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OfflinePsilicon
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Agar - what, why and how? Everything a beginner needs to know. * 60
    #21176491 - 01/26/15 05:37 AM (7 years, 8 months ago)

Let's face it: agar is scary.  There's no good reason to be afraid of agar, but the fear of agar persists, mainly because it's a tool professional scientists use and many amateur mycologists have the mistaken impression that it requires a scientific background and years of knowledge or experience.  Nothing could be farther than the truth, and in fact most agree that agar is much easier than working with liquid culture.  Many people, myself included, are convinced that agar is a prerequisite for making LCs, because no other inoculant is as reliable as agar by a long shot.

This is an explanation of the basics of agar.  This wasn't intended for experienced or well-read users, so if this bores you, good!  For everyone else, you can expect to come away from this knowing exactly what agar is, what we use it for, how it's prepared, and the terms to use in the Shroomery search engine if you want to read more about any of the materials or methods I talk about here.  I have peppered this document with links to things I find interesting, including some very helpful teks, but you won't need to click them to achieve these goals. 

My last and most important goal is to tell you how to formulate your own recipes out of whatever you have on hand.  Think of an agar plate like a prepackaged lunch for your fungi:  there are guidelines and parameters for what you want to have in there for them to be healthy, but they're just guidelines. If you put too much mustard on your sandwich it isn't going to kill you, and for the most part the same goes for your fungi. For this reason I've resisted the urge to just say, "Here's the fucking recipe.  Just make it like this."  I don't want you freaking out about the precision of your scales or anything like that, although precision does help if you can manage it.


What is agar?


This is not heroin.  Do not put it in your arm.


Agar is a gel. Extracted from seaweed, it acts just like the gelatin in Jello or the pectin in fruit jelly in that it dissolves easily in hot water, and upon cooling it turns the entire solution into a solid, locking anything inside of it into place.  The main molecule responsible for this property is agarose, but there are other associated molecules that contribute to or lessen the gelling properties.  Agar comes in many grades and purities, and the purer grades solidify at higher temperatures and have fewer minerals.  Scientists even use pure agarose for some purposes like electrophoresis and blotting, and it will work fine for our purposes as well, but it's much too expensive for what we need to accomplish. Some here use research grade agar formulated specifically for this purpose, but usually it's in the form of one of several premixed powders which can be easily measured out, dissolved and sterilized. These are also kind of expensive, but excellent, and their only drawback is a lack of flexibility. A big tub of potato dextrose agar (PDA) is never going to turn into light malt extract agar (LMEA). The stuff we use around here is usually food grade agar-agar, shown above. You can buy it at Oriental markets in the dessert section (or sometimes the Thai section), or just about anywhere on the internet.  Telephone Brand, shown below, is the most commonly used, and it comes in 25g packages which are identical to the 25g packages of its competitors except for the fact that this one has a large and inexplicable rotary phone on the front. If your packet has a smaller rotary phone, or even no phone at all, it'll be fine. Either way, you'll end up spending $2 for enough agar-agar to make about 60 plates.


Call your friends over, boys.  We're seaweed slime for dessert tonight!

There are two main advantages agar has over the other easily obtainable gelling agents.  One is that is turns into a gel at a much higher temperature, so you don't have to stick it in the fridge like your grandmother's Jello mold.  The other is that the air is full of bacteria and fungi which can break down pectin and gelatin with enzymes called pectinase and gelatinase.  Most contaminant species (and maybe the species you're trying to grow, since many, many species have pectinases) will simply break down gelatin or pectin and your beautiful petri dish will turn back into a liquid.  But while bacteria, molluscs and fungi which can break down agar with an enzyme called agarase do exist, they're rare and are about as likely to get into your agar plates on any given day as you are to get seaweed in your hair.  This means that an agar gel will almost always stay solid and continue to provide a stable platform even when it's hopelessly contaminated.  This is important. More on that later.

Why agar? (What's in it for me?)


Looking at this open plate hurts my soul.

There are a lot of benefits to agar.  Most of them involve the fact that on agar, you have a two-dimensional work surface which stays perfectly dry so you can see everything growing on it, but also allows the culture access to as much food and water as it wants.  This means you can easily see any contaminants like pin molds or the parasitic Trichoderma.  Healthy mushroom mycelium will stand out as clear as day.


