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rave420
open minded




Registered: 07/20/10
Posts: 694
Loc: Vancouver Island
Last seen: 14 years, 2 months
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I want to share my Agar Technique.
#12973985 - 07/29/10 07:31 PM (14 years, 5 months ago) |
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To get you a basic idea
what you're about to read takes a big dump on sterile procedure, and is not intended to replace or amend to any proper agar teks.
It's for the every day guy that rather buys stuff from the convenience store than order stuff online. It's for those that think agar is some kind of black magic that will only be successful if you work in a clean room.
It does not involve - Petri dishes - Flow hoods - Glove boxes - Ovens, lysol and bleach - anything that can't be bought at a convenience store
What it DOES involve is - a pressure cooker - tiny mason jars (as the dishes)
First off, you want to clean. If anything moldy has ever been in the room you consider doing this, think about maybe choosing another room. I for one do it in my bathroom, and spray every nook and cranny with alcohol. My bathroom is a VERY clean place, so if you clean your bathroom once a month kinda thing then i suggest you move to your bedroom.
Anyway, go ahead and cook up the agar according to recipe. Any recipe will do. Nothing has to be sterile right now. I mean, the cleaner you work the more you reduce the chance of contaminants. I boil my agar and put it in a glass bottle. If it cools i can stick it in the microwave (about 5 minutes for 1 l of agar at 1000W), it liquefies it right up.
Then, i use 125ml mason jars. I like to bleach my jars for a few minutes, and of course you want to make sure there is no debris inside of them (read CLEAN, not sterile). I then go ahead and pour my agar into the jars, maybe about 1/2" to 1" deep. I seal them up with rubber side up and load them into my PC. Also, give them a tinfoil lid to prevent dripping water to form a puddle on your agar. I then proceed to PC them for 90 minutes (i recon 20 will do, but i am NEVER in a rush when working on stuff like this), and let it cool down naturally.
And that is all there is to it. When i have to place spores in it or make transfers, i douse the jar in a thin mist of alcohol from the outside, alcohol on my hands, and then i slightly lift up the lid, just enough for my utensils to fit inside.
Contamination is rarely an issue for me. I made 12 dishes about 7 days ago, and none of them show contamination, and i opened every single one to inoculate with spore syringe. Here and there i get an odd jar that shows contamination, but it's less than 10%. and that is something i can live with.
I don't have to buy polystyrene dishes that are non-reusable, because i can reuse my mason jars just fine. Sure, you pay 10$ for 12 of em, but they will last you the rest of your career.
The only thing that needs improvement is the lid in my opinion. Gas exchange is limited to the metal on glass seal. Maybe i could put some Tyvek squares on the top.
And that's about it, i thought i share my quick and painless (even though a bit sloppy) way of doing agar dishes. This is by no means superior to a proper sterile procedure, but if you work clean you will do good. this has worked for me many times, and i hope it will work for someone else too.
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we all breathe the same air, drink the same water, and draw our strength from the same giant fireball.
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hugger-mugger


Registered: 10/14/09
Posts: 201
Loc: Dallas
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Re: I want to share my Agar Technique. [Re: rave420]
#12974685 - 07/29/10 09:48 PM (14 years, 5 months ago) |
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No one has anything to say about this??
Sounds great to me, wish someone would chime in on this!
I love keepin it simple.
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rustycobwebs
Green mold cultivator

Registered: 12/17/05
Posts: 799
Last seen: 9 years, 9 months
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Re: I want to share my Agar Technique. [Re: rave420]
#12974835 - 07/29/10 10:18 PM (14 years, 5 months ago) |
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Quote:
rave420 said: I then go ahead and pour my agar into the jars, maybe about 1/2" to 1" deep.
That's probably too deep. I mean, you can't have too deep but the growth will only be on the top. Agar is a 2 dimensional growing medium, unlike jars, which is why it can be incubated at higher temperatures because the jars growing in every direction puts off way more heat.
This isn't bad overall though, i suppose. Make RTV silicone ports and put a few drops of a spore syringe on there.
This is fine for germinating the spores. Though, if anyone intends to do this you should know that it took me awhile to find agar agar powder and it wasn't at a convenient store. However it was readily available and so are the supplies to make a glovebox.
Glovebox would be highly recommended for when you transfer the colonized agar into substrate jars, or clean agar plates/jars.
-------------------- and let it be so Praise Psilocybe
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jokefox
Top of the chain



Registered: 12/22/09
Posts: 6,231
Loc: never where I should be
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Re: I want to share my Agar Technique. [Re: rustycobwebs]
#12974867 - 07/29/10 10:25 PM (14 years, 5 months ago) |
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you dont need to bleach anything if its going in the PC only do a small cm layer of agar i do as thin as possible becuase im jewish like that
and bathrooms are dirty also you dont need to spray alcohol , just clean the bathroom or room with warm soapy water i use mister clean
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13shrooms
Lightning Shaman



