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Violet



Registered: 12/06/11
Posts: 4,205
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These guys have shot it to you pretty straight. I would only say it a little differently myself, so will save my breath for a more important matter.
Though I will answer your original question more directly - Yes, we need the 2D surface of agar to be able to sector and separate strains to reach the final point of isolation. Once we have an isolate, however, grain petris can indeed replace agar plates for the specific purpose of transfers and master grain jars. It's important to keep our master cultures on agar, which has significantly less mycelium expansion per radial growth, but also having a side-chain of culture on grain petri allows us to make tweezer transfers to new dishes, and a colonized grain petri can be broken up in seconds to be a powerful inoculant for grain containers, far more effective than agar wedges, especially after the trouble of slicing up and spreading an agar plate.
Next post, I'll describe how I organize my culture tech projects to easily select and show the cultures which shine thru...
-------------------- Intentionally or not, here in mushcult we are purveyors of love culture and enlightenment movement. Let's try to act like it! PODS TEK - Growing Invitro with BRF/verm or Grass Seed containers The simplest, quickest, safest tek! For beginners, culturers, lazy people, stealth lovers, contam haters, and alternative seekers! • Violet's Teks and Posts •
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GoodyMcLuvin
Shamanic Level Dimitri Proponent


Registered: 01/12/15
Posts: 74
Loc: Hippie Town, NC
Last seen: 8 years, 4 months
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Re: Culturing • GRAIN 'PETRI' TEK [Re: Violet]
#21190160 - 01/28/15 07:15 AM (9 years, 3 days ago) |
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Yes!! I love how you posted the desired genetics. That was the most thorough list that I've seen on the subject. Thank you all so much. And thanks, V. When you going to get around to doing that? And how long does it take for one of your grain Petri's to pin, if it is just an average colonizing subvariety?
-------------------- *I graduated from the corner, my respect be my diploma,I went OG for the dollars, Js thought I went to college*
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Violet



