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teal
Art of Troll

Registered: 04/14/11
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Easy Antibiotic Agar Tek For The Absolute Beginner 6
#17356799 - 12/08/12 06:04 PM (11 years, 1 month ago) |
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Hello Shroomery,
I would like to share with you an antibiotic agar (AA) technique I have used numerous times and had great success with. It's main advantage over all other growing teks is that is does not require absolute sterility in its preparation. Nowhere in this process will you need to use gloves, pressure cookers, flow hoods or flame/alcohol sterilization.
Introduction
AA is a great way to start a culture going, free from contaminants that may linger inside spore syringes. A colonized plate may be used to inoculate grain or to create a liquid culture for inoculating PF jars.
If you already use PF Tek, using syringes made from AA can jump start colonization by a week and help colonization finish before other contaminants such as lingering bacteria can take over. You also do not need to keep buying fresh spore syringes or keep taking prints.
Materials
To prepare AA, you will need the following materials
 Antibiotic: first and foremost you will need an antibiotic. The one I'll be using in this tek is called Furan-2. It's cheap, works well and is not significantly degraded by heat. Check a pet store that sells fish; antibiotics are used to treat fish diseases. Even Walmart sells fish, so they might carry it. Another alternative is Ebay. I found a pack for <$10. Not all antibiotics are made equal, so I can't say how well tetracycline or others will work.
 Vinegar/Oil Pouring Jar: This jar is excellent for pouring agar. The glass is not heat resistant pyrex, but if you know how to handle PF jars without breaking them, then you should be fine. We are not going to be using the cap. You can buy it at any Walmart.
 Petri Dishes: will hold the solidified agar. If you can buy them locally that's great, otherwise get them off ebay.
 I prefer to use the Falcon 60x15mm pretri dishes. These are about half the diameter of the regular dishes. The smaller surface area helps reduce contamination. The plastic also doesn't melt if you pour really hot agar (which is a requirement of this technique). You can easily buy them on ebay, here's 20 for <$10.
 Agar: You will need the powdered form. Any health food store should sell these, just ask for it. Ebay is another alternative.
 Dextrose: also known as corn sugar, get it at any health food store. Walmart will probably not carry it. Do not use regular cane sugar.
 Potato: we are preparing potato dextrose agar (PDA), so you will need a potato.
Preparation
The following measurements will produce enough AA for 8-10 60mm petri dishes or 4 150mm dishes.
Weigh out ~1.5 g of dextrose and place it in your jar. You can eyeball all the measurements used here.
 1.5 g of dextrose in a tea spoon.
Add 1.0g of agar powder to your jar.
 1.0 g of agar in a tea spoon.
Add a very small quantity of antibiotic to your jar. The rule of thumb I follow is to add enough Furan-2 to visibly color the solution yellow.
 A quantity of Furan-2 used to sterilize 100mL of agar.
Cut one potato (the size doesn't matter) and boil it with half a cup of water for 15 minutes. Let the water boil down to 25-50 mL.
 Potatoes boiling in water.
 Prepared potato broth.
Pour the potato broth into the jar. Fill the jar ~1/3 with the potato broth.
 Potato broth, dextrose, agar and antibiotic in the vinegar jar. Notice the yellow color due to the antibiotic.
Add tap water to the jar until it looks like the picture below (~3/4 of the way).
 Final AA solution before sterilization.
Now you need to heat the solution to boiling for it to become liquid. You can probably microwave it, but here I steam sterilize it for 20 minutes. Cover the top with aluminum foil before steam sterilization.
 Agar ready to be steam sterilized. 20 minutes is enough to fully sterilize it. Avoid pressure cooking as the intense heat could degrade the antibiotic.
After sterilizing the agar, take the jar out and place it on a towel (avoid placing hot glass directly on cold surfaces). With the top covered with aluminum foil, swirl the jar to get all the agar dissolved. It will not dissolve on its own, but sit at the bottom of the jar as a sticky paste.
Next pour the agar onto your plates. Do not let the agar cool down, pour it while it's HOT. The plates should be washed clean but do not have to be sterile. The heat of the agar will kill all fungal spores and the antibiotic will kill all the bacteria.
 9 60x15mm AA plates prepared using all the solution from the vinegar jar.
Go ahead and let the jars cool down and solidify. At this point you have your finished sterile AA plates. At this point, sterile technique must be used. Note that any bacteria you introduce at this point may be able to grow. I have found that bacterial colonies will grow for 1-3 days and then stop growing. Fungi on the other hand will continue to grow
Results
PDA can be used to grow just about any fungi. Here is some Claviceps paspali growing on slants.
 Claviceps paspali growing on AA
You can use AA to clean contaminated syringes. Here I bought a syringe from a Shroomery sponsor I won't name and put some drops on AA. Notice the orange bacterial colonies are exactly in the shape of the water puddle. While the bacteria did grow a little bit, growth stopped soon after, but the fungi kept growing.
 Contaminated spore syringe solution injected onto AA. The orange spots are bacteria and the white is the germinated spores.
 Contaminated spore syringe injected onto AA. Bacterial growth was impeded by the antibiotic and fungi mycelium was able to colonize the rest of the plate. Clean samples could now be isolated.
Speed up your PF jars with mycelium syringes
When you isolate a clean sample of fungal mycelium onto a fresh plate, you can easily make a clean mycelium syringe.
To prepare one, pour boiling water into a cup. While the water is still boiling hot, fill a syringe with the boiling water (the syringe need not be clean). Empty the syringe and repeat this step several times. This will help clean out any contaminants. Some bacterial endospores can survive this process, but they won't pose any harm.
Fill the syringe with fresh boiling water and let it cool to room temperature.
Flame sterilize the tip of the syringe and let it cool.
Inject the cool water on top of a colonized agar plate.
Flame sterilize an innoculating loop (can be made with a piece of wire). Use the inoculate loop to scrape mycelium off the plate into the water. If you don't have an innoculating loop, suck up all the water with the syringe and spray onto the plate again. Do this several times. The agitation from the water will help remove mycelium from the plate.
Refill the syringe with the water from the agar plate. You now have your mycelium syringe ready to inncolulate grain or PF jars.
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mukhail
Creeper

