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InvisiblebodhisattaM
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BOD's Comprehensive Agar TEK * 895
    #21922023 - 07/09/15 09:21 PM (5 years, 1 month ago)

This tek is part of bod's cultivation is easy as fuck link list



AGAR IS EASY learn to cultivate amazing mushrooms AGAR IS EASY


Bod's Comprehensive Agar TEK

Here's the TL;DR TEK by enlightenment

and here's all the agar resources and TEKs compiled into one spot

Here are my cultivation videos, I encourage you to watch the agar ones there.


What is agar and why is it useful for the mushroom cultivator


Quote:

Workman said:
I use agar and I love it.

The idea is that you are growing on a 2-dimensional plane (the agar surface) instead of a 3-dimensional substrate (PF cake, grain in jars, etc.) On the flat surface of the agar, contaminates cannot hide from you. You can isolate clean mycelium from dirty prints. You can also clone fragments of tissue, do breeding experiments by isolating single spores and select and propagate specific isolates. Agar is also often used for the storage of favored strains. Just pop them in the fridge for several months.

Agar culture is just a more powerful and precise way of doing things. If you like control, agar is for you. If not, just inject your spores and cross your fingers :wink:



workman 1999


Quote:

c10h12n2o said:
why make a spore syringe in 2017? are you making a documentary about how people used to grow shrooms? :lol:

agar, agar, and agar




LC vs GLC vs Agar


Agar-Agar is a gel forming ingredient, extracted from seaweed, that is used to make petri dish media.
You could use gelatin but mycelium will digest gelatin and you'll be left with a useless goopy mess. Agar-Agar enables mycelium to grow on a solid plate surface so that you can cut wedges out, and use them to inoculate things. The solid 2-dimensional media also makes for good visual indication that your culture is either clean or not clean. Agar being on a 2d surface, rather than 3d(cake or grain jar) or in liquid, allows for easy salvaging of most contamination problems before it's ever a problem. In some cases, if a contaminant appears on a dish, you can re-capture mycelium. Then use that clean wedge to create a new 100% clean dish, and entirely save a culture. 

Agar-Agar itself is not nutritious. This is confusing as "agar" refers to the ingredient in "agar". Agar sometimes means the whole media prepared with agar(the gel forming powder) and then nutrition and water.
So from here on I will use A-A when I mean agar-agar the non nutritious powder

this stuff
and I'll use agar, when I mean the stuff that you pour into the dishes that mycelium will grow on.

Agar is useful for germination of spores. Spore syringes are inherently not 100% clean, this means if you inject spores into a jar, PF cake, grain spawn, LC, etc.... it has a chance of germinating a contamination. On agar you will soon see you can obtain clean mycelium from a spore syringe. This isn't to say you'll never have success using spores to directly inoculate, but using agar first will make sure you don't waist your time. This is a natural step for any serious cultivator

Agar is also, basically, a must for cloning mushrooms.
And agar is a tool for the cultivator to store cultures long term.

Here's the TEK let's get started


Supply list

  • Petri Dishes (I use 100x15 in sleeves of 20, I buy a box of 500 (25 sleeves)for $50-75)
  • Agar-Agar powder (I use telephone brand it comes in 25g packets for about $1-$2)
  • Nutrients to add the the agar (They're listed below in the TEK)
  • Bottle to pour agar from (I use 500mL media bottles with GL45 screw caps)
  • Funnel that fits into your bottle nicely and stands on its own
  • premade agar : this takes the place of agar-agar + nutrients. I almost never use premade agar.
  • Gram Scale
  • Pressure Cooker
  • Microwave(optional, for melting pre-sterilized bottles of prepared agar)
  • Thermometer NSF certified and calibrated regularly an infrared thermometer is more useful but a normal digital stick thermometer works.
  • Still Air Box(SAB) OR a Flow Hood(FH)
  • A torch (I use a bernzOmatic UL100)
  • Scalpel (I use x-acto knife with #11 blades)
  • Inoculation loop (I use disposable pre-sterilized ones)
  • Parafilm or Cling Wrap (I prefer cling wrap, more info below)