Registered: 12/06/11
Posts: 4,205
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Re: Culturing • GRAIN 'PETRI' TEK [Re: Violet] 3
#21190631 - 01/28/15 10:04 AM (9 years, 3 days ago) |
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Here I outline exactly how I utilize this culturing technique to select the best cultures from a round. It's gonna be wordy... but pictures wouldn't help much and they'd be exactly the pictures I already have in the tek, so.
I inoculate all grain petris at the same time, as close to dead-center of the grain circle as I can. I keep a batch of grain petris made with a single strain together. I keep them organized by strain and date, but don't bother labeling all the individual containers. That will come later as they begin to perform.
As some of them get close to fully colonizing to the very edge of the grain (yes, this takes quite some time) I keep a close eye on them so that I know within half a day at least when they complete. (Having them a bit off-center can make this require your best judgment.)
When a container is done, I THEN label the container... NUMERICALLY, starting from 1 - This way the containers are labeled numerically in order that they were fully colonized, pretty much numbering them from fastest to slowest. (Typically at this point I'm open to the possibility of ignoring the ones slower than the first 5-8 containers. If I don't need containers for something else, I'll water them and let them flush out just for the bit of yield) It's unnecessary to note the timeframe it took them to colonize initially. Though not a bad bit of info to take note of in one's notebook, it's not necessary to clutter one's mental organization of the cultures. The order of colonization is all that really matters.
In addition to labeling them by full colonization NUMBER, I also write the DATE which represents when I "initiated fruiting conditions" ... this is because I unscrew the lid a bit (about 1 inch) so that they can push out excess CO2 and have the small amount of gas exchange they need to pin. THIS date IS important, because it's necessary to know how long it took the containers to pin from full colonization time which is going to be at least a bit different for most of the containers.
Time until pins form will vary - that's the point. The idea is to find the fastest pins that exemplify the qualities we're looking out for. I wouldn't just automatically clone the first pins... they'll be fast, but will a single lone pin be what we're looking for? Maybe, but chances are something else would beat it out.
We don't want just the fastest strain... we want the fastest prolific strain. If it makes a strain a day slower (once isolated) for those qualities, it's BEYOND worth it, and we still will have speed! So we select pins of a size that look like they won't be tiny fruits, coming from areas with many other pins, possibly indicative of prolific fruiting.
What else? * I think it is good to take these pins from the outside portions of the cake, near the periphery, because those areas were colonized most recently. * If using grass seed as I suggest for this technique since it has a greater proclivity for fruiting directly, there's a good chance that you'll get fruits on the inside. If they occur VERY quickly, then they're worth selection if the sample looks good. But if they pin there at the same timeframe as near the edge, favor the edge since it was colonized more recently.
The young pins are transferred to an agar dish as outlined in the tek. I label the pins cloned to agar by the numbered grain petri (1, 2, 3, etc.), and ALPHABETICALLY if I took more than one pin per original grain petri. Such as 1A, 1B, 1C, 2A, 2B, 3A, 3B etc.
We take the fast healthy growth from the pins and transfer to new plates. It's at this point that we may start seeing sectoring. It may take another transfer or two, but I have found that pin clone transfers get to the sectoring point quite quickly, which is nice!
Sectoring is, of course, when growth from an agar piece transfer begins dividing itself into different appearances, with thin 'gaps' between more dense healthy points. Obviously we want the thickest and fastest growth to transfer.
Any such transfers, when it involves in more than one split gene to test in the future, get their own alphabetic label in sequence of the original. Such as, if I get 2 sectors that I like and want to test from 1B and already have a culture running called 1C, I will call the second sector 1D.
So now we have several isolates! It's good to make more than 1 dish for each culture once we know we have isolated cultures. One to keep as a long-term master culture in case we love it, and the others to do the initial test and potentially a further test grow-out if it makes it to the final contest :] It's at this point that I end up with a LOT of dishes, using a LOT of containers... many growers might not have this many. I wouldn't suggest ordering petris just for this reason but it certainly might help someone in a bind. Personally I just like to have lots of containers around. But there are options... you can take the chance at dropping the ones you have good reason to think aren't as promising as others (such as containers other than the 5 fastest) or you can just do 1 dish of each, and use it for your master even after taking from it for the tests, making expansion plates from it for storage and for the future if/when you decide to keep it.
They need to be tested to see how well they exemplify fruiting qualities we cloned them to hopefully have. I choose to test them by doing pretty much the same thing- I inoc at least 2 grass seed grain petris from each culture, regardless of whether I used grass seed or brown rice for the culture selection. This is important because grass seed has the best results fruiting directly as well as the least amount of nutritive mass per volume so we can really see which produces the most yield from the least substrate. This time we don't have to inoculate in the middle. We can use several agar wedges and shake the inoculated containers like crazy to spread the mycelium from the wedge all over the grains, then let it sit to colonize. When fully colonized, crack the lids as normal to initiate pinning.
When pins form, have a notebook ready to write the dates that the pins were seen. This gives a good (although not perfect, due to potentially varied times colonizing depending on inoc) indication as to how quickly the specific isolate fruits.
Most importantly, take notes as to the size of the yields. The first flush MAY be limited due to limited water content, so the TRUE results will appear once you start watering. You can choose to water when you see the first pins if you want to try reducing this effect, but regardless, your overall winner will be made clear within 3 "flushes".
It's also definitely worth taking note of the dates between each harvest. Some cultures may have shorter time periods from one flush to the next, which is appreciable.
After all is said and done... the 2 or 3 most prolific, quick-fruiting, quick-colonizing cultures are tested with a full grow out to see how the yields and qualities fare when really expanded. Those fruits are tested for potency as best as we can! Depending on those results, the ease of picking (particularly from casing layers) and other qualities listed in the "About selecting ideal cultures" post are also taken into consideration for the selection of our one champion culture!
-------------------- Intentionally or not, here in mushcult we are purveyors of love culture and enlightenment movement. Let's try to act like it! PODS TEK - Growing Invitro with BRF/verm or Grass Seed containers The simplest, quickest, safest tek! For beginners, culturers, lazy people, stealth lovers, contam haters, and alternative seekers! • Violet's Teks and Posts •
Edited by Violet (01/28/15 02:06 PM)
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Guardian187
Neophyte


