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verum subsequentis
seeker of truth



Registered: 03/22/16
Posts: 8,732
Last seen: 1 year, 8 months
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Penis Envy isolation 24
#24741888 - 10/27/17 06:03 PM (6 years, 4 months ago) |
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Aloha friends and fellow pursuers of enlightenment. I'm well on my way toward attaining a shit load of isolates and thought I'd start a thread to share the journey and hopefully get some educated perspectives on isolation.
I've been advised by many to give up on isolation and just stick with clones but I've never been very good at not going all the way and I just can't help myself. I need to know what pure isolated genetics have to offer. Don't worry yourself, I've got plenty of clones going as well.
So, I'm 19 transfers in and have reached the point of around 60 plates of Penis. All of said plates were started from the same ms plate. I believe i have a handful of isolates and that most of my plates will be isolated on the next transfer. I'll be doing my 20th transfer tomorrow.
14th transfer
 17th transfer

18th tranfer

19th transfer

Now a couple 19th transfer close ups.

Sorry, the pictures aren't the best but if you expand them I think you can see clearly enough.
This is my first "try" at isolation. I started with thousands of questions and found that using the search function (properly) answered almost all of them. That and just going for it and learning along the way. At the moment i have just a few questions that really boil down to the same question.
Are mono cultures always symmetrical?
I know they are supposed to be but in real life if you set the agar wedge on it's side are you going to end up with a "perfect" circle? Also, is it possible for the rhizomorphs to make the outer edge imperfect?
Let us refer to the above close ups as 1-15, top to bottom left to right. (my friend says that "top to bottom, left to right" is confusing. I say he's confusing but, any who, ENGLISH mother fuckers. Number them the same way you would number words in a sentence. He says i should say left to right, top to bottom.
close up 1. Is it possible that the left side is just as isolated as the right? I noticed that in some plates the agar solidified at a slight angle and that the thin side looks a little more rhizomorphic than the thick side. i suppose that could starve the myc out a little resulting in more rhizo growth.
close up 4. Lookin pretty sexy until about 8 o'clock. I'm thinking that this could be the result of setting the wedge in sideways. It makes sense in my mind. The myc that's closest to the agar gets a head start while the shit that has to grow over the wedge doesn't actually start growing on the plate for a few days. Image 7 and 11 show similar activity.
That's it for now. I Love this shit. Thank you so much to all ya'll who've put your heart and soul into this community. I appreciate it.
Edit 10/28/17 Transfer 20. Should be 90% isolates. Sixty plates.

Edit- 10/29/17. While doing transfers yesterday I noticed something interesting. A good number of plates where very uniform on one side and very rhizo/not so uniform on the other. Upon further inspection it became clear that every plate behaving in this manner had solidified at an angle. EVERY plate with angled agar was doing this.

I think that this is very interesting and I'm curious if any of you more experienced folks have ever experimented with thin vs thick agar and myc appearance. edit 11/1/17
In an effort to better understand This shit, I've decided to take pictures of a few of these as they grow out. I'll be taking a picture of these six every day or two and watching how they develop.

Plate p20-8bm (sw) had the transfer wedge intentionally placed on it's side. The myc covered side of the agar is facing the "(sw)" and the underside of the wedge is facing the "x". i expected it to take longer to grow toward the "x".
edit 11/2/17 here are those six plates again.

Edit 11/4/17

And this is the whole lot of them. Good lord, I was pretty certain that I'd finally have a bunch of isolates in the mix but, I guess I'm not there yet. A few are probably mono but not many.

Looks like I got some more genetics to filter through. It amazes me. twenty transfers in and not isolated yet. Some of these plates have been "for sure" isolates on the next transfer for three or four plates and there're always more genetics to work through. What a project.
If any of ya'll have opinions on the best looking sectors or how many sectors I've got on these guys please let me know. Also, if you want a close up of any plates, just ask. Peace.
Edit 11/19/17 ROUND 22 I was to lazy to photograph all 60 so I just took a couple of good shots. It's looking pretty good. I Have a handful that I'm pretty sure are isolated and the rest will be soon.

Round 22 sneak preview.

Any opinions on how many sectors are present? edit 12/1/17
Alright, here are a couple plates from Transfer twenty two.

Edit 12/12/17
Here a a randomly selected handful from this round.

Definitely getting closer but not there yet.
Edit 12/19/17
I've decided to do a few transfers real quick to save plates and time. I should have continued doing three transfers per plate longer but this is my first isolation and I got excited when I started seeing "sectoring" and wanted to let them grow out a bit more. Here are a few from this round. Transferred to new plates last night,

Edit 12/27/17
Time to do some more transfers.