How to make Agar and Petri Dishes


here's a link to some popular agar recipes


I personally like to use, in give or take this order
  • Grain Soak Water agar(save water from hydration of grain spawn add 20g/L Agar-Agar powder)
  • BRF agar (20g/L BRF and Agar-Agar)
  • MEA, malt extract agar. Use light malt extract for homebrewing at 20g/L
  • YPD, yeast peptone dextrose agar, I buy this premade and use as is
  • PDA, I just kind of wing this one every time I use it


To make the best agar I like to add all the dry ingredients into a media bottle

these are 500mL pyrex media bottles, they're made for agar, they're about 15 dollars shipped, and they're the only bottle I will personally use for agar. Completely unmodified as is these are bar none the best vessel for pour agar.

These bottles have a removable polypropylene(PP/recycling code 5 plastic) ring at the lip of the bottle so agar doesn't dribble down the side of the bottle.
the lids are replaceable and the rings are replaceable too and both easily found for sale. (search: GL45 caps/rings)

after the dry ingredients are added I fill with room temperature tap water until I reach a hair over 400mL in volume
That's enough for 20 plates, a sleeve of 100x15mm dishes

put the cap on and shake, then into the microwave with the cap very loose
bring to a boil. careful don't let it boil over and make a fucking huge mess.
I find it best to not walk away during this step, just keep an eye on it.

after you hit a boil put the cap on tight and give it a shake again. Some recipes like BRF agar will have sediment issues, I just don't care. MEA and grain soak water will be pristinely clear.

slightly loosen the cap and cover with foil.

then place into your pressure cooker, I like to make the water in the PC come up to the level of the agar in the bottle. this will make it take longer to hit pressure and cool down but will cut down on boil over.

Sterilize at 15PSI for 20m.

agar is sterilized in 20m at 15psi. I always usually go to 20m I would not go longer than 30-45m you risk caremelization of sugars in your media.

IF you have sediment in your media you may need to use the 30-45m time frame. IF you filter or have completely clear, dissolved media then 20m should be be sufficient


***
If you don't have a pressure cooker agar media can be sterilized by steam alone, steam your agar bottle like you would a PF jar for no less than 45 minutes. Not having a PC/autoclave is no reason to avoid agar. Although I HIGHLY recommend using a PC for agar. And in general do not suggest doing agar without a PC
***

after the pressure drops to 0 you can remove the lid of your PC and tighten down the cap on the bottle and take the foil off.

At this point you can store the bottle in the fridge to use later or start to bring it down to pour temperature.

if you store the bottle in the fridge all you have to do to use it again is microwave it until it's liquid again. don't try to microwave it on high for 8 minutes. do a few minutes then shake, then a few more then shake.

Solid

half melted

fully melted

thanks microwave :thumbup:, and that's the only thing a microwave is good for in this hobby

now you have Sterilized agar in your media bottle, but it's burning hot after coming out of the PC or the microwave if you're using a previously sterilized bottle


Let's pour some plates


you have to cool it down to pour it without burning your hand, but how cool is too cool before it turns into a solid again?

FACT TIME

Quote:

Agar exhibits hysteresis, melting at 85 °C (358 K, 185 °F) and solidifying from 32–40 °C (305–313 K, 90–104 °F). This property lends a suitable balance between easy melting and good gel stability at relatively high temperatures.



I find it starts to gel back up at about 42C(107F) most of the time at the gel strength used for plates. At 47C(117f) you can pour a whole sleeve of plates before it gels up on you. and pouring at as low of a temperature as possible minimizes condensation a lot.

to get agar to melt again you have to go all the way back up to 85C(185F), once it is gel simply heating it 47+(117+F) won't melt it, you have to get it all the way back up to 85C(185F).

Using more or less A-A in your recipe will make these temperatures and the stiffness of the agar plates a little bit different.