Registered: 11/29/14
Posts: 716
Loc: Center of the Universe
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Re: Culturing • GRAIN 'PETRI' TEK [Re: Violet]
#21191037 - 01/28/15 11:36 AM (9 years, 3 days ago) |
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Nice summary Violet. This is EXACTLY what I've been interested in learning more about and implementing.
I have quite a few petris of two different 'strains' I'm isolating out (originally from a spore syringe). I'm working with two different medias as well.
Would it work for me to transfer sectors from my petris to the grass seed containers as you do? I'm assuming it would, but thought I'd check first
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Violet



Registered: 12/06/11
Posts: 4,205
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Re: Culturing • GRAIN 'PETRI' TEK [Re: Guardian187]
#21191190 - 01/28/15 12:07 PM (9 years, 3 days ago) |
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You already have your cultures running on agar, so there's no since in doing so to clone pins, but it would be an excellent way to test their colonization times, pinning times, and some fruiting qualities!
Don't transfer right off a sector; make sure you have a fully grown-out place that's shown to be an isolate, and keep an original master of it!
-------------------- Intentionally or not, here in mushcult we are purveyors of love culture and enlightenment movement. Let's try to act like it! PODS TEK - Growing Invitro with BRF/verm or Grass Seed containers The simplest, quickest, safest tek! For beginners, culturers, lazy people, stealth lovers, contam haters, and alternative seekers! • Violet's Teks and Posts •
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blindingleaf
blue collar underworld


Registered: 07/19/13
Posts: 22,008
Loc: sub-surface unseen
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Re: Culturing • GRAIN 'PETRI' TEK [Re: Violet]
#21191220 - 01/28/15 12:12 PM (9 years, 3 days ago) |
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Cool stuff violet!
-------------------- A few thoughts on cultivation MICROBIAL HUSBANDRY!!!! The whole is greater than the sum of its parts
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GoodyMcLuvin
Shamanic Level Dimitri Proponent


Registered: 01/12/15
Posts: 74
Loc: Hippie Town, NC
Last seen: 8 years, 4 months
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Ya I love how intricately she describes everything. And yes thank you, V. That's much more clear than all other things I've read on the subject.
-------------------- *I graduated from the corner, my respect be my diploma,I went OG for the dollars, Js thought I went to college*
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blindingleaf
blue collar underworld


Registered: 07/19/13
Posts: 22,008
Loc: sub-surface unseen
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I've always appreciated the way she articulated her ideas even if I don't use them. I like that she is passionate enough about it to really dive that deep.
-------------------- A few thoughts on cultivation MICROBIAL HUSBANDRY!!!! The whole is greater than the sum of its parts
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GoodyMcLuvin
Shamanic Level Dimitri Proponent


Registered: 01/12/15
Posts: 74
Loc: Hippie Town, NC
Last seen: 8 years, 4 months
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I went and got agar, and I'm about to go get the things for my SAB. But I did find some filters at Sam's clubs, four pack that were some how statically charged and supposedly attracted molecules. Not only did they down to the required .3 microns they filtered down to .1!! Four pack for something like thirty six dollars. Perfect for a flo hood.
Sorry if this was off topic but considered it semi appropriate since it pertained to agar work. Plus thought it was a good deal so I wanted to share.
-------------------- *I graduated from the corner, my respect be my diploma,I went OG for the dollars, Js thought I went to college*
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Guardian187
Neophyte


Registered: 11/29/14
Posts: 716
Loc: Center of the Universe
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Re: Culturing • GRAIN 'PETRI' TEK [Re: Violet]
#21192441 - 01/28/15 04:29 PM (9 years, 2 days ago) |
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Quote:
Violet said: You already have your cultures running on agar, so there's no since in doing so to clone pins, but it would be an excellent way to test their colonization times, pinning times, and some fruiting qualities!
Don't transfer right off a sector; make sure you have a fully grown-out place that's shown to be an isolate, and keep an original master of it!
Right on! Thanks
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Jerome09
Alchemist