Edit 1/4/18
Alright, First off, HAPPY NEW YEAR to all you wonderful fags. Looks like it might be a doozy of a year. Second, I'd Like to thank all of you that voted for me in the 12 ROCKETS deal. I appreciate it very much and am honored to have been selected.
Well, i just sat down at the old flow hood and got fifty plates out of a liter of MEA.
Now it's time for my next transfers. on to transfer 27. Here are the 26th transfers for your viewing pleasure.
 
edit 1/13/18
ok, here's the lot of em.
 
AND, now that i was selected for the twelve rockets thing, i don't have to try and take it easy on uploads. so...
  
And a couple of back-lit-back-door action photos for all you line of division spottin mother fuckers.

EDIT 1/21/18
alright, 1 liter of bright red LMEA in the AA and time to show you all the latest batch. and then to the flow hood. Plate 28. if your curious about the labeling it's from the top and counts clockwise. ie: top, bottom right, bottom left. the 28 on the first plate denotes the plate number of all cultures.
                       
culture 10 and 19 were so small (invisible) that i got insecure and transferred a bigger slice to it's own plate. if you look you'll notice that 10 didn't take and 19 did.
culture 3 is FINALLY isolated (I THINK). i believe I am finally arriving. Here she is from the back.

Same shot with a little editing to make details more visible.

Edit 1/26/18
I'm not sure when, but soon i'll switching back to giving every culture it's own plate. i find it easier to distinguish sectors when i let them grow out a little. until then I'll be putting a few per session in their own plate as to properly ascertain where I'm at.
Here are the two that got their own plates last time (plate 28). back-lit, front and back. PENIS ENVY CULTURE 10 (B) PENIS ENVY CULTURE 19  Edit 1/28/18 This is why I'm thinking about switching back to one culture per plate That's just another picture of culture 10 that I took up close and edited a bit to show more detail.edit 1/29/18 PLATE 29                     notice that little trich colony on 19,20,21? Good thing i still have my sexy little SAB edit 1/30/18 I've been taking pictures of my plates after they have been transferred out of in hopes of being able to look back on this project and learn a little something about which transfers turned into what. I also figure that this could be useful to anyone that is new to transferring from agar.
                     edit 2/1/18 these are the two that got their own plates last time (p 29) A little more grown out. Culture 3
 
Culture 19
edit 2/7/18
culture 3 and 19 all grown up


ALRIGHT BITCHES, PLATE 30 You get front lit front and back lit back this time.
Seems to me that the farther isolated i get the less rhizo the culutre is. It also seems that the appearantly isolated transfers grow more slowly than the ones with genetics still fighting for nutrients.
I also noticed a bit more water in my dishes this time. The water was always sitting right on the transfers, almost like the transfer was sweating. I've seen this before but never so extreme. It fucked up a couple of the cultures a little. You'll see it if you're looking. It's probably just cosmetic though. Does anyone know why this happend? maybe i transferred to my new plates while they were still a bit warm and the temperature differential caused the sweat.
Edit 2/14/18
PLATE 31

plate 32,33,34 was dropped 1 day after transfering. This dislodged the trasnfer, which then bounced around and made it's new home the cover. All satellites appear to by mycelium left over from the tumble.
I believe that a good many of these are isolated So I'm moving to 1 plate per culture for plate 32. It'll be nice to let them grow out a bit more and see what I'm working with.
I'm also upping my malt concentration back to 30 grams per liter. That's the ratio i used for my first 16 transfers or so. I've since lowered it to 20 grams per liter because i read that lower nute ratios would help in identifying sectors. I don't know if it's a good idea or not but i had a gut feeling about it and i tend to follow my gut.
EDIT 2/25/18
the site would not let me make this post any longer. so, we can carry on here !!!!!!!!!!!!!!!!!!!!Penis Envy Isolation Part 2!!!!!!!!!!!!!!!!!!!!
Edited by verum subsequentis (02/25/18 11:08 PM)
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mushboy
modboy



Registered: 04/24/05
Posts: 32,501
Loc: where?
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im waiting
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LemurLemur
Pray for Boog



Registered: 01/30/17
Posts: 6,004
Loc: Drinking on the roof
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Re: Penis Envy isolation [Re: mushboy]
#24742104 - 10/27/17 07:59 PM (6 years, 4 months ago) |
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--------------------
 (when my data is fast play Lemur in chess at chess.com)[ [gradient:#D40B29,#18C418]Any1 expecting a trade from me i havent forgot about you pinky promise, i fr promise shits just shit rt now[/gradient]
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Drayce
Spidget Finner



