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OfflineVooch
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Agar noobie questions
    #23593653 - 08/30/16 01:09 PM (7 years, 5 months ago)

Ok, so this is my first time trying agar out. I have always just been an inoculate with multispore syringe casual grower. I want to reduce contam and colonization time. so i bought 250 grams of Malt Extract Agar from mycosupply, and i just want to make sure i am doing this right before i use it and waste it...

Here is what i think i am supposed to do, please tell me if it is right or wrong as i havent really found a guide for the pre mixed stuff.

1. follow directions on bottle and mix 25g mea with 500 ml water and in a mason jar and then sterilize for 35 mins at 15 psi... duh

2. after it is sterilized though, do i just pour enough to coat the bottom of the petri dishes? (also i will be doing the pouring inside of a glovebox i made to reduce contams)

3. After i let the agar cool in the petri dishes, i was going to knock them up with multispore syringes, about .05 CC's per dish. is that enough per dish?

4. let the petri dishes colonize for a few days, then in a sanitary environment cut off pieces of the agar to inoculate grain spawn jars.


I am an amateur mycologist trying to learn, and appreciate any help the community can give me on these questions, thank you.

Vooch

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Invisibleh0ldthedoor
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Re: Agar noobie questions [Re: Vooch]
    #23593689 - 08/30/16 01:23 PM (7 years, 5 months ago)

Why would you make the agar, PC it for sterilization, then proceed in a manner designed to reduce contams?

Wouldn't it make more sense to make the agar, put it where it needs to go, and sterilize the plate/jar/whatever as a whole?

Just a thought.

Oh and you don't need to worry about cc measurements if you're incoulating from syringe to agar. You'll be working in drops. A drop or two to cool your loop/needle and a single drop for the plate.


--------------------
Always keep your foes confused. If they are never certain who you are or what you want, they cannot know what you are like to do next. Sometimes the best way to baffle them is to make moves that have no purpose, or even seem to work against you.

– Petyr Baelish

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OfflineVooch
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Re: Agar noobie questions [Re: h0ldthedoor] * 1
    #23593695 - 08/30/16 01:25 PM (7 years, 5 months ago)

So make it, then pour it into the petri dishes, and then PC the petri dishes? and i dont know, like i said, ive never done agar, i thought you were supposed to PC it after it is made and then pour it into the dishes?

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OfflinePsilocyBen17
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Re: Agar noobie questions [Re: Vooch] * 1
    #23593705 - 08/30/16 01:27 PM (7 years, 5 months ago)

Hey man, welcome to the shroomery....

sounds like you have a good handle on things, I just wanted to point a few things out.

1. Don't use a mason jar to pour from. That would be messy. Use something with a long neck, like a wine bottle, to make the pouring easier....

2. 500ml of agar will make a ton of plates....I usually do 1/2 cup (125ml) at a time for about 20 plates. Just make sure you do the math accordingly. (25g per 500ml....12.5g per 250ml....etc)

3.Make it a little on the wet side if you plan on germinating spores, and be prepared to wait atleast a week to see germination.

4. If you let your plates grow out and then transfer that plate into a grain jar you will be putting the spores right on the grains, and this is what you are trying to avoid. First, transfer some clean growth to a second plate. then let that plate grow out. Only put it on grain once you are sure its clean. That way each new generation of plates will be cleaner. Most people do 3 or 4 transfers (or generations of plates) before they are sure their spores or clean. it could take many more if cloning from a dirty fruit found in the wild.


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OfflinePsilocyBen17
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Re: Agar noobie questions [Re: h0ldthedoor] * 1
    #23593711 - 08/30/16 01:29 PM (7 years, 5 months ago)

Quote:

h0ldthedoor said:
Why would you make the agar, PC it for sterilization, then proceed in a manner designed to reduce contams?

Wouldn't it make more sense to make the agar, put it where it needs to go, and sterilize the plate/jar/whatever as a whole?

Just a thought.

Oh and you don't need to worry about cc measurements if you're incoulating from syringe to agar. You'll be working in drops. A drop or two to cool your loop/needle and a single drop for the plate.




There are both "pour" and "no pour" teks when it comes to agar....don't confuse the guy. He is on the right track.

I'm really happy to see this because IMO everyone should start with agar. its simple.


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OfflineVooch
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Re: Agar noobie questions [Re: PsilocyBen17]
    #23593720 - 08/30/16 01:31 PM (7 years, 5 months ago)

This reply was so helpful thank you! im glad i asked. cause i was just going to knock up a bunch of plates at once.

So I should knock up one first with my spores, make sure there are no contams, then use that to knock up more dishes before even thinking about going the grain jars correct?

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Invisibleh0ldthedoor
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Re: Agar noobie questions [Re: Vooch]
    #23593721 - 08/30/16 01:31 PM (7 years, 5 months ago)

Never done what you're doing with actual petri dishes. Could be (and likely am) completely wrong. After working with just pasty plates, that order of operations seemed a bit unusual.

Wait for someone else to chime in

Edit: Ben came to save the day. Again, only worked with pasty plates here. Any benefit to pour vs no-pour agar for beginners? From reading up on pour agar, it looks like there's more vectors for contamination.

Edit-Edit: How many plates do you have? Inoculate more than one plate and work from the one(s) with the best growth. First go-round, only inoculated four, one showed noticeable growth and even that was shit. Now, dozens are sitting, doing their thing.


--------------------
Always keep your foes confused. If they are never certain who you are or what you want, they cannot know what you are like to do next. Sometimes the best way to baffle them is to make moves that have no purpose, or even seem to work against you.