Registered: 02/27/15
Posts: 28
Last seen: 6 months, 3 days
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Re: Culturing Tech • Agar containers, "Grain Petri" Tek, Invitro growing & Sterile Spores! [Re: SpitballJedi]
#21418743 - 03/17/15 12:05 AM (8 years, 10 months ago) |
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Violet or other people...
Do you reccomend doing violet's invitro tek with a ms syringe?
If it is all you have as a... nube
?
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Violet



Registered: 12/06/11
Posts: 4,205
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Re: Culturing Tech • Agar containers, "Grain Petri" Tek, Invitro growing & Sterile Spores! [Re: Jerome09]
#21418751 - 03/17/15 12:11 AM (8 years, 10 months ago) |
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Which tek? The invitro tek with grass seed, or starting grain petris with spore syringe?
Syringe works for both. It actually can work rather nicely on grain petris, skipping the first step of germination on agar for transfer. For the invitro grow tek, spore syringes work fine, little differently than starting grain jars with spore syringe. I still recommend using a still-air box to inoculate, and keeping your containers unmodified - squirt in your spore solution under the edge of the lid in still-air box or as clean and still-air of a condition as you can get.
-------------------- Intentionally or not, here in mushcult we are purveyors of love culture and enlightenment movement. Let's try to act like it! PODS TEK - Growing Invitro with BRF/verm or Grass Seed containers The simplest, quickest, safest tek! For beginners, culturers, lazy people, stealth lovers, contam haters, and alternative seekers! • Violet's Teks and Posts •
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Jerome09
Alchemist


Registered: 02/27/15
Posts: 28
Last seen: 6 months, 3 days
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Re: Culturing • INVITRO GROWING [Re: Violet]
#21422909 - 03/17/15 06:44 PM (8 years, 10 months ago) |
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With Violet's invitro tek...
do you keep a 12/12 light cycle throughout the entire grow?
What about temperature? Do you lower it to say 75 for fruiting? I do know she writes "Fruiting conditions are initiated by cracking the lid moderately to very loose"
Excited about this tek as a way to ensure a reliable set and forget grow. I thought mini monotubs would be the ultimate set and forget grow but after my first one was dominated by Trich a few days ago I HAD TO MAKE A CHANGE...
thoughts??? I am hoping Violet's invitro tek will be a solution in a contaminated house. I have had nothing but success with 2 seperate pf tek grows so I figure this invitro grow will be just as successful in colonization.
Any thoughts on this method and/or others that work in a contaminated house??
I just plain don't feel like cleaning all the clothes off my floor and trying to "sanitize" my whole room. Not going to go to great lengths to try and make another monotub work because... what if it contaminates again..uggghhh. Would rather do small pf tek or invitro grows that do work and don't require me to change my house...
That's my advice and how I see it anyways, hopefully my first VIolet's invitro grows work out.. I'm using RGS and a ms syringe.
Also Violet has some very unique and opinionated philosophies that she feels strong about and shes obviously had a lot of success so I will tread down her path and hoping to see what she sees...
ohh the magic!
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Jerome09
Alchemist


Registered: 02/27/15
Posts: 28
Last seen: 6 months, 3 days
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Re: Culturing • INVITRO GROWING [Re: Jerome09]
#21422974 - 03/17/15 06:49 PM (8 years, 10 months ago) |
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Oh Thanks violet for the response about using a ms syringe for the RGS invitro tek. Glad to hear it works well. Sounds like it should replace the pf tek as the very first tek for beginners.
WHAT'S the VERDICT on casing a 32oz RGS invitro grow??? I know it's not necessary to fruit it, but is it worth the time to case it??
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Violet



