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OfflineLTSwoomz
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Registered: 02/16/13
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About to try Agar for the first time...
    #18996961 - 10/18/13 07:10 PM (10 years, 4 months ago)

So I got some Agar and Barley Malt Powder from the local HF store. Got 20 x-plate petri dishes in the mail today too. After my last few attempts at innoc'ing sterilized grain jars from a syringe it was decided that my syringe must be contaminated, which is unfortunate because it's new. I have another one I haven't used yet. The plan is to use agar to either outrun contam and get useable cultures or conclude that my syringe is completely bad. I'm going to do a 1/4th batch and just do 5 plates for now. I had some questions as I'm hesitant.

What I'm Working With
20 X-plate Agar plates (Y-plates were out of stock)
Agar and Barley Malt Powder
Glovebox /w Tyvek sleeves instead of gloves.
Pressure Cooker
WBS
SFD lids, foregoing SHIPs since the intention is to innoc with wedges.

Questions
Am I correct in assuming that I can possibly get healthy myc from contaminated syringe using agar? I guess in my head I imagine cutting slices of healthy "sectors" on one agar plate and transferring it to another, creating healthy, clean samples.

I'm nervous about doing this right and being sanitary. I understand that while working in the glovebox, I should always use direct but smooth hand movements to avoid extra airflow, and open lids towards me and keep my hands away from the tops of the plates and jars. Do I need to PC the poured petri dishes afterwards and before I knock them up with the spore syringe?

I'm new to agar and am reading through a LOT of material, but I was wondering if anyone had any suggestions on guides (preferably with pictures or videos) on it? Specifically instructions on storing knocked up agar plates, sectioning, isolation, and anything to do with handling agar in a glovebox without a HEPA filter.

I've read around the forums. The consensus seems to be that barley malt powder is light malt extract, or at least works exactly the same. I'm asking for confirmation that I can use it for MEA?

If I can't get anything useable to grow on this agar, what's the next most feasible step to producing useable myc? I really, really want to do monotubs but I've tried ~ 12 WBS jars over the last 3 months and not a single one has grown anything but contams, and this is over the course of 3 or 4 different syringes, bought at seperate times, with the utmost care given to sanitation and sterilization. I'm getting a little discouraged at this juncture.


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Edited by LTSwoomz (10/18/13 07:21 PM)


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Offlineblueconfusion
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Re: About to try Agar for the first time... [Re: LTSwoomz]
    #18997009 - 10/18/13 07:18 PM (10 years, 4 months ago)

:takingnotes: I am too doing agar for the first time and want as much info as possible


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InvisiblePastywhyteMDiscord
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Re: About to try Agar for the first time... [Re: blueconfusion]
    #18997047 - 10/18/13 07:26 PM (10 years, 4 months ago)

When it comes to agar many things can be used as nutes. I usually do PDA with potato flakes, but I have also used grain soak water (just filtered the gypsum out), dog food, Malt extract, honey and karo. As for technique in your SAB that will only come with practice. A good idea (and you might feel dumb doing it) is to practice your movements in there beforehand.

Agar is awesome and once you get over the initial uncertainty you will love it :awesomenod:


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OfflineStupendous-Yappi
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Re: About to try Agar for the first time... [Re: LTSwoomz]
    #18997323 - 10/18/13 08:23 PM (10 years, 4 months ago)

Quote:


Questions
Am I correct in assuming that I can possibly get healthy myc from contaminated syringe using agar? I guess in my head I imagine cutting slices of healthy "sectors" on one agar plate and transferring it to another, creating healthy, clean samples.



Yeah, you just keep on transferring until you get a clean dish.

Quote:

Do I need to PC the poured petri dishes afterwards and before I knock them up with the spore syringe?



Not if the agar and empty dishes were already PC'd. I'm guessing it would make a big mess trying to PC the poured dishes.


