope! I'm always glad to see people benefiting from something I shared.
-------------------- 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
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb] 1
#28387004 - 07/06/23 11:19 AM (10 months, 7 days ago) |
|
|
School was hectic, but I'm on the fluffy side now with summer break. Still, I had just enough time to keep things going.
GMEN Update:
3/8 of the control pairs have been fruited now, with 2 more on grain and the other 3 about to hit grain. Originally, I had 4 in fruiting, but I lost 1 sub to jumping the gun on spawning.
As a brief refresher, they are labeled by nuclear type (A or B) in Gm:En order, and further divided by mitochondrial type (mt). For example:
ABmtEN =
GoldMember type A Enigma type B Mitochondria type EnigmaThe 3 I have fruited already are AAmtEN, BAmtEN, and BBmtEN, and the results have been unexpected. What is fascinating is that AAmtEN and BAmtEN are presenting pseudocarp fruits. BBmtEN had a shorter colonization and fruiting time, with wild type fruit- nominal in both cases. Summer was already in full swing when BBmtEN started fruiting, and some fruit flies got into the sub, so that one is closed up rn. The others are in sealed bags, so pics aren't great.  Bottom fruit in AAmtEN and BAmtEN
 AAmtEN - blobs with caps?
 AAmtEN - Yes. Blobs with caps.
 BAmtEN - Stranger blobs with less pronounced cap-like areas
 BAmtEN - Previous fruits from opposing angle to give a sense of shape
Next on the docket is ABmtEN which is about ready to spawn, and BAmtGM which still has a week or so until spawning. The remaining AAmtGM, ABmtGM and BBmtGM are hitting grain in a couple minutes here. So here are my preliminary observations: I expected pseudocarp formation to be a stacked set of recessive alleles, meaning the genes for it would have to be in both nuclear types of a dikaryon to consistently produce pseudocarps. This might not be the case. The fact that AAmtEN and BAmtEN are producing pseudocarps indicates that the "Enigma A" monokaryon contains the mutations involved. The fact that BBmtEN has wild type fruit where the other two are only producing pseudocarps confirms this to some degree. For counterpoint, this could be an error if Enigma A is actually dikaryotic. Since all 3 strains are from the Enigma side of the formed dikaryon, if Enigma A was a dikaryon to start it would consistently throw Enigma fruits. It is, admittedly, the simpler explanation. I don't believe it to be the case, however. From the appearance of the pseudocarps alone, they are clearly not "Enigma," even given that Enigma "shape shifts" from cauliflower to finned florets in general shape. I have to regenerate Enigma A from my slant and check again for certainty, but I spent a lot of time agonizing over the confirmation of those cultures. Additionally, if AAmtGM and BAgmtGM DON'T form pseudocarps, it will not only indicate that Enigma A is monokaryotic, but also that Enigma A is required in conjunction with the Enigma mitochondria type. Neither AB culture (mtGM or mtEN) should have anything remarkable about them by this model (expecting generally wild type phenos), but they will still be fruited because I won't know until I know. I'll be printing everything that prints regardless, because all the cool kids are in the F2. *** Meanwhile, I'm still technically searching for my prize in the GCKS cross, but proof of concept level has been achieved IMO. I have produced squat phenos with gold spores using the Swinger tek. Additionally, I have done an intragenerational cross with Swinger tek to try and combine traits I want from two different F2 cultures. On the back end, I will be dedikaryotizing both cultures and running them through the same tests as GMEN. I have all summer to, so why not? The intragenerational cross predicts less variability than the current generation, but still more than the next full generation. For that reason, I consider this to be an interstitial generation and have labeled it F2b instead of F3. The clones were selected for body shape (Allum Sativum, a crenelated garlic-shaped fruit with small cap) and stature/spore color (Hotei, a short, stocky, gold-spored fruit).  Allum Sativum (GCKS.F2)
 Hotei (GCKS.F2)
 GCKS.F2b ASxH
 ASxH #3 "Tall Goomba"
 ASxH #5 (Gold Print)The swinger portion of this is already complete and dedikaryotization has already been started. As predicted, the fruits produced (dubbed GCKS.ASxH) are an intermediary range of phenotypes. Something neat happened that I wasn't expecting- roughly half the phenotypes produced gold spores. If you refer back to punnet squares, that means gold spore is heterozygous recessive in Allum Sativum. It's a small bonus, but it increases the odds of finding my prize pheno in the F3 (fatty squat with smallish cap and gold spore).  Inheritance pattern for homozygous x heterozygous*** Additionally, I've been passively running F4 multispore of my GOGH project and I found a really neat new pseudocarp. It's on grain and I'll be spawning soon (a day or two). I'm calling it Dragon Eggs, because there is a central oblate spheroid mass with patches of smaller, densely grouped nub growths over the surface, that look a bit like scaled eggs to me. I can't wait to see it in isolation.
-------------------- 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
|
trippleblack
Stranger

Registered: 12/01/19
Posts: 373
Last seen: 2 months, 6 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: Muad.Dweeb] 1
#28390199 - 07/09/23 10:16 AM (10 months, 4 days ago) |
|
|
di di mating.. or what appears to be didi mating is my way to go.
extreme potency range is why i prefer creating cultures this way; i'll first create or use(like tw2) a strong proven culture and cross it with another strong proven culture.
tw2 x 118



ape melamc 
did various crosses at the least -6 times; got my best cultures because of this. I did link a few studies and lightly explained how i go about it, you can search a shroomery thread titled "Hybrid Vigor in Cubensis F1 crosses (AKA “Heterosis" )"
..
using some the teks outlines in this thread offer great control over pinpointing and crafting phenotypes; i will be using.
|
Muad.Dweeb
Seer



Registered: 04/13/17
Posts: 148
Last seen: 8 months, 26 days
|
Re: The Weirding Way - Advanced Bene Gesserit Techniques [Re: trippleblack]
#28392278 - 07/11/23 09:42 AM (10 months, 2 days ago) |
|
|
I just want to reiterate that I don't believe Swinger tek functions through "di-di" mating, which I don't think is a thing. Obfuscation of the actual outbreeding event just makes it appear to be di-di mating because we don't casually observe the event that causes one or both cultures to dedikaryotize. I think that cases of people claiming that two dikaryotic cultures crossed on agar or in grains can be explained by the same process (when demonstrable outbreeding has occurred), and I'm currently building a data set that I think will ultimately support this model I'm calling Trauma Induced Promiscuity.
The basic working theory is that there is a threshold of trauma to the mycelium that causes dedikaryotization to enable recombination in hopes of a new combo having some genetic skill set that will allow species survival. My first trial with grains already supports the model and I have several variations in the works. Currently, I'm only running Gold Member and Enigma so there is direct comparison with previous experimental/control grows, but I'll be repeating the whole series of tests with several other combinations, including intragenerational crosses to show results for closely related pairings. It'll be a lot of work, but that comes with making bold claims, I suppose.
-------------------- 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
|
|