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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
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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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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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InvisibleNumeroEno
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Re: Giving Isolating a shot. [Re: X2QE]
    #23627257 - 09/08/16 09:16 PM (7 years, 4 months ago)

The dog food agar is just really easy and it grows beautiful cultures. It seems to help with making the plates pin too. I had some tampanensis fruit inside the petri dish and I never expected that to happen.

Anyway it can take as little as a week after full colonization. It depends on the mushroom. Cubes usually pin 2 weeks to a month after the plate gets fully colonized. Those invitro pins are also nice for transferring from a contaminated plate.


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

: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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InvisibleBoogieman47
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Re: Giving Isolating a shot. [Re: NumeroEno]
    #23627390 - 09/08/16 09:57 PM (7 years, 4 months ago)

I have noticed using a gram or two less of the nutrients and pouring thin agar I'll get pins in a couple weeks and it helps with nice rhizo growth


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OfflinePsilocyBen17
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Re: Giving Isolating a shot. [Re: Boogieman47]
    #23627487 - 09/08/16 10:33 PM (7 years, 4 months ago)

The sooner you can transfer after you see clean growth the better......you aren't SUPPOSED to let a petri colonize fully but I dont always get around to things as fast as I should *sigh*


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Offlinetombosley8
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Re: Giving Isolating a shot. [Re: PsilocyBen17]
    #23627662 - 09/09/16 12:07 AM (7 years, 4 months ago)

well if your using the plate to noc up masters and your sure your culture is clean then letting it grow almost all the way would be ideal.

oh yeah and dog food agar is awesome
got this info from blinding leaf:

"u can use dry dog or cat food plus agar for a recipe.  10g GROUND UP KIBBLE, 10g agar powder, 500ml water.  i just use as is straight into PC, but some people simmer it and strain.  whatever works for u.  seriously a great recipe though."


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


Edited by tombosley8 (09/09/16 12:10 AM)


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Offlinejust_curious
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Re: Giving Isolating a shot. [Re: mushboy]
    #23627875 - 09/09/16 03:14 AM (7 years, 4 months ago)

Quote:

just_curious said:
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,



Quote:

mushboy said:
no water?




Lol yes, 1/2 cup of water for the recipe above. Just repeat for 16, 24, etc. Also, one time I only had jell food coloring. This is perfectly fine, just cut back on the honey if you have to use it. Use like 1.3 grams of honey if you use the gel food coloring.


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InvisibleNumeroEno
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Re: Giving Isolating a shot. [Re: tombosley8]
    #23628341 - 09/09/16 08:19 AM (7 years, 4 months ago)

Quote:

tombosley8 said:
well if your using the plate to noc up masters and your sure your culture is clean then letting it grow almost all the way would be ideal.

oh yeah and dog food agar is awesome
got this info from blinding leaf:

"u can use dry dog or cat food plus agar for a recipe.  10g GROUND UP KIBBLE, 10g agar powder, 500ml water.  i just use as is straight into PC, but some people simmer it and strain.  whatever works for u.  seriously a great recipe though."




Yeah blindingleaf turned me onto it too. I feed my dog the nature's domain grain free salmon formula from costco and use that in my agar. When reading the ingredients I noticed that sweet potato is the first ingredient, and the mushrooms seem to love it. Here's a dog food agar plate that I let fully colonize and pin. The pin in the middle was transferred to a new plate. I left this plate on the shelf for a few more days and the pins got pretty big.



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

: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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OfflinePsilocyBen17
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Re: Giving Isolating a shot. [Re: tombosley8]
    #23628373 - 09/09/16 08:37 AM (7 years, 4 months ago)

Quote:

tombosley8 said:
well if your using the plate to noc up masters and your sure your culture is clean then letting it grow almost all the way would be ideal.

oh yeah and dog food agar is awesome
got this info from blinding leaf:

"u can use dry dog or cat food plus agar for a recipe.  10g GROUND UP KIBBLE, 10g agar powder, 500ml water.  i just use as is straight into PC, but some people simmer it and strain.  whatever works for u.  seriously a great recipe though."




How does this smell when you PC it? Id like to try.


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InvisibleNumeroEno
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Re: Giving Isolating a shot. [Re: PsilocyBen17]
    #23628395 - 09/09/16 08:47 AM (7 years, 4 months ago)

It doesn't smell bad at all. It smells like dog food but it isn't overpowering.