Registered: 01/01/09
Posts: 26,719
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Re: I want to share my Agar Technique. [Re: rustycobwebs]
#12974900 - 07/29/10 10:33 PM (14 years, 5 months ago) |
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yeah a GE hole with Synthetic Filter Discs, tyvek or polyfill and silicone injection ports would be good.
baby food jars work well also (if you have those around).
I would use a 1/8" - 1/4" layer of agar though.
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A M U
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silas simon
get it



Registered: 03/21/10
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Re: I want to share my Agar Technique. [Re: 13shrooms]
#12974988 - 07/29/10 10:54 PM (14 years, 5 months ago) |
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this is me abusing the thread saving feature of the forums for later use and also saying nice idea
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ever so curious about psilocybe baeocystis. do please pm me if you can facilitate some hands on learning!
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2ndChancesRDivine
I slayed the Jabberwock!



Registered: 04/19/10
Posts: 1,100
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Re: I want to share my Agar Technique. [Re: silas simon]
#12975111 - 07/29/10 11:33 PM (14 years, 5 months ago) |
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I use babyfood jars. I want to get some tri sectioned plates. That would be cool!
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All your RC are belong to me.
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rave420
open minded




Registered: 07/20/10
Posts: 694
Loc: Vancouver Island
Last seen: 14 years, 2 months
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Re: I want to share my Agar Technique. [Re: silas simon]
#12975142 - 07/29/10 11:45 PM (14 years, 5 months ago) |
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keeping it simple while adhering to stringent rules is what it's all about. I compromise my sterility at certain points, but that is a calculated risk i am taking whose likely outcome i can directly influence to a certain point wich lies way below my not-acceptab e thresold.
Basically the point where you crack the jars to put stuff in or take stuff out is the most critical point. I try making sure that everything that gets into contact with the jar is sterile, such as hands, the screw part of the lid and any tool entering. I spray them with alcohol because that greatly lowered my contamination. I did a lot of experimenting with this, and it shows that it's way to easy for spores to land on the outside of the jar and then are somehow transfered during handling, knocked off the lid during opening etc. I founf without spraying i got an average 45% contam (based on a 48 sampe size), and that with spraying i got below 10% (based on ~500 sample size). This can be CRUCIAL if you are trying to isolate strains for example, and you loose the one dish you REALLY needed for something. Like, for what i am doing i don't really need that kind of accuracy and precision, i always use one sample i am trying to propagate on several jars, so i can afford to loose one. But there really isn't any reason to unless you somehow get contaminants in there. In my opinion and experience, misting the jar from the outside with 99% isopropanol does help(mixed 100:1 with water 100alc 1water, reason for that is that "wet" alcohol kills a lot better than almost dry alcohol). If you have to have reliable results then nothing beats a proper sterile procedure though.
I suppose that the synthetic filter disks would really make things convenient. Alternativley, you can use two layers of Tyvek and leave the metal lid off and just screw the outer part down with the tyvek on it.
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we all breathe the same air, drink the same water, and draw our strength from the same giant fireball.
Help me with my collection (trades only)
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fastfred
Old Hand



Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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Re: I want to share my Agar Technique. [Re: rave420]
#12975642 - 07/30/10 05:31 AM (14 years, 5 months ago) |
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Well, jars are hard to work with and 10x the cost of a dish. Including sterilization costs you'd have to use them at least 15x to even come close to breaking even compared to the cost of petri dishes.
The frustration, extra work, and increased failure associated with poor agar containers like jars is just not worth it.
-FF
-------------------- It drinks the alcohol and abstains from the weed or else it gets the hose again. -Chemy
The difference between the substances doesn't matter. This is a war on consciousness, on our right to the very essence of what we are. With no control over that, we have no need to speak of freedom or a free society. -fireseed
"If we are going to have a war on marijuana, the least we can do is pull the sick and the dying off the battlefield." -Neal Levine (MPP)
I find the whole "my drug should be legal but yours should be illegal" mindset disgusting and hypocritical. It's what George Bush and company do when they drink a cocktail and debate the best way to imprison marijuana users. -Diploid
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Doc_T
Random Dude




Registered: 03/06/09
Posts: 42,395
Loc: Colorado
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Re: I want to share my Agar Technique. [Re: fastfred]
#12975715 - 07/30/10 06:34 AM (14 years, 5 months ago) |
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Agreed. Jars work, and it's ok for getting started. But not great.
-------------------- You make it all possible. Doesn't it feel good?
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fungus_tao
Hah Zah!