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Re: Easy Antibiotic Agar Tek For The Absolute Beginner [Re: teal]
#17356848 - 12/08/12 06:17 PM (11 years, 1 month ago) |
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Me likey. +5 for you. I think I may try this out next month.
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PussyFart
Retired Cultivation Extrodinaire



Registered: 04/08/12
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Re: Easy Antibiotic Agar Tek For The Absolute Beginner [Re: teal]
#17356853 - 12/08/12 06:18 PM (11 years, 1 month ago) |
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Quote:
teal said: You can probably microwave it, but here I steam sterilize it for 20 minutes.
You cannot sterilize anything in a microwave oven.
--------------------
THIS HOBBY IS NOT FOR THE IMPATIENT! PLEASE BE PATIENT, DON'T BE A PATIENT! A Tale of 10 Isolates, GT Cluster Clone Monotubs, RR's Let's Grow Mushrooms DVD, SGFC(Shotgun Fruiting Chamber), Monotub Tek, Damion5050's Coir Tek, TL's Tek List, Frank's Tek List, EvilMushroom666's Pasteurization Tek, How It Should & Shouldn't Look - NEW CULTIVATORS GUIDE *** *** AFGHAN KUSH GROW LOG *** ***
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cloudboy
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Re: Easy Antibiotic Agar Tek For The Absolute Beginner [Re: PussyFart]
#17356906 - 12/08/12 06:32 PM (11 years, 1 month ago) |
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Can you add AA to grain spawn jars?
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teal
Art of Troll

Registered: 04/14/11
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Re: Easy Antibiotic Agar Tek For The Absolute Beginner [Re: PussyFart]
#17356967 - 12/08/12 06:45 PM (11 years, 1 month ago) |
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Quote:
cloudboy said: Can you add AA to grain spawn jars?
Yes, just cut a wedge and drop it in. You could make a mycelium syringe too, but watch how much water you add.
Quote:
Notahacker420 said:
Quote:
teal said: You can probably microwave it, but here I steam sterilize it for 20 minutes.
You cannot sterilize anything in a microwave oven.
The idea is that once the agar is hot enough, the combination of heat and antibiotic can sterilize anything it touches. So the purpose of the microwave is just to heat the agar to boiling. I haven't tested it, but I have seen other AA teks that just use a microwave.
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Stool_Sample
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Re: Easy Antibiotic Agar Tek For The Absolute Beginner [Re: teal]
#17357098 - 12/08/12 07:18 PM (11 years, 1 month ago) |
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-------------------- "wise words here"
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rubycon
Qui the Peacebringer



