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Offlinejust_curious
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Giving Isolating a shot.
    #23625178 - 09/08/16 09:21 AM (7 years, 4 months ago)

So, I took a clone and placed it to agar. I want to ensure that I have the best genetics, but I also want to make sure I do it correctly.

I understand to find the fastes, healthiest growth on your agar plate, and pull from the outter edge as soon as possible after identifying sectors.

What I'm confused about is, are you just doing one plate per transfer until you have an even growing isolate and the splitting up between multiple plates at that point? Or, are you making a couple dishes with each transfer.

Lets say I did one by one until I finally has a nice, even, isolated plate. Would I transfer to, say, 10 plates and then keep one to the side as a master?

Why isnt scenescence an issue when working with agar?


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InvisibleThadeous
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Re: Giving Isolating a shot. [Re: just_curious]
    #23625309 - 09/08/16 10:16 AM (7 years, 4 months ago)

I'm no expert with isolates, but I would imagine it's up to you what to do with your isolate. That only really leaves the issue of senescence; an excellent question.


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Offlinejust_curious
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Re: Giving Isolating a shot. [Re: Thadeous]
    #23625323 - 09/08/16 10:23 AM (7 years, 4 months ago)

Also, I never have "apparent" bacteria. It never reveals itself and I use colored agar. Which leads me to think that my agar is clean. What when my jars are fully colonized, there's no more color. Does this happen to you guys, or is it definitely a contam. It's almost like the color fades out.


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Invisibler.lutece
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Re: Giving Isolating a shot. [Re: Thadeous]
    #23625330 - 09/08/16 10:25 AM (7 years, 4 months ago)

My understanding from reading what RogerRabbit has to say on the topic is that your first plate should be transferred as soon as the mycelium starts to grow so that you can hopefully outrun any potential contams. After that, you go one by one until you have clean growth on a single plate (it might only take that one transfer), and from there you make 10-20 plates from it. The exact amount is up to you. Basically you take a rice-grain sized sample from leading edges all around the plate so that you can start isolating sectors. Once those 10-20 plates start growing out, keep transferring them until you have isolates (all growth coming from a single sector) and then those isolates get spawned and fruited individually. (So you're spawning 10-20 jars at the same time and then they will all be fruited.) That way, you can determine which isolate performs the best. Keep that one as your master and take it to bulk.

As you can tell, labeling is very important in this.


--------------------
One goes into an experiment knowing one might fail.
But one does not undertake an experiment knowing one HAS failed.


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Invisibler.lutece
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Re: Giving Isolating a shot. [Re: just_curious]
    #23625343 - 09/08/16 10:30 AM (7 years, 4 months ago)

When you say that your color disappears after the jars fully colonized, are you referring to your spawning jars or are you using jars as petris that are fully colonized?


--------------------
One goes into an experiment knowing one might fail.
But one does not undertake an experiment knowing one HAS failed.


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InvisibleNumeroEno
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Re: Giving Isolating a shot. [Re: just_curious]
    #23625361 - 09/08/16 10:36 AM (7 years, 4 months ago)

Quote:

just_curious said:
Also, I never have "apparent" bacteria. It never reveals itself and I use colored agar. Which leads me to think that my agar is clean. What when my jars are fully colonized, there's no more color. Does this happen to you guys, or is it definitely a contam. It's almost like the color fades out.




Whenever I put food coloring in agar the color disappears once the plate is fully colonized. I've never given it too much thought but it would be cool to know why that happens. I'm guessing that the mycelium's digestive enzymes break down the food coloring.


--------------------

:gd_icon:  Let it grow! Let it grow! Greatly yield!  :gd_icon:
What shall we say, shall we call it by a name
As well to count the angels dancing on a pin
Water bright as the sky from which it came
And the name is on the earth that takes it in

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Offlinebuttermycoface


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Re: Giving Isolating a shot. [Re: r.lutece]
    #23625367 - 09/08/16 10:38 AM (7 years, 4 months ago)

my colored agar also looses its color when its fully colonized.