Registered: 12/06/11
Posts: 4,205
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Re: Culturing • INVITRO GROWING [Re: Jerome09]
#21425027 - 03/18/15 12:38 AM (8 years, 10 months ago) |
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Yes, 12/12 ambient or semi-direct fluorescent lighting, or indirect daytime sunlight, is fine. Also, 450nm LED is good too, for varying shorter lengths depending on the LEDs intensity. I use ambient lighting in the room and from windows - it does plenty. I have suspended a fluoro above them on the shelves before, that did nicely too.
Any room temperature is fine for Cubensis. 70-80 degrees is ideal, especially 72-75. The mushrooms release heat, and in the retaining environment of invitro they may warm themselves and grow in somewhat different ways.
These methods should be excellent for you as a way to have strong successful grows with the least susceptibility to environmental contam issues.
As for casing grass seed invitro... I have found it to be mostly or completely unnecessary. Safe the material and effort for when you move on to whole brown rice (or other grains) should you choose to. I think the only perceived advantage to casing grass seed is that it may allow you to unscrew your lid even looser for fruiting conditions. Not a big deal, if even helpful at all.
I encourage you to post such questions in the invitro seed tek thread from now on - I'd like that one to be bumped and for questions like these
-------------------- Intentionally or not, here in mushcult we are purveyors of love culture and enlightenment movement. Let's try to act like it! PODS TEK - Growing Invitro with BRF/verm or Grass Seed containers The simplest, quickest, safest tek! For beginners, culturers, lazy people, stealth lovers, contam haters, and alternative seekers! • Violet's Teks and Posts •
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KauaiOrca
Waterman


Registered: 08/12/08
Posts: 3,131
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Re: Culturing • INVITRO GROWING [Re: Violet]
#21924614 - 07/10/15 01:45 PM (8 years, 6 months ago) |
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Is anyone using this tek that can give an objective report on results? How well did it work for you using on RGS as the substate, no casing no other additives?
How well did fruiting invitro in the container work for you? Good yield? Is it really strain dependent? Any thoughts from people that have done this tek and like or dislike it would be really appreciated.
-------------------- "The universe is endless, limitless and infinite. Any effort to define it's boundaries is an attempt to overcome ignorance. We are physical, mental and spiritual beings ... there is no beginning and there is no end. There is only memory. Our repeated loss of memory experiences create the illusion of beginnings and ends. Immortality is the ability to retain full memory through all consciousness transformations. Loss of memory is man's greatest curse and, in very real terms, death." -- Ancient Taoist Master
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bodhisatta 
Smurf real estate agent


Registered: 04/30/13
Posts: 61,889
Loc: Milky way
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Re: Culturing • INVITRO GROWING [Re: KauaiOrca]
#21924918 - 07/10/15 02:44 PM (8 years, 6 months ago) |
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Quote:
KauaiOrca said: Is anyone using this tek that can give an objective report on results? How well did it work for you using on RGS as the substate, no casing no other additives?
How well did fruiting invitro in the container work for you? Good yield? Is it really strain dependent? Any thoughts from people that have done this tek and like or dislike it would be really appreciated.
http://www.shroomery.org/forums/showflat.php/Number/19035259/fpart/1/filter/i/vc/1#19035259
you can filter a thread to show only images. there's only 3 pages worth with the last one posted over a year ago. even violet's own results with this TEK are lack luster. and why show us only 4-5th flush results and not take any pictures of 1st 2nd or 3rd flush results?
as far as peer review, repeatability of claims, and standing the test of time.... you tell me, compared to the other TEKs out there.
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Violet