How to get your agar to 47C(117F) to make it ready to pour.

the water bath
every real lab will have a water bath that can be set to 47C to keep agar liquid and ready to pour. As well it can cool down hot agar from the sterilizer or microwave. They're expensive so this is how to do it at home

Fill a Tupperware with hot water (must be at least 120F)
put your agar bottle in the hot water so that the water is level with the agar inside the bottle

the water in the tupperware will heat up as the bottle cools down, when the water in the bath once again has cooled down to 120F you can assume the bottle is now also at 120F and ready to pour.

you can also do it in your PC


add some cool water till the temperature reaches 150F or so then wait until the thermometer hits 120F or so

alternatively you can buy an infrared thermometer to check as well
Quote:

Kenetic said:






when you pour your agar at as near the temperature it starts to solidify you minimize condensation, if you pour your agar at 140F when you can handle the bottle but it's still a bit too warm you will end up with condensation

Take your cooled bottle of agar over to your work area

A SAB in my case


you can do this at home without fancy equipment in one of these(four examples)
ONE
TWO
THREE
FOUR(my favorite one)
don't let the size of tubs be a limitation, build one from something else if you want a big roomy SAB you can have one.

pour your plates. use your wrist, don't let your hand ever go above anything sterile. this isn't a sterile technique TEK but make sure to use good technique. Pour from the bottom up, make smaller stacks of 5-10 high.

 



if you have leftover agar put the cap back on, make sure you don't contaminate your cap when you pour. I put mine on a piece of foil


if you pour your dishes correctly at the low temperature you'll end up with something like this

near the top of the stack you might get a few dishes with some condensation, I actually like to pour stacks tall, so I do them 10 high, if you do a bunch of short stacks you get a bunch of dishes with a little bit of condensation if you do a stack of 10 you get 7-8 perfect dishes and 2 you may have to fix.

how do you fix them?

use a cup with warm water and a flat bottom, put it on top of the dish for a few seconds-minutes (wont hurt if the water is warm-hot)
and boom your dishes are crystal clear again.


Make extra dishes to have around. The sleeve the dishes come in is great storage.




You can fit all 20 back in and take it up, store in the fridge. I don't bother wrapping them at this stage.


Glass petri dishes, Pyrex or Flint glass and re-use/sterilization


Pyrex dishes withstand dry heat and steam sterilization for the most part indefinitely they cost more than flint glass dishes that might not take extremes forever and also is inherently more fragile


these dishes are borosilicate ie pyrex

https://en.wikipedia.org/wiki/Dry_heat_sterilization

technically your glass dishes should be surgically clean before sterilization in an autoclave or oven. That way the sterilization actually sterilizes the glass not the debris on the glass and the glass.

steam sterilization is just fine but we have pressure cookers not autoclaves. you can run a dry cycle on an autoclave and it pumps the chamber to a vacuum and sucks the water out of the chamber and leaves tubes,instruments,tools,lab clothes,petri dishes dry when you take them out. We don't have fancy autoclave bags either so we wrap things in foil.

when you take foil wrapped glass dishes out of your PC the foil is going to be wet and I find it nicer to have everything including the package the dishes in dry so wrapping them in foil and baking them is preferred.

That concludes the making of agar and making of petri dishes part of the TEK.
now let's see what can be done with the poured dishes.


Using agar dishes

spore germination, cloning, strain isolation, use as an inoculant and long term storage


you may notice I don't use parafilm very often, I prefer saran wrap myself, you can use either or. to use saran wrap take the whole roll out of the carton. and cut the roll into one inch wide mini rolls. use each mini roll to make at least two revolutions around the dish and pull it to snap it off which will make a nice seal. When you unwrap the dishes the saran wrap makes a good surface to put in between two dishes so that they don't slide around on each other as much. Saran wrap is quicker for me, I don't have to pre-prepare cutouts of it like parafilm aside from the original making of the mini rolls. It's also quite a bit less expensive. although parafilm works out to be very cheap per dish anyway.