Quote:

r.lutece said:
When you say that your color disappears after the jars fully colonized, are you referring to your spawning jars or are you using jars as petris that are fully colonized?




his referring to his food coloring disappearing when the plate is fully colonized


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Offlinejust_curious
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Re: Giving Isolating a shot. [Re: buttermycoface]
    #23625417 - 09/08/16 11:05 AM (7 years, 4 months ago)

Quote:

buttermycoface said:
my colored agar also looses its color when its fully colonized.

Quote:

r.lutece said:
When you say that your color disappears after the jars fully colonized, are you referring to your spawning jars or are you using jars as petris that are fully colonized?




his referring to his food coloring disappearing when the plate is fully colonized




^^This^^ I don't know why I said jars lol. I just don't understand where my contams are coming from. My agar appears clean, my jars appear clean, but as soon as I spawn to bulk, BOOM, green. So braise of this, I'm starting from scratch with an isolated strain (once I get one). I started with MS to agar and then to CVG (no contams), then cloned live tissue to agar, then to CVG, no issue. I made like 6-7 monos as a complete noob and had no problems, but as of lately...FML!!. I figured I would scratch everything and start back over with the basics. I just wanted to confirm that my color being gone wasn't actually a contam.


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Invisibler.lutece
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Re: Giving Isolating a shot. [Re: just_curious]
    #23626477 - 09/08/16 05:08 PM (7 years, 4 months ago)

I don't think you're supposed to allow dishes to colonize completely. I'm not sure if it increases contamination potential, but I know it makes isolating sectors more difficult.


--------------------
One goes into an experiment knowing one might fail.
But one does not undertake an experiment knowing one HAS failed.


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OfflineX2QE
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Re: Giving Isolating a shot. [Re: r.lutece]
    #23626505 - 09/08/16 05:19 PM (7 years, 4 months ago)

I have read that the walls of the petri harbor contams.:shrug:


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InvisibleNumeroEno
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Re: Giving Isolating a shot. [Re: X2QE]
    #23626537 - 09/08/16 05:31 PM (7 years, 4 months ago)

There are advantages to letting your petris get overgrown, like pins. If you wrap your plates right after they solidify and let them cool in front of the flowhood they stay clean. Letting a plate pin and dropping the pin on a new plate is a quick way to a nice clean proven fruiting culture.


--------------------

:gd_icon:  Let it grow! Let it grow! Greatly yield!  :gd_icon:
What shall we say, shall we call it by a name
As well to count the angels dancing on a pin
Water bright as the sky from which it came
And the name is on the earth that takes it in

DOG FOOD AGAR

MY ELECTRIC INOCULATION LOOP


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OfflineX2QE
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Re: Giving Isolating a shot. [Re: NumeroEno]
    #23626723 - 09/08/16 06:32 PM (7 years, 4 months ago)

Quote:

NumeroEno said:
There are advantages to letting your petris get overgrown, like pins. If you wrap your plates right after they solidify and let them cool in front of the flowhood they stay clean. Letting a plate pin and dropping the pin on a new plate is a quick way to a nice clean proven fruiting culture.




I didn't know pins fruiting in the petri was a practice, I thought it was just an anomaly. :wowz:

I have some extra petris (pasty plates) that have been colonizing for about 2-3 weeks now. They are pretty overgrown. Do I just let them keep growing? Or do I need to introduce some sort of fresh air exchange?


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Offlinespore-ty
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Re: Giving Isolating a shot. [Re: X2QE]
    #23626784 - 09/08/16 06:47 PM (7 years, 4 months ago)

Is agar agar, potato flakes, and malt extract a good agar recipe?


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Invisibler.lutece
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Re: Giving Isolating a shot. [Re: spore-ty]
    #23626824 - 09/08/16 06:56 PM (7 years, 4 months ago)

Ingredients seem good, not sure about measurements.

Here are some tested agar recipes: What are some good agar media recipes?


--------------------
One goes into an experiment knowing one might fail.
But one does not undertake an experiment knowing one HAS failed.


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OfflineX2QE
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Re: Giving Isolating a shot. [Re: spore-ty]
    #23626855 - 09/08/16 07:02 PM (7 years, 4 months ago)

I have never tried it. I am still new. I have only used MEA which has worked for me so far.

Usually do 1/2 of this recipe. If I remember correctly it makes about 15-20 pasty plates.