Registered: 10/07/17
Posts: 260
Loc: Gaia
Last seen: 3 months, 11 days
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Re: Penis Envy isolation [Re: LemurLemur]
#24742137 - 10/27/17 08:12 PM (6 years, 4 months ago) |
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Holy smokes.... that's a lot of plates.
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nubgrower
Dude



Registered: 08/27/17
Posts: 274
Loc: Moon 13 the Moon.
Last seen: 6 years, 2 months
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very cool
-------------------- Great teks here. And here. Fortunately, I’m adhering to a pretty strict, uh, drug regimen to keep my mind, you know, uh, limber
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van hatton
Still a noob



Registered: 11/23/14
Posts: 5,617
Loc: Michigan
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Re: Penis Envy isolation [Re: nubgrower]
#24742225 - 10/27/17 08:50 PM (6 years, 4 months ago) |
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Impressive as hell
-------------------- If I ever give out misinformation please inform me so I can have the correct information. Tmethyl said: Chuck Norris once roundhouse kicked a monotub that wasn't pinning fast enough. The force of the kick rearranged the genetics of the mushrooms, we now call them Penis Envy. Caps McGee said:
Fun part is figuring out what works best for you
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CataSTRAWfic_Grows
RIP Tom Petty the GOAT



Registered: 09/12/17
Posts: 139
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Re: Penis Envy isolation [Re: van hatton]
#24742697 - 10/28/17 04:05 AM (6 years, 4 months ago) |
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bookmarked
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Fungus Mountain
Poke-N-Squirt



Registered: 10/16/14
Posts: 1,188
Loc: Front row @ the Freakshow
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Impressive work. Think I'll hang around and watch the show.
-------------------- “Until they became conscious they will never rebel, and until after they have rebelled they cannot become conscious.” ― George Orwell, 1984 "If you can't explain it simply, you don't understand it well enough." ― Albert Einstein
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mushhiehunter
Trichodermatologist



Registered: 10/11/16
Posts: 837
Loc: Česká republika
Last seen: 3 years, 3 months
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Quote:
verum subsequentis said: Aloha friends and fellow pursuers of enlightenment. I'm well on my way toward attaining a shit load of isolates and thought I'd start a thread to share the journey and hopefully get some educated perspectives on isolation.
I've been advised by many to give up on isolation and just stick with clones but I've never been very good at not going all the way and I just can't help myself. I need to know what pure isolated genetics have to offer. Don't worry yourself, I've got plenty of clones going as well.
So, I'm 19 transfers in and have reached the point of around 60 plates of Penis. All of said plates were started from the same ms plate. I believe i have a handful of isolates and that most of my plates will be isolated on the next transfer. I'll be doing my 20th transfer tomorrow.
14th transfer
 17th transfer

18th tranfer

19th transfer

Now a couple 19th transfer close ups.

Sorry, the pictures aren't the best but if you expand them I think you can see clearly enough.
This is my first "try" at isolation. I started with thousands of questions and found that using the search function (properly) answered almost all of them. That and just going for it and learning along the way. At the moment i have just a few questions that really boil down to the same question.
Are mono cultures always symmetrical?
I know they are supposed to be but in real life if you set the agar wedge on it's side are you going to end up with a "perfect" circle? Also, is it possible for the rhizomorphs to make the outer edge imperfect?
Let us refer to the above close ups as 1-15, top to bottom left to right. (my friend says that "top to bottom, left to right" is confusing. I say he's confusing but, any who, ENGLISH mother fuckers. Number them the same way you would number words in a sentence. He says i should say left to right, top to bottom.
close up 1. Is it possible that the left side is just as isolated as the right? I noticed that in some plates the agar solidified at a slight angle and that the thin side looks a little more rhizomorphic than the thick side. i suppose that could starve the myc out a little resulting in more rhizo growth.
close up 4. Lookin pretty sexy until about 8 o'clock. I'm thinking that this could be the result of setting the wedge in sideways. It makes sense in my mind. The myc that's closest to the agar gets a head start while the shit that has to grow over the wedge doesn't actually start growing on the plate for a few days. Image 7 and 11 show similar activity.
That's it for now. I Love this shit. Thank you so much to all ya'll who've put your heart and soul into this community. I appreciate it.
Dude, you should consider a microbiology career... beautiful dishes!
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Antimatter
Nobody Important


Registered: 03/12/09
Posts: 144
Last seen: 5 years, 5 months
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Very nice!
Mad props!
-------------------- BEANS GREENS POTATOES TOMATOES
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verum subsequentis
seeker of truth



Registered: 03/22/16
Posts: 8,732
Last seen: 1 year, 8 months
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Re: Penis Envy isolation [Re: Antimatter] 1
#24744267 - 10/28/17 08:47 PM (6 years, 4 months ago) |
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Transfer 20. Should be 90% isolates. Sixty plates.
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Snazz
Polymath