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

: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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OfflinePsilocyBen17
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Re: Giving Isolating a shot. [Re: NumeroEno]
    #23628417 - 09/09/16 08:59 AM (7 years, 4 months ago)

Girls don't like pressure cooker smells....


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InvisibleNumeroEno
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Re: Giving Isolating a shot. [Re: PsilocyBen17]
    #23628437 - 09/09/16 09:06 AM (7 years, 4 months ago)

The best thing I ever did was buy that 25x. No PC noises or smells in the kitchen. My girlfriend was grateful :yesnod:


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

: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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OfflinePsilocyBen17
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Re: Giving Isolating a shot. [Re: NumeroEno]
    #23632438 - 09/10/16 01:35 PM (7 years, 4 months ago)

no noise? No smell? Cool, didn't know that about the 25x....doesn't the steam need to escape somehow?


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OfflineMushierage
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Re: Giving Isolating a shot. [Re: PsilocyBen17]
    #23632447 - 09/10/16 01:38 PM (7 years, 4 months ago)

Wife walks into the kitchen while PC is going.

"That thing stinks."

Me: "Well so do I sometimes, but you don't say anything about it and pretend everything is fine."

Wife smiles in knowing agreement and walks away.


--------------------
Don't like researching posts? Read this! . Also, if you're new and your posts contain the words: Humidifer, incubator, air-stone, or heater, then you need to read and UTFSE before asking people to review your setup.  OR... You should be cultivating reptiles and fish, not mushrooms.



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InvisibleNumeroEno
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Re: Giving Isolating a shot. [Re: PsilocyBen17]
    #23632485 - 09/10/16 01:51 PM (7 years, 4 months ago)

Quote:

PsilocyBen17 said:
no noise? No smell? Cool, didn't know that about the 25x....doesn't the steam need to escape somehow?




The only time you let it vent steam is when you're starting your run, and it vents steam quietly. It uses a stopcock instead of a weight, and you control the heat and pressure with a thermostat. Once you start your run it builds pressure, and does an emergency release of the pressure goes over 25. As long as you keep your pressure between 15 and 20 the run is completely silent. When if cools off a vacuum forms so if you want to avoid pulling a vacuum you need to open the stopcock when it's at about 1psi.


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

: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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Offlinetombosley8
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Re: Giving Isolating a shot. [Re: Mushierage]
    #23632538 - 09/10/16 02:05 PM (7 years, 4 months ago)

yeah dog food is awesome but I think most of the dog food I use for agar is mostly corn, meat/meal, wheat, soybean etc. and since I also use it for mudafuka's bottle tek I went with the one with the lowest fat as he recommended(I think it was him) and the cheapest stuff you can get.

I don't feed my dogs that crap.

They are grain free and salmon eaters too.

Good to know that the good stuff works for agar too.

Could be another good mixup of nutes to keep the cultures from getting lazy.

The cheap stuff is also a good changeup from potato agars since it's mostly corn.

and about the AA electric sterilizers they are very quiet and let off very little steam/smell while cooking, but I always vent the steam immediately after the cycle while still at full pressure so that part is very loud.

I'm just too impatient and busy (and not sure the proper procedure) to come back once it gets down to 1 psi to open the valve before it cools down.

Also when doing muda's bottle tek the dog food does smell quite bad even with the sterilizers so they do let some smell out for sure.

Agar dog food doesn't seem to smell bad probably because it's not being cooked as long.

edit:
Quote:

NumeroEno said:

The only time you let it vent steam is when you're starting your run, and it vents steam quietly. It uses a stopcock instead of a weight, and you control the heat and pressure with a thermostat. Once you start your run it builds pressure, and does an emergency release of the pressure goes over 25. As long as you keep your pressure between 15 and 20 the run is completely silent. When if cools off a vacuum forms so if you want to avoid pulling a vacuum you need to open the stopcock when it's at about 1psi.




good to know about the 1 psi on opening the valve.




Edited by tombosley8 (09/10/16 02:11 PM)


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Re: Giving Isolating a shot. [Re: tombosley8]
    #23633203 - 09/10/16 05:23 PM (7 years, 4 months ago)

I like to let it cool all the way and pull a vacuum. I wrap a clorox wipe around the stopcock and open it in front of the flowhood. With spawn bags it needs upwards of 8 hours to cool anyway, so I usually do my run before bed. I have a countdown timer that I plug it into, so I just let it get up to pressure and set the timer for 4 hours and go to bed. By the time I get home from work the next day the bags are ready to inoculate.


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DOG FOOD AGAR

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