Some idiot heard pleurotus grew well on coffee and decided to make an agar plate out of it.



Wedges of colonized agar can be cut out with flame-sterilized scalpel or hobby knife and then transferred onto a clean plate to isolate.  Mushrooms, both wild and cultivated, can be cut open and a tiny piece of tissue can be placed on the gel to recover, return to a vegetative state and begin to colonize again in a process called cloning.  Spores from dirty syringes or prints can be germinated, and contaminants can be left on the old agar as clean growth is transferred to a new plate.  Remember what I said earlier about how important it was for a culture medium to avoid turning into a nutrient soup?  You could never do this if your mycelium and sixteen bacterial colonies shared a puddle. The genetics of any multispore grow can be limited by selecting only the fastest-growing mycelium.  This will start you off with genetics that you know will have the ability to colonize quickly, vastly improving the speed of your grows.  Cultures can be refrigerated for storage, on petris in the short term or slants in the long term.  In short, if you want to consistently grow mushrooms that are more potent, bigger, faster, fruit more prolifically or which carry some cool mutation, or if you want to trade some random in the marketplace for his dirty sporeprint taken from a cool species or variety, or take prints of your own fruits and save them for later use, agar is the only reliable game in town.


HOW TO AGAR


Bet you already had one of these.


Two things you need to get together before doing agar work: a still air box and a pressure cooker/sterilizer/autoclave. Still air boxes are essential for a lot of other stuff in this hobby, like making sporeprints, inoculating grain jars and so on.  Ditto for pressure cookers.

There are two types of agar preparation teks:  pour and no-pour.  With a poured agar tek, you prepare the solution by boiling the ingredients to dissolve them, bottle it, sterilize it and then pour it into sterile petris to let it cool.  With a no-pour tek, you prepare the agar in the same way, distribute it among your containers (typically small jars or pp5 containers available at any supermarket, but never petri dishes, which can't survive a PC cycle!), let it cool, and then sterilize it.  There are benefits and drawbacks to both, but your decision on which of these methods to use depends on your circumstances. There isn't really a wrong decision.  I've done a lot of both, and I can't say I really prefer one over the other. 

-Poured agar takes a little skill to get right, although not much.  The petri dishes give you excellent visibility and they're broader and flatter than no-pour dishes, which means less frequent transfers due to the greater surface area of the agar and they take up less space in the fridge.

-No-pour agar requires absolutely no skill or competence to prepare without contamination, but I haven't encountered a pp5 or glass container that can rival the clarity of polyurethane petris, so it's harder to ask questions about the appearance of a culture here on the Shroomery.  Sealing and opening them isn't nearly the chore it is with petris, however, and in some places it can be really expensive to get petri dishes shipped.  All no-pour dishes are reusable.

Agar recipes can vary a LOT, but don't be daunted.  It just goes to show the huge variety of stuff that mushroom mycelium can grow on when you don't give it any choice. There's no magic formula and no hard-and-fast rules here, just a bunch of parameters.  Many experienced growers believe that rotating your formula frequently will prevent senescence, and some believe that senescence is caused by growing cultures on sugary media.  It might be that both are right, or that neither is. There are a lot of big names on both sides of those arguments, and the take-home is that nobody knows for sure so don't worry about it until they figure it out because it hasn't affected us much so far.

WHAT DO I PUT IN IT?


These are ranges for what fungi find acceptable.  Take any of these ingredients and use more than the minimum and less than the maximum and your recipe will be just fine. If you're freaking out about what your fungi think about the lunch you're making for them, keep in mind that what most species you'll cultivating usually grow on either rotten logs or half-dried turds.

1. Starch 0-8%*
2. Sugars: 0-2% (as long as it doesn't push the total of sugars and starches over 8%)
3. Agar 1-3%
4. Water to make up the remainder
5. Nitrogen is entirely optional, but does seem to help in small amounts.

Starches are easy to come by, no matter where you are.  The molecules are large, but simple--basically just a daisy chain of simple sugars--and they're a very efficient source of both carbon and energy for our little fungal friends.  Sources that I've used include, but are not limited to, grain water, potato flakes, the water left over after you boil potatoes, wheat flour, WBS flour, oat flour and corn starch.  I haven't made dog food agar myself, but kibble has a lot of starch in it.  *It should be noted that some sugars, like those in high fructose corn syrup or honey, don't perform as well without starch.  Others, like light malt extract, do just fine.