Registered: 11/24/15
Posts: 1,584
Loc: Canada
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verum subsequentis
seeker of truth



Registered: 03/22/16
Posts: 8,732
Last seen: 1 year, 8 months
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Re: Penis Envy isolation [Re: Snazz]
#24745944 - 10/29/17 04:43 PM (6 years, 4 months ago) |
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Updated.
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Antimatter
Nobody Important


Registered: 03/12/09
Posts: 144
Last seen: 5 years, 5 months
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Quote:
verum subsequentis said:

I think that this is very interesting and I'm curious if any of you more experienced folks have ever experimented with thin vs thick agar and myc appearance.
Does the "uphill" (thick) slant have more fuzz while the "downhill" (thin) has rhizomorphic mycelium?
-------------------- BEANS GREENS POTATOES TOMATOES
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verum subsequentis
seeker of truth



Registered: 03/22/16
Posts: 8,732
Last seen: 1 year, 8 months
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Re: Penis Envy isolation [Re: Antimatter]
#24746050 - 10/29/17 05:35 PM (6 years, 4 months ago) |
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Yeah. I wouldn't call it fuzz. But yeah, the thin side is always less uniform and more obviously rhizomorphic. The pictures are both oriented in the same way. Left is thin and right is thick.
Edited by verum subsequentis (10/29/17 05:37 PM)
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Antimatter
Nobody Important


Registered: 03/12/09
Posts: 144
Last seen: 5 years, 5 months
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My bad, "less uniform" is what I should have said.
That's interesting, in passive observation, I have seen someone post side by side images of dishes with different nutrition ratios demonstrating similar differences (6-8% dishes had more compact uniform growth compared to 1-2% dishes). However, it doesn't really make sense that the thinner side would have less nutrients available to it than the thicker side
-------------------- BEANS GREENS POTATOES TOMATOES
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mushboy
modboy



Registered: 04/24/05
Posts: 32,501
Loc: where?
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Re: Penis Envy isolation [Re: Antimatter]
#24746093 - 10/29/17 06:04 PM (6 years, 4 months ago) |
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it doesnt.
a culture on thicker media has more of a food source to draw from, but that doesnt change the nutritional content. you pour thin because it uses less media and its easier to stab and pick up a thin cut than i thick one.
thicker media can be used to store a culture longer though. i dont do slants yet so i store cultures in the fridge on really thick pastyplates. its not the best way but it works.
you also pour thin cause its gangsta'
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Ajaxx_2
OG


Registered: 10/19/17
Posts: 12
Last seen: 6 years, 3 months
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absolutely beautiful! i wish i had a flow hood. i'd like to do a project like this this winter. stuck with a SAB
what is your agar mix recipe? i used to grow years back, and im getting back into it. i've tried agar a few times recently with zero luck...
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van hatton
Still a noob



Registered: 11/23/14
Posts: 5,617
Loc: Michigan
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Re: Penis Envy isolation [Re: Ajaxx_2]
#24746213 - 10/29/17 06:53 PM (6 years, 4 months ago) |
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I'm thinking it has to do with the mycelium colonization slightly under the surface of agar.
The deeper it is the further down it'll have to go. The opposite for thin.
That's the only thing I can think of.
Still doesn't exactly explain the difference. It's a sweet observation tho.
Once you have a true monoculture you should do a thick and thin comparison. I think Tha may possibly yield different results but who knows.
-------------------- If I ever give out misinformation please inform me so I can have the correct information. Tmethyl said: Chuck Norris once roundhouse kicked a monotub that wasn't pinning fast enough. The force of the kick rearranged the genetics of the mushrooms, we now call them Penis Envy. Caps McGee said:
Fun part is figuring out what works best for you
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verum subsequentis
seeker of truth



Registered: 03/22/16
Posts: 8,732
Last seen: 1 year, 8 months
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Re: Penis Envy isolation [Re: van hatton]
#24746391 - 10/29/17 08:23 PM (6 years, 4 months ago) |
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I'll definitely test this out on some mono cultures. it's interesting. I'd dismiss it if i wasn't this far along in isolation and if it only happened on a plate or two. The fact that it happened on ten plates that are all isolated or one transfer away from being isolated raises my curiosity.
I think it is a pretty observable fact that the myc isn't only colonizing the agar that it's visible on. I've noticed myc growing out of "uncolonized" agar many times. I believe that there are little "threads" of myc, that are not visible to the naked eye, within the agar. My theory thus far is that thinner agar equals less nutrition which equals more rhizos.
my recipe is always either standard MEA or oat grain soak water diluted 50%.
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