– Petyr Baelish

Edited by h0ldthedoor (08/30/16 01:44 PM)

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OfflineGreg
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Re: Agar noobie questions [Re: h0ldthedoor]
    #23593779 - 08/30/16 01:47 PM (7 years, 5 months ago)

OP, your procedure sounds correct to me.

Couple of things though:
25g sounds a bit much for normal MEA premix. I use 18g or 20g.
I guess if it says to use 25g on the package you could use that much, but that's going to be some very stiff agar.

Make sure you wrap your petris after inoculating, especially if you like to pick them up and look at them fairly often like I do.
What you want is called parafilm, several of the sponsors here have it.
Some people also use saran wrap I've noticed, but I don't think it would work as well.

Here's a Youtube vid on how to use it:


If your petris get lots of condensation on the lids, you can put a jar of hot water on top of the stack as it's cooling. You can also try pouring after the agar has cooled slightly.

One other thing:
You mentioned a glovebox? That's old school and not really used anymore. Typically what people use now is a still air box (SAB) or a flowhood.

SAB tek: https://www.shroomery.org/forums/showflat.php/Number/20048771

Edited by Greg (08/30/16 01:53 PM)

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OfflineVooch
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Re: Agar noobie questions [Re: Greg]
    #23593813 - 08/30/16 01:55 PM (7 years, 5 months ago)

I say glovebox, although i guess technically it is a still air box. still learning all the correct terminology.and i have 25 petri dishes. I am going to try to take pictures of the entire process from start to finish and hopefully be able to make a guide for people who are confused like i am about working with the pre mixed MEA. yall have been super helpful so far, so glad i decided to ask questions before proceeding with this.

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OfflineKenetic
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Re: Agar noobie questions [Re: Vooch]
    #23593871 - 08/30/16 02:11 PM (7 years, 5 months ago)

The pour method is awesome.  I've never tried the pasty tech, but it seems like you can do way more dishes with way less work and space.  plus it's fun and easy.

Don't try to sterilize it in the petri dishes, you'll either melt them or render them useless by boil-over.

Try to get about 3/16ths to a 1/4 inch in each dish, any less and they will dry out quicker.  Any more and you are just wasting it.

Your plan sounds pretty good overall, but if you are gonna use a spore solution try to smear the drop in a line in the center using an inoculation loop.  That way if the drop is contaminated, you'll have a better chance of growing myc that doesn't have a contam growing right on top of it.


--------------------
Todo Cambia
   

               
                                                :cookiemonster::elmo:



DMT said: Everyone know's me, they just don't know it yet

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Invisibleh0ldthedoor
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Re: Agar noobie questions [Re: Kenetic]
    #23593942 - 08/30/16 02:40 PM (7 years, 5 months ago)

Pasty Plates are nice because you can decide "hey, let's start working with agar" and actually work with agar in the same day with supplies obtained locally and paid for with cash. There is no need to order any supplies over the internet to be delivered to your home. Plus, if you've attempted pf tek and are moving onto agar, the widemouth half-pint jars work well as plates.

Petri dishes, if they are single-use plastic, yes definitely do not PC.

If they are glass, you can reuse them by using a PC to sterilize them when you're done. However, it is indeed inadvisable to PC filled petri dishes.


--------------------
Always keep your foes confused. If they are never certain who you are or what you want, they cannot know what you are like to do next. Sometimes the best way to baffle them is to make moves that have no purpose, or even seem to work against you.

– Petyr Baelish

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OfflinePsilocyBen17
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Re: Agar noobie questions [Re: Vooch]
    #23594297 - 08/30/16 04:36 PM (7 years, 5 months ago)

Quote:

Vooch said:
This reply was so helpful thank you! im glad i asked. cause i was just going to knock up a bunch of plates at once.

So I should knock up one first with my spores, make sure there are no contams, then use that to knock up more dishes before even thinking about going the grain jars correct?




Hi Vooch,

Yes, this is correct. Just be aware that contams can sometimes hide on plates, meaning they are dirty even if they look clean....

As a rule I do at least 3 transfers even if it looks clean on the first transfer....better to waste $0.10 worth of agar than a $1 worth of grain (and all the work involved in cleaning out contamed jars).

When it comes to plates just be aware that a little agar goes a long way. I  only knock up 3 or 4 jars with clean agar, and then use these as master jars for grain to grain transfers to make up to 400 jars of spawn. Instead of using 400 agar wedges to knock up 400 jars....

not only is the first way less labour intensive, it is also faster. Speed is important cuz it gives the contams less of a window...

Hope you can understand what I mean. Post up if you don't :thumbup:


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Edited by PsilocyBen17 (08/30/16 04:39 PM)

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OfflineKenetic
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Re: Agar noobie questions [Re: PsilocyBen17]
    #23594720 - 08/30/16 06:25 PM (7 years, 5 months ago)

I think you guys are giving some great advice.  I like the way the pasty tek is illustrated, it's brilliant and is like the pf tek version of agar.  And it's a great point that you don't have to order anything online.  People notice.
I was intimidated at first using the pour method for my first time but overall I had a great success.  I've gotten farther with my spawn jars than ever before.  I've been doing 12 transfers at a time and getting a lot of practice. I'm looking forward to fruiting (in a month or so). 
I like the grain master method but in my case I had ten jars sterilized so I just cut out ten wedges from four plates and gave each jar one.  A little more work than g2g but not backbreaking.  I'm gonna save one jar to master the next batch, and go from there.


--------------------
Todo Cambia
   

               
                                                :cookiemonster::elmo:



DMT said: Everyone know's me, they just don't know it yet

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