Registered: 12/06/11
Posts: 4,205
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Re: Culturing • INVITRO GROWING [Re: bodhisatta] 1
#21926622 - 07/10/15 11:12 PM (8 years, 6 months ago) |
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Hi Orca. I'm sure you're aware that you're asking your questions in the culturing thread, so must be asking about the results with the culturing method, as opposed to a grow-out tek. I love this culturing method, I have kept around master cultures selected by using it from the very first times I did it, now like 2yrs old. It has given me 3 or 4 cultures that I know I can throw at a lot of seed and get awesome production keeping with the best-done of any methods. The cultures also do excellently, even ideally, with pretty much any method.
Now, this isn't uncased or invitro (which I mostly only do both when culturing) but I have the first flushes of an early round of tests with cultures taken via this method. http://www.shroomery.org/forums/showflat.php/Number/19414571#19414571 I don't grow much Cubensis these days, as I prefer shiitake and grow less in general now too, as is reflected by my infrequency on the forums. But when I do, I'm typically casing brown rice invitro and being very lazy about things.
hi Bod, happy to see you're still tirelessly up to the same tired trolling and logical fallacies etc., and spending droves of your time on here playing big. Like the townie that graduated a year after me that's still living there trying to get a rapping career off the ground. Fun stuff. As before, I'd be mindful of how you insult my grows, as I get far better results on average than anything you've ever shown at your best. Not to mention, it's just bad character of you. In fact, last I knew about the matter, you weren't even supposed to be posting in my threads anymore, since you never do anything but start shit, on purpose according to your own admission... You must be enjoying my abscence and the false sense of victory it allows you to award yourself.
-------------------- Intentionally or not, here in mushcult we are purveyors of love culture and enlightenment movement. Let's try to act like it! PODS TEK - Growing Invitro with BRF/verm or Grass Seed containers The simplest, quickest, safest tek! For beginners, culturers, lazy people, stealth lovers, contam haters, and alternative seekers! • Violet's Teks and Posts •
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bodhisatta 
Smurf real estate agent


Registered: 04/30/13
Posts: 61,889
Loc: Milky way
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Re: Culturing • INVITRO GROWING [Re: Violet]
#21926805 - 07/11/15 12:28 AM (8 years, 6 months ago) |
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You really have nothing better to do than pop in every time you're mentioned. We're trying to help people grow here with the least effort and best results.
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KauaiOrca
Waterman


Registered: 08/12/08
Posts: 3,131
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Re: Culturing • INVITRO GROWING [Re: Violet]
#21927768 - 07/11/15 07:47 AM (8 years, 6 months ago) |
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Quote:
Violet said: Hi Orca. I'm sure you're aware that you're asking your questions in the culturing thread, so must be asking about the results with the culturing method, as opposed to a grow-out tek. I love this culturing method, I have kept around master cultures selected by using it from the very first times I did it, now like 2yrs old. It has given me 3 or 4 cultures that I know I can throw at a lot of seed and get awesome production keeping with the best-done of any methods. The cultures also do excellently, even ideally, with pretty much any method.
Now, this isn't uncased or invitro (which I mostly only do both when culturing) but I have the first flushes of an early round of tests with cultures taken via this method. http://www.shroomery.org/forums/showflat.php/Number/19414571#19414571 I don't grow much Cubensis these days, as I prefer shiitake and grow less in general now too, as is reflected by my infrequency on the forums. But when I do, I'm typically casing brown rice invitro and being very lazy about things.
Thanks Violet. That's helpful. I'm nowhere near as experienced as you are so the distinction between culturing and just growing is more clear. To summarize your advice and PLEASE correct me if I'm wrong …
1. Use invitro RGS for culturing, meaning finding a good fruiting strain because it produces really clean prints and pins for agar transfers?
2. Use brown rice and casing (do you grind the rice BRF cake style) for the actual grows invitro to give as good a yield as possible? I'm mostly interested in invitro PP5 jar grows for everything.
Your advice is really appreciated. Also, if you were to pick one strain from vendor spores to get started as a good one for this method, any thoughts? Your openness, desire to help and really clear spirit are an inspiration on these threads!
-------------------- "The universe is endless, limitless and infinite. Any effort to define it's boundaries is an attempt to overcome ignorance. We are physical, mental and spiritual beings ... there is no beginning and there is no end. There is only memory. Our repeated loss of memory experiences create the illusion of beginnings and ends. Immortality is the ability to retain full memory through all consciousness transformations. Loss of memory is man's greatest curse and, in very real terms, death." -- Ancient Taoist Master
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