How I wrap my dishes Video

Munchauzen's cultivation and sterile technique videos
if you are uncomfortable with sterile technique these videos should help tremendously. I won't cover agar to agar transfers in this TEK as it's better covered by sterile technique TEKs than an agar TEK

Spore germination


you can inoculate with a spore syringe
by placing a drop in the center of the dish
or by making a streak plate

here's just drops on a plate, germination occurs all over the plate


spore syringes are inherently not 100% clean
a more fool proof technique to ensure success is to properly plate the spores
a streak plate
Quote:

enlightenment said:
Without description:


Spore solution:






thanks enlightenment these are awesome as fuck

here's how I do it from start to finish

my DIY Inoculation Loop TEK

Have a sterilized shot glass or a blank petri dish(inside is sterile still)
flame your needle on the spore syringe then move into the SAB
squirt out a drop to cool the needle and then squirt a little bit into your dish
take your inoculation loop (I use disposable plastic ones) otherwise flame it
bring into the SAB then dip in agar(if flamed) then dip in the spore solution in the dish
then make a Z pattern on the dish with the inoculation loop
remember in a SAB no hands above sterile surfaces. only sterile things above sterile surfaces.
then repeat as needed. I usually do about 5-10 dishes with spores to ensure something viable comes.


once spores germinate you should take a transfer to another dish
video 3 here


Cloning


There may be more than one way to skin a cat, but when it comes to cloning the only right way to do it is with agar.
Like all things in this hobby there's more ways than one to get away with things. People have used the 9er tek and many other techniques to clone without agar. I'm going to tell you they all suck ass, the only right way to clone is with agar :shrug:

Agar is entirely too easy, cheap, and reliable to spend your time trying to do tissue cultures any other failure ridden way.



frank's proper cloning TEK
also covered in let's grow mushrooms by RR

My favorite way to clone is to wait for a dish to pin



a little too mature :lol:


more like this

and move those pins to agar like the pictures above.
the little pins grow very quickly and vigorously, the pins from another petri dish are going to have no contamination on them so cloning is even easier than taking a biopsy from a fruit growing in a tub. Biopsy clones are perfectly fine too.


if you leave a dish about 6-8 weeks usually it will start to knot/pin and then a few more days and you'll have a pin to clone.

Strain Isolation


Many people get overly concerned with finding an isolate. Many isolates suck.
any two spores that successfully mate make a strain.

thus with spores on a petri dish
Quote:

RogerRabbit said:
It's very normal not to see the rhizomorphic growth until you make  few transfers.  With multispore inoculation on agar, you have hundreds if not thousands of strains growing together and all over on top of each other.  Remember, any two spores that germinate and 'pair up' by forming a clamp connection is the very definition of a 'strain'.  Just dig in and take a very small piece of mycelium no larger than a grain of rice.  Move this to a new dish. 

When this dish begins to grow out, you may or may not begin to see sectors.  It might take a second transfer before you get few enough strains to see the individual sectors.  Take the best looking growth again and move it to a fresh petri dish.  Eventually, when you hold a dish up the the light and look from the back(agar) side, you will see individual sectors like the spokes on a wheel.

In the picture below, the dish on the left is the result of two transfers from the original spore swipe. Prior to the second transfer, the rhizomorphic mycelium was buried in the mass of strains and couldn't be differentiated, just like yours are.  The rhizomorphic sections are now clearly showing up at ten o'clock and two o'clock.  Each of those sections has four or five individual sectors that will be isolated out next time I do agar work in a day or two. The remainder of the dish will be discarded.  The dish on the right is a single sector isolate, as you can see the entire dish is rhizomorphic and when held up to the light, there is no further sectoring.  I fruit out each individual sector to determine the best fruiting isolate.  Many isolates are mediocre fruiters, but if you want to find the one that will deliver those monster flushes time and time again, this is how it's done.  I keep a small piece from each petri dish in the refrigerator until I've found the best fruiting isolate, then I get that piece out of the fridge and grow it out.  I always keep a piece from the original petri dish in test tubes in the refrigerator so I can go back to that strain for years to come with no fear of senescense.  It's a lot of work, but hey. . .It's a hobby right? 
RR




https://www.shroomery.org/forums/showflat.php/Number/4377475#4377475

also there's this thread STRO's cleaning and isolating on agar TEK

so you see a clone has proven it's potential by growing, with isolates your going blind you need to test every isolate, some will suck
here's a good example
https://www.shroomery.org/forums/showflat.php/Number/18219379

Also a lot of noobs worry about rhizomorphic growth. Worry about healthy growth. you do not know if rhizomorphic growth will be any good till you test it. so tomentose growth may be a better fruiter or more potent, you won't know so don't concentrate on only rhizomorphic mycelium on dishes.