Sugars are even easier to find.  For these, I've used table sugar, sugar in the raw, high fructose corn syrup, honey, maltose, light malt extract, maple syrup, and dextrose/corn sugar for sold for bottling beer. Some have reported honey's antifungal properties can persist through a sterilization cycle and hinder spore germination, but I've never experienced this.

For nitrogen, I've used a small amount of autolyzed yeast extract, brewer's yeast, bread yeast, mushroom blanching water and the crap from the bottom of a beer carboy after racking.  Dog kibble also has a bunch.

For water, I use the tap.  An interesting thing about water is that its density is almost exactly 1g/mL, which means 1mL of water weighs 1g.  This is useful because if you're trying to measure water you can do it by either weighing it or using a measuring cup.

The stiffness of your agar is another consideration.  1% agar is very, very soft and 3% is very, very stiff.  Softer, wetter agars are better for germinating spores, but stiffer agars are easier to transfer and better for clones and isolation.  I find a solution of 1.5-2.5% agar-agar to be a happy medium. 

See what I did there? :awesomenod: 

A decent sized agar plate which won't dry out too quickly but won't waste resources is about 20-25mL, so if you're doing 10 you want at least 250mL of medium to account for spillage.  This translates to 0-20g of starch, 0-5g of sugar, and 2.5-7.5g of agar, and then however many grams of water you need to make the whole thing weigh 250g. Never forget that you aren't married to any agar recipe; you can always switch up your recipe based on what you expect to be doing with your plates or what you have on hand. All that remains is to decide how agar can help you, decide whether you want to try poured dishes or no-pour plates first, get an idea of how the medium is prepared, make a run Wal-Mart for the PC and then to either the Asian market or the sponsor pages for agar-agar and plates, and go scavenging around in your cupboard for the rest. 

I'd like to thank my two excellent editors for this article, blindingleaf and Pastywhyte. And I'd like to thank you for taking the time to read this. :grin:


Edited by Psilicon (12/05/17 08:38 PM)


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InvisiblePastywhyteMDiscord
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Re: Agar - what, why and how? Everything a beginner needs to know. [Re: Psilicon] * 5
    #21176501 - 01/26/15 05:40 AM (7 years, 8 months ago)

So agar is easy! Who knew! This should be required reading for anyone looking to step their game up, and should also make it clear just how simple this all is. Fantastic writeup my friend :congrats:

:justastonishing: :kaneclap:


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Invisiblemycomattie
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Re: Agar - what, why and how? Everything a beginner needs to know. [Re: Pastywhyte] * 1
    #21176557 - 01/26/15 05:55 AM (7 years, 8 months ago)

Awesome!


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InvisibleMudaFuka
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Re: Agar - what, why and how? Everything a beginner needs to know. [Re: mycomattie] * 1
    #21176612 - 01/26/15 06:06 AM (7 years, 8 months ago)

:stayfunky:


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AMU
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InvisibleImperfect Iam
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Re: Agar - what, why and how? Everything a beginner needs to know. [Re: MudaFuka]
    #21176793 - 01/26/15 06:48 AM (7 years, 8 months ago)

Excellent, TY Van!


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All you touch, and all you see, is all your life will ever be- Pink Floyd

Life is what happens while you're busy making other plans- John Lennon


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Invisiblewowimflabbergasted
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Re: Agar - what, why and how? Everything a beginner needs to know. [Re: Imperfect Iam]
    #21176972 - 01/26/15 07:31 AM (7 years, 8 months ago)

:likeaboss:


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Offlinemushpunx
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Re: Agar - what, why and how? Everything a beginner needs to know. [Re: wowimflabbergasted]
    #21177312 - 01/26/15 09:05 AM (7 years, 8 months ago)

Yea dude what a really nice guy!


Seriously great write up!!


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InvisibleMunchauzen
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Re: Agar - what, why and how? Everything a beginner needs to know. [Re: mushpunx]
    #21177316 - 01/26/15 09:06 AM (7 years, 8 months ago)

This gives me some great ideas!

Thanks for taking the time to write this info down and share it!