Malt Extract Agar (3%)(MEA)(General Microbiology)(FDA M93)
-----------------------------------------------------------------
30 g Malt extract
20 g Agar
1 L Distilled water

Boil to dissolve ingredients. Avoid overheating, which causes softening of agar and darkening of medium color. Autoclave 15 min at 121?C. Dispense 20-25 ml into sterile 15 x 100 mm petri dishes. Final pH, 5.5 ? 0.2.

This medium is recommended as a general maintenance medium. [1]


This is the post I got the recipe from: https://www.shroomery.org/forums/showflat.php/Number/3077006


Edited by X2QE (09/08/16 07:03 PM)


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Offlinespore-ty
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Re: Giving Isolating a shot. [Re: X2QE]
    #23626860 - 09/08/16 07:03 PM (7 years, 4 months ago)

Regular malt extract or light malt extract thanks for the links ill check em out when im home


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Offlinejust_curious
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Re: Giving Isolating a shot. [Re: spore-ty]
    #23626907 - 09/08/16 07:14 PM (7 years, 4 months ago)

For 8 "pasty plates", I use 2.5 grams agar, 2 teaspoons of potato flakes, heat until mostly dissolved. Add in 1.8 grams of honey, and prior to pouring Into plates, put 2 drops of any dark colored food coloring. Pour into your pasty plates and PC at 15psi for 45 minutes,


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InvisiblemushboyMDiscord
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Re: Giving Isolating a shot. [Re: just_curious]
    #23626929 - 09/08/16 07:19 PM (7 years, 4 months ago)

no water?


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InvisibleNumeroEno
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Re: Giving Isolating a shot. [Re: X2QE]
    #23627006 - 09/08/16 07:44 PM (7 years, 4 months ago)

Quote:

X2QE said:
Quote:

NumeroEno said:
There are advantages to letting your petris get overgrown, like pins. If you wrap your plates right after they solidify and let them cool in front of the flowhood they stay clean. Letting a plate pin and dropping the pin on a new plate is a quick way to a nice clean proven fruiting culture.




I didn't know pins fruiting in the petri was a practice, I thought it was just an anomaly. :wowz:

I have some extra petris (pasty plates) that have been colonizing for about 2-3 weeks now. They are pretty overgrown. Do I just let them keep growing? Or do I need to introduce some sort of fresh air exchange?




Yep just let the plate sit there and it will pin eventually. If you use poured plates wrapped in parafilm they get pretty decent gas exchange, but you don't want FAE.

Also, speaking of agar, why doesn't anyone ever talk about dog food agar? It's way the fuck better than PDA. I like to rotate between dog food agar and MEA.


--------------------

:gd_icon:  Let it grow! Let it grow! Greatly yield!  :gd_icon:
What shall we say, shall we call it by a name
As well to count the angels dancing on a pin
Water bright as the sky from which it came
And the name is on the earth that takes it in

DOG FOOD AGAR

MY ELECTRIC INOCULATION LOOP


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OfflineX2QE
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Re: Giving Isolating a shot. [Re: NumeroEno]
    #23627086 - 09/08/16 08:07 PM (7 years, 4 months ago)

Quote:

NumeroEno said:
Quote:

X2QE said:
Quote:

NumeroEno said:
There are advantages to letting your petris get overgrown, like pins. If you wrap your plates right after they solidify and let them cool in front of the flowhood they stay clean. Letting a plate pin and dropping the pin on a new plate is a quick way to a nice clean proven fruiting culture.




I didn't know pins fruiting in the petri was a practice, I thought it was just an anomaly. :wowz:

I have some extra petris (pasty plates) that have been colonizing for about 2-3 weeks now. They are pretty overgrown. Do I just let them keep growing? Or do I need to introduce some sort of fresh air exchange?




Yep just let the plate sit there and it will pin eventually. If you use poured plates wrapped in parafilm they get pretty decent gas exchange, but you don't want FAE.

Also, speaking of agar, why doesn't anyone ever talk about dog food agar? It's way the fuck better than PDA. I like to rotate between dog food agar and MEA.




How long does it typically take to pin? Idk about the dog food agar. I will give it a go next. What do you like about it?


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