people get strains and varieties confused
A variety is a name on a syringe
A strain is two spores that have mated together

B+, GT, Cambo, Creeper, Penis Envy, KSSS, AA+, or any variety can grow weak, strong, tall, short, retarded, fast, etc...
B+(variety) is basically a mom and a dad(the name itself is the parent) and every time you squirt B+ into a jar they the name on the syringe has sex and has kids. your jars have 100s of kids your b+ parents made. Some of those kids are awesome some are stupid. Each of those kids is a strain.
workman clears up how varieties are made


isolates of leuistic orissa India psilocybe cubensis

use as an inoculant


Quote:

stareatclouds said:




Yes it's that fucking easy folks.

if you're going to inoculate agar is the way to go. from spawn to LC if you want to make sure it's clean and you're not rolling the dice you need to work with agar inoculations.


inoculation of spawn with wedges

sometimes you lose your blade :mad2:


inoculation of LC with agar wedges

Long term storage

agar dishes can be stored in a regular refrigerator for extended periods of times, longer than a couple years.

For best results you will want a dedicated refrigerator set at ~35-37F right above freezing

A dedicated refrigerator solves the temperature swing problem since it's not opened and closed but a few times a year.

if you must use a refrigerator that does get regular openings then the best way to ensure your dishes stay good is to put them into zip lock bags, preferably individually.
then put them into an insulated lunch box to hold the temperature, fill any void space with ice packs to act as a thermal capacitor which buffers against temperature swings.

any insulation you can the better.

the dishes can be stored for years but it's best to make transfers to new dishes at least once a year, take a small rice sized grain from the master plate and put it to a new plate let it colonize the plate nearly completely then put it to refrigeration, this way you can keep your clone cultures or isolates for decades without worrying about senescence occurring. 

you may also refrigerate slants

which are another technique of using agar for long term storage.
here's more on that




The end


did you know
70% alcohol sanitizes better than 90+ or higher concentrations
more info about that here




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OfflinePurePleasure
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Re: pour agar TEK [Re: bodhisatta] * 1
    #21922119 - 07/09/15 09:35 PM (5 years, 1 month ago)

First.


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Re: pour agar TEK [Re: PurePleasure]
    #21922200 - 07/09/15 09:45 PM (5 years, 1 month ago)

Nice pics, good description :thumbup:

10-15 min is a little short of a PC run tho, for sure if your using grain soak water or the like I would up that.


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Re: pour agar TEK [Re: PurePleasure]
    #21922204 - 07/09/15 09:45 PM (5 years, 1 month ago)

nice way to show how simple it really is! water bath idea was a nice touch :thumbup:

Now wait for unknown number on pages of how to mix agar with water :strokebeard:


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InvisiblebodhisattaM
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Re: pour agar TEK [Re: Pastywhyte]
    #21922317 - 07/09/15 09:59 PM (5 years, 1 month ago)

Quote:

Pastywhyte said:
Nice pics, good description :thumbup:

10-15 min is a little short of a PC run tho, for sure if your using grain soak water or the like I would up that.



for grain soak agar probably yes
for most other agar recipes with amino acids and some sugars you don't want to cause maillard reactions and you also don't want to caramelize any sugars.



that's the directions for MEA PDA YPD etc..

liquid media is much much easier to sterilize so even with grain soak I would imagine the time could be very short to get a acceptable SAL.



these are the stages of agar melt during microwave. I don't like to melt the agar on full blast, I do it in shorter intervals or with the power turned down.


Edited by Trusted cuItivator (07/09/15 10:02 PM)


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InvisiblebodhisattaM
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Re: pour agar TEK [Re: bodhisatta]
    #21922343 - 07/09/15 10:08 PM (5 years, 1 month ago)

even if we had these nasty friends

in agar media since it's liquid the processing time is shorter.


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Re: pour agar TEK [Re: bodhisatta] * 1
    #21922353 - 07/09/15 10:10 PM (5 years, 1 month ago)

I usually go for 20 most of the time and 45 for grain water. Never really had issues but I don't work with yeasts or peptone. Usually just work with whats laying around the house :tongue:

I have had grain water LC go bacterial if I don't run a full 45 min.