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OfflineSarah Tonin
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Re: Agar - what, why and how? Everything a beginner needs to know. [Re: Munchauzen]
    #21177422 - 01/26/15 10:18 AM (7 years, 8 months ago)

:cheer:  van knocked it out of the park  :cheer:  love all the links  :cheer:


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OfflineMushroom_J
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Re: Agar - what, why and how? Everything a beginner needs to know. [Re: Sarah Tonin]
    #21177517 - 01/26/15 11:08 AM (7 years, 8 months ago)

Anyone try coffee in their agar mix?


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Invisibleblindingleaf
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Re: Agar - what, why and how? Everything a beginner needs to know. [Re: Mushroom_J]
    #21177672 - 01/26/15 12:51 PM (7 years, 8 months ago)

nice VDG!


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MICROBIAL HUSBANDRY!!!!

The whole is greater than the sum of its parts


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Offlinespacechildo
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Re: Agar - what, why and how? Everything a beginner needs to know. [Re: Mushroom_J]
    #21177704 - 01/26/15 01:21 PM (7 years, 8 months ago)

A happy medium! :thumbup:

great read, I'll be sure to link a lot of people to this as I've had so many Q's about exactly these things! :yesnod:


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Invisibleblindingleaf
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Re: Agar - what, why and how? Everything a beginner needs to know. [Re: spacechildo]
    #21177706 - 01/26/15 01:23 PM (7 years, 8 months ago)

what is mushroom blanching water?  u mean like when u cook mushrooms in water, u use that leftover water?  cause thats pretty genius


--------------------
A few thoughts on cultivation
MICROBIAL HUSBANDRY!!!!

The whole is greater than the sum of its parts


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OfflinePsilicon
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Re: Agar - what, why and how? Everything a beginner needs to know. [Re: blindingleaf] * 1
    #21177792 - 01/26/15 02:22 PM (7 years, 8 months ago)

Quote:

blindingleaf said:
what is mushroom blanching water?  u mean like when u cook mushrooms in water, u use that leftover water?  cause thats pretty genius




Yup, that's exactly what I mean.  Thanks for the kind words, guys.  :smile:


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Agar - what, why and how?  Everything a beginner needs to know.
Oat Prep Tek
Bored?  Please take one of my experiments off my hands.


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Invisibleblindingleaf
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Re: Agar - what, why and how? Everything a beginner needs to know. [Re: Psilicon]
    #21177795 - 01/26/15 02:23 PM (7 years, 8 months ago)

WTF!!  ur a nut!  so wait, if i wanna try that, am i substituting the whole 500ml tap for blanch water?  how long are u blanching?


--------------------
A few thoughts on cultivation
MICROBIAL HUSBANDRY!!!!

The whole is greater than the sum of its parts


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OfflinePsilicon
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Re: Agar - what, why and how? Everything a beginner needs to know. [Re: blindingleaf] * 1
    #21177801 - 01/26/15 02:27 PM (7 years, 8 months ago)

Just 1-2 minutes, as if you were going to freeze them.  And yeah, I used the water from that in place of all the water.  If it tastes mushroomy then it probably is, right?


--------------------
Agar - what, why and how?  Everything a beginner needs to know.
Oat Prep Tek
Bored?  Please take one of my experiments off my hands.


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InvisibleSpitballJedi
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Re: Agar - what, why and how? Everything a beginner needs to know. [Re: Psilicon]
    #21178286 - 01/26/15 06:34 PM (7 years, 8 months ago)

:congrats:very nice. Hopefully this will encourage more beginners to get in to agar.


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Invisibleblackdust
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Re: Agar - what, why and how? Everything a beginner needs to know. [Re: Pastywhyte]
    #21178786 - 01/26/15 08:35 PM (7 years, 8 months ago)

One of the best set-up teks I have seen.

:billymaythumbup:


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Offlinestreamer

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Re: Agar - what, why and how? Everything a beginner needs to know. [Re: blackdust] * 1
    #21178860 - 01/26/15 08:57 PM (7 years, 8 months ago)

Great write up VDG. I'm definitely running out of reasons for not getting into this!


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OfflinecronicrMFacebook
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Re: Agar - what, why and how? Everything a beginner needs to know. [Re: blackdust]
    #21179305 - 01/26/15 10:52 PM (7 years, 8 months ago)

Quote:

blackdust said:
One of the best set-up teks I have seen.

:billymaythumbup:



gonna agree with BD here...don't  hate me


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