Registered: 05/10/06
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Re: I want to share my Agar Technique. [Re: Doc_T]
#12975948 - 07/30/10 09:00 AM (14 years, 5 months ago) |
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Ok, you've walked us through how you pour and inoculate, but what's next? How do you make transfers without a glovebox? What is the point of germinating spores on agar? I mean you could go from agar -> LC, or agar -> grain but if you skip the isolation step you might as well go directly from spores -> grain. Isolating will take at least 100 dishes and a 10% contam rate would be unacceptable for that purpose. IMO you would be miles ahead to pour dishes in front of a flow hood or in glove box. Nothing wrong with using jars to get your feet wet, but obviously petri dishes are far better for agar work. Tao
-------------------- Follow the light
The Light is your guide.
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Doc_T
Random Dude




Registered: 03/06/09
Posts: 42,395
Loc: Colorado
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Re: I want to share my Agar Technique. [Re: fungus_tao]
#12975996 - 07/30/10 09:16 AM (14 years, 5 months ago) |
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Quote:
Isolating will take at least 100 dishes
Cloning doesn't.
-------------------- You make it all possible. Doesn't it feel good?
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fungus_tao
Hah Zah!



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Re: I want to share my Agar Technique. [Re: Doc_T]
#12976093 - 07/30/10 09:52 AM (14 years, 5 months ago) |
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Well, that's true. He was talking about inoculating agar with spores though so I was just kind of wondering what's the point Tao
-------------------- Follow the light
The Light is your guide.
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FooMan



Registered: 02/02/05
Posts: 8,957
Loc: Earth
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Re: I want to share my Agar Technique. [Re: rave420] 1
#12976120 - 07/30/10 10:01 AM (14 years, 5 months ago) |
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I've done that in the past, but mainly for cloning whole pins. I've posted this before, but I'll be damned if I can find it:







I PC the jars with the agar mixed inside already with the lids loose to let the steam in/out during cooking. Let cool completely, then open the PC and tighten the lids. When you're ready to clone, just drop the first pin that comes up in a flush into peroxide for a few seconds, then quickly drop the pin into the jar.
Before you peroxide nazis start yapping... please just save it for someone else. This works, despite whatever minimal tissue damage may occur.
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Quick WBS Prep
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Doc_T
Random Dude




Registered: 03/06/09
Posts: 42,395
Loc: Colorado
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Re: I want to share my Agar Technique. [Re: fungus_tao]
#12976127 - 07/30/10 10:03 AM (14 years, 5 months ago) |
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Clean up a print, one obvious answer. That wouldn't necessarily take 100 jars either. 3 jars with spores, transfer likely myc to 3 more. Do the same once you have fruitbodies. Dirty print to clean clone culture in a dozen jars.
-------------------- You make it all possible. Doesn't it feel good?
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fastfred
Old Hand



Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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Re: I want to share my Agar Technique. [Re: Doc_T]
#12976163 - 07/30/10 10:16 AM (14 years, 5 months ago) |
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> Dirty print to clean clone culture in a dozen jars.
You guys are over complicating this. You can clean a print in 1-2 transfers and isolation only takes a few transfers also.
-FF
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Doc_T
Random Dude




Registered: 03/06/09
Posts: 42,395
Loc: Colorado
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Re: I want to share my Agar Technique. [Re: fastfred]
#12976202 - 07/30/10 10:24 AM (14 years, 5 months ago) |
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"dozen, max" I meant. Not "hundreds!!1!
-------------------- You make it all possible. Doesn't it feel good?
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fungus_tao
Hah Zah!



Registered: 05/10/06
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Re: I want to share my Agar Technique. [Re: Doc_T]
#12976243 - 07/30/10 10:34 AM (14 years, 5 months ago) |
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Nice and to the point FooMan!
Doc, It's just the whole idea of transferring agar wedges through the open air of a bathroom/bedroom that bothers me. A 90% success rate is very good considering that's what the OP is doing. Tao
-------------------- Follow the light
The Light is your guide.
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Doc_T
Random Dude




Registered: 03/06/09
Posts: 42,395
Loc: Colorado
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Re: I want to share my Agar Technique. [Re: fungus_tao]
#12976447 - 07/30/10 11:33 AM (14 years, 5 months ago) |
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Open air, Five dollar glovebox.
-------------------- You make it all possible. Doesn't it feel good?
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13shrooms
Lightning Shaman



Registered: 01/01/09
Posts: 26,719
Loc: IN ETHERS TORSION FIELD
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Re: I want to share my Agar Technique. [Re: rave420]
#12977393 - 07/30/10 03:26 PM (14 years, 5 months ago) |
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Quote:
rave420 said: To get you a basic idea
what you're about to read takes a big dump on sterile procedure, and is not intended to replace or amend to any proper agar teks.
I understood you.
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A M U
Click here ^ for the AMU forum
VVV AMUs Free Active/Edible/Exotic Spore Print or Syringe or Edible Culture Trade Thread VVV
"Man is the sex organ of the machine world" ~ Marshall McLuhan
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