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Re: pour agar TEK [Re: Pastywhyte]
    #21922360 - 07/09/15 10:12 PM (5 years, 1 month ago)

that's strange, the indicator strips that are made with the most hard to sterilize endospores only take a 15m cycle, liquid media should be sterilized without such a lag time like grains do. either way do what works for you


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Re: pour agar TEK [Re: bodhisatta] * 2
    #21922367 - 07/09/15 10:15 PM (5 years, 1 month ago)

Maybe its the elevation I'm at but when grain water LC shows bacteria on the test plate, I don't argue with it.


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Re: pour agar TEK [Re: Pastywhyte]
    #21922453 - 07/09/15 10:46 PM (5 years, 1 month ago)

Yea to each their experience. I do my grains for 2 hours rather than the 90 standard


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Re: pour agar TEK [Re: bodhisatta]
    #21922466 - 07/09/15 10:51 PM (5 years, 1 month ago)

:popcorn:


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Re: pour agar TEK [Re: invitro]
    #21922511 - 07/09/15 11:02 PM (5 years, 1 month ago)

Back on topic I like the idea of the bath to bring down to temp. I am a cheap fucker and prefer to by tetracycline from the pet store than gentamiacin. The bath would speed up the wait for the agar to cool enough to add the tetra.


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Re: pour agar TEK [Re: Pastywhyte]
    #21922755 - 07/10/15 12:26 AM (5 years, 1 month ago)

Makes me mad I still don't have access to rare and crazy antibiotics and anti fungals. I would never get bored.


--------------------
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Re: pour agar TEK [Re: maddchef]
    #21922758 - 07/10/15 12:28 AM (5 years, 1 month ago)

:jackiechanofapproval:


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Re: pour agar TEK [Re: cronicr]
    #21923402 - 07/10/15 07:30 AM (5 years, 1 month ago)

The tetracycline route sure is interesting. I may have to try that out.


--------------------
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Lets Grow Mushrooms Videos
PastyWhyte's Easy Agar TEK
Agar's Liquid Inoculant TEK


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Re: pour agar TEK [Re: FreeWorldOrder]
    #21923961 - 07/10/15 10:39 AM (5 years, 1 month ago)

I may have to add this to my tek bod. I wanted to make a section on pouring dishes, but yours seems way better than what I could have done haha. I'm a pasty plate guy anyway


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Re: pour agar TEK [Re: FreeWorldOrder]
    #21923970 - 07/10/15 10:42 AM (5 years, 1 month ago)

:takingnotes:

I never thought of precooking agar and storing it in the fridge for later heating and pouring. Brilliant!


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Re: pour agar TEK [Re: bodhisatta] * 1
    #22085395 - 08/13/15 08:56 AM (4 years, 11 months ago)

Quote:

bodhisatta said:
you can prepare multiple bottles of agar, use a foil cap during sterilization for added precaution. Then put the spare bottles in the fridge. re melt in a microwave when you want to use them. you can consolidate agar if you have a marginal amount in a couple of your bottles, do it just like you would do a g2g transfer as long as you do it in your SAB or with a FH you won't contaminate your agar.



these are the stages of agar melt during microwave. I don't like to melt the agar on full blast, I do it in shorter intervals or with the power turned down.





I got a few of these media vessel bottles in 1000ml size. What size do you have pictured here?

I was thinking about mixing up and sterilizing a few bottles to store in the fridge to remelt when needed. But was wondering, do they actually need to be stored in a fridge if they are going to be remelted and used in a few weeks to a month?


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Re: pour agar TEK [Re: Juiceh] * 1
    #22085435 - 08/13/15 09:08 AM (4 years, 11 months ago)

Almost did this writeup a while back after we talked about it. Didn't have pics though. I've been rockin pasty plates lately. Good stuff :rockon:


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Re: pour agar TEK [Re: Grey]
    #22085572 - 08/13/15 09:52 AM (4 years, 11 months ago)

500ml. The fridge is mostly for some of the lab grade shit with things that degrade like some stains. Mea and pda should be a-ok out of fridge


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