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OfflineStromriderM
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Re: About to try Agar for the first time... [Re: Stupendous-Yappi]
    #18997349 - 10/18/13 08:29 PM (10 years, 4 months ago)

Agar rocks :rockon:

Just watch the let's grow mushrooms video series and it will explain all the ins and outs of using agar much better than any of us ever could


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InvisiblePastywhyteMDiscord
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Re: About to try Agar for the first time... [Re: Stromrider]
    #18997420 - 10/18/13 08:40 PM (10 years, 4 months ago)

Quote:

Stromrider said:
Agar rocks :rockon:

Just watch the let's grow mushrooms video series and it will explain all the ins and outs of using agar much better than any of us ever could



:whathesaid: I love those videos :awesomenod:


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OfflineLTSwoomz
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Re: About to try Agar for the first time... [Re: Pastywhyte]
    #18997457 - 10/18/13 08:48 PM (10 years, 4 months ago)

Won't moving the PC'd dishes to the GB contaminate them?

And thanks for your answers guys!


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Edited by LTSwoomz (10/18/13 08:48 PM)


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Offlinellama_police
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Re: About to try Agar for the first time... [Re: Pastywhyte]
    #18997461 - 10/18/13 08:48 PM (10 years, 4 months ago)

Don't over complicate your recipe if you're starting out. 2g agar + 2g malt extract per 100ml of water. Distilled preferred, but I use tap water. I boil my water first to take out chlorine etc and it also helps with dissolving the powders. Just mix it together and PC for 25 minutes in a bottle (I use an Absolut Vodka bottle), let it cool (in a SAB) until it nearly solidifies again then pour into your dishes and leave it to set.


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OfflineLTSwoomz
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Re: About to try Agar for the first time... [Re: llama_police]
    #18997481 - 10/18/13 08:51 PM (10 years, 4 months ago)

Quote:

llama_police said:
Don't over complicate your recipe if you're starting out. 2g agar + 2g malt extract per 100ml of water. Distilled preferred, but I use tap water. I boil my water first to take out chlorine etc and it also helps with dissolving the powders. Just mix it together and PC for 25 minutes in a bottle (I use an Absolut Vodka bottle), let it cool (in a SAB) until it nearly solidifies again then pour into your dishes and leave it to set.




Yeah I don't intend to complicate the recipe but I don't have direct access to LME and so I looked up if Barley Malt Powder works as well and from what I've read from a forum search it's basically LME called something different.

How long should I boil the water? I'll be using tap water as well.

As for letting it try until it nearly solidifies... Does that mean I just shake it up really well when ready for the pour?


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OfflinebEelzeBosS
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Re: About to try Agar for the first time... [Re: LTSwoomz]
    #18997533 - 10/18/13 09:04 PM (10 years, 4 months ago)

Here's what I do...this way you wont get any or very few flakes in your agar:

-Put dry ingredients into an empty long-neck rum bottle.

-Measure out some distilled water, microwave until boiling.

-Using a funnel, pour about a third of the water right from the microwave into the rum bottle.

-Swirl around for about 20 seconds

-Repeat until all the water is well mixed with the dry ingredients

-Drill a small hole in rum bottle lid, stuff with polyfil, put lid on bottle, and cover with foil.

-Swirl the bottle one more time then pressure cook @ 15 psi for 20-30 minutes.

-Let cool for awhile, but not all the way.

-Move to SAB before removing the foil to pour into the petris.


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OfflineLTSwoomz
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Re: About to try Agar for the first time... [Re: bEelzeBosS]
    #18997552 - 10/18/13 09:09 PM (10 years, 4 months ago)

Thanks, that's very informative.


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InvisiblePastywhyteMDiscord
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Re: About to try Agar for the first time... [Re: llama_police]
    #18997574 - 10/18/13 09:15 PM (10 years, 4 months ago)

Quote:

Pastywhyte said:
When it comes to agar many things can be used as nutes. I usually do PDA with potato flakes, but I have also used grain soak water (just filtered the gypsum out), dog food, Malt extract, honey and karo.




Quote:

llama_police said:
Don't over complicate your recipe if you're starting out.




I don't use them all together :lol: What's simpler than a spoon of potato flakes :shrug:


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OfflineStupendous-Yappi
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Re: About to try Agar for the first time... [Re: LTSwoomz]
    #18997633 - 10/18/13 09:30 PM (10 years, 4 months ago)

Quote:

LTSwoomz said:
Won't moving the PC'd dishes to the GB contaminate them?

And thanks for your answers guys!


Wrap them in foil, then remove the foil in the Glove Box.


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InvisiblePastywhyteMDiscord
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Re: About to try Agar for the first time... [Re: bEelzeBosS]
    #18997651 - 10/18/13 09:34 PM (10 years, 4 months ago)

Don't pc plastic petris :smbfacepalm:


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OfflineStupendous-Yappi
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Re: About to try Agar for the first time... [Re: Stupendous-Yappi]
    #18997660 - 10/18/13 09:36 PM (10 years, 4 months ago)

Are the petri dishes glass, or the per-sterilized plastic?

The plastic ones will probable melt in the PC, but if their per-sterilized then just open the bag in the Glove Box.


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Offlinellama_police
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Re: About to try Agar for the first time... [Re: LTSwoomz]
    #18997743 - 10/18/13 10:04 PM (10 years, 4 months ago)

Quote:

LTSwoomz said:
Quote:

llama_police said:
Don't over complicate your recipe if you're starting out. 2g agar + 2g malt extract per 100ml of water. Distilled preferred, but I use tap water. I boil my water first to take out chlorine etc and it also helps with dissolving the powders. Just mix it together and PC for 25 minutes in a bottle (I use an Absolut Vodka bottle), let it cool (in a SAB) until it nearly solidifies again then pour into your dishes and leave it to set.




Yeah I don't intend to complicate the recipe but I don't have direct access to LME and so I looked up if Barley Malt Powder works as well and from what I've read from a forum search it's basically LME called something different.

How long should I boil the water? I'll be using tap water as well.

As for letting it try until it nearly solidifies... Does that mean I just shake it up really well when ready for the pour?




I just boil the kettle. And yes, wait until it's kind of starting to gel, give it a swirl and pour it. If it's too chunky, heat it up again in a bowl of warm water. I believe it sets at around 40 celsius


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OfflineMidnight Cyclone
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Re: About to try Agar for the first time... [Re: bEelzeBosS]
    #18997880 - 10/18/13 10:49 PM (10 years, 4 months ago)

Quote:

blueconfusion said:
:takingnotes: I am too doing agar for the first time and want as much info as possible




Right there with ya, all of my agar supplies should be arriving some time next week. So I'm gettin my read on.

Quote:

chaotic646 said:
Here's what I do...this way you wont get any or very few flakes in your agar:

-Put dry ingredients into an empty long-neck rum bottle.

-Measure out some distilled water, microwave until boiling.

-Using a funnel, pour about a third of the water right from the microwave into the rum bottle.

-Swirl around for about 20 seconds

-Repeat until all the water is well mixed with the dry ingredients

-Drill a small hole in rum bottle lid, stuff with polyfil, put lid on bottle, and cover with foil.

-Swirl the bottle one more time then pressure cook @ 15 psi for 20-30 minutes.

-Let cool for awhile, but not all the way.

-Move to SAB before removing the foil to pour into the petris.




Nice, I like this a lot. Your rum bottles have metal lids I assume?
Like I could use an empty Kraken bottle?


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OfflineLTSwoomz
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Re: About to try Agar for the first time... [Re: Pastywhyte]
    #18998091 - 10/19/13 12:12 AM (10 years, 4 months ago)

Well that fuckin sucks because when I got them I opened them to store em in two stacks. I figured they'd be PC-able because they're from a lab equipment website. Shit.


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OfflineStupendous-Yappi
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Re: About to try Agar for the first time... [Re: LTSwoomz]
    #18998116 - 10/19/13 12:23 AM (10 years, 4 months ago)

Quote:

LTSwoomz said:
Well that fuckin sucks because when I got them I opened them to store em in two stacks. I figured they'd be PC-able because they're from a lab equipment website. Shit.



It depends on the type of plastic so it may still be able to be PC'd. I would ask the vendor.

You can also do a fake Sterilization of the dishes. Just do a search for sterilizing plastic petri dishes. Many people claim to have good luck with "Sterilizing" their dishes. But since it's not truly sterile you may want to avoid it and any possible contam headaches.


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InvisiblePastywhyteMDiscord
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Re: About to try Agar for the first time... [Re: Stupendous-Yappi]
    #18998117 - 10/19/13 12:24 AM (10 years, 4 months ago)

Quote:

Stupendous-Yappi said:
You can also do a fake Sterilization of the dishes. Just do a search for sterilizing plastic petri dishes. Many people claim to have good luck with "Sterilizing" their dishes. But since it's not truly sterile you may want to avoid it and any possible contam headaches.



:lolwut:


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