Registered: 11/19/12
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Re: Easy Antibiotic Agar Tek For The Absolute Beginner [Re: Stool_Sample]
#17357274 - 12/08/12 07:51 PM (11 years, 1 month ago) |
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Thanks man I'm moving to Agar and I find this highly useful.
-------------------- Credulous at best, your desire to believe in angels in the hearts of men. Pull your head on out your hippy haze and listen; I shouldn't have to say it all again. The universe is hostile and So impersonal. Devour to survive; So it is. So it's always been.
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tabiyeh



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Re: Easy Antibiotic Agar Tek For The Absolute Beginner *DELETED* [Re: rubycon]
#17357376 - 12/08/12 08:11 PM (11 years, 1 month ago) |
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Post deleted by tabiyehReason for deletion: Life is lowly anonymity.
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st1llnox
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Re: Easy Antibiotic Agar Tek For The Absolute Beginner [Re: tabiyeh]
#17360404 - 12/09/12 11:42 AM (11 years, 1 month ago) |
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Looks... delicious!
-------------------- Back, bitches. st1lln0x: so i'm on weed, temazepam, adderall, dexedrine, dxm, dph, alcohol, nicotine, caffeine, tryptophan, GABA, and kratom Cavemen_savemen: st1lln0x, do you feel like a robot yet? st1lln0x: I feel like a fucking Gundam Click to friend me on Steam for Counter-Strike
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Drunk3n Duck
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Re: Easy Antibiotic Agar Tek For The Absolute Beginner [Re: st1llnox]
#17360673 - 12/09/12 12:23 PM (11 years, 1 month ago) |
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i just ordered some petri dishes i think ill try out this tek for them thanks!
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teal
Art of Troll

Registered: 04/14/11
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Re: Easy Antibiotic Agar Tek For The Absolute Beginner [Re: Drunk3n Duck]
#17381291 - 12/12/12 05:23 PM (11 years, 1 month ago) |
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bump
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The.Medicine.Man
Ethnobotanical Enthusiast


Registered: 01/04/11
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Re: Easy Antibiotic Agar Tek For The Absolute Beginner [Re: teal]
#17607624 - 01/25/13 08:10 AM (11 years, 4 days ago) |
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Quote:
teal said:
Cut one potato (the size doesn't matter) and boil it with half a cup of water for 15 minutes. Let the water boil down to 25-50 mL.
Pour the potato broth into the jar. Fill the jar ~1/3 with the potato
.
I can't find this same bottle, I know you said boil down to 25-50m l, but how many ml do you use in the final solution?
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hippopotamu5
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Re: Easy Antibiotic Agar Tek For The Absolute Beginner [Re: The.Medicine.Man]
#17607778 - 01/25/13 09:04 AM (11 years, 4 days ago) |
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It looks like he boils one half cup of water with potatoes down to 25-50 ml and then uses ALL of it to mix in with agar, dextrose, and antibiotic before adding more plain water (judging from the picture it looks like he uses about 2 or 3 parts plain water to 1 part potato water).
Edited for: You're right, half cup rather than a cup. I misread.
--------------------
 karode13 said: Don't be a pussy, they're only glochids. From a Reviewer on Amazon.com: Sounds like your cacti was already dying do to your crappy care of it..... probably some kind of rot. Or maybe it hated you and commit seppuku.
Edited by hippopotamu5 (01/25/13 12:24 PM)
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The.Medicine.Man
Ethnobotanical Enthusiast


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Re: Easy Antibiotic Agar Tek For The Absolute Beginner [Re: hippopotamu5] 1
#17607971 - 01/25/13 09:59 AM (11 years, 4 days ago) |
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He starts with half a cup (4oz) of water. Reducing it to 25-50ml (.84-1.7 oz). I'm thinking I will try this with 1oz liquid and see how it turns out. What would happen if I used too little liquid when making agar? Would it still colonize?
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The.Medicine.Man
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Re: Easy Antibiotic Agar Tek For The Absolute Beginner [Re: The.Medicine.Man]
#17607976 - 01/25/13 10:01 AM (11 years, 4 days ago) |
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Quote:
The.Medicine.Man said: He starts with half a cup (4oz) of water. Reducing it to 25-50ml (.84-1.7 oz). I'm thinking I will try this with 1oz liquid and see how it turns out. What would happen if I used too little liquid when making agar? Would it still colonize?
Looking at this glass, and its intended purpose, it is probably a 4oz container. He fills it approximately 1/3, so just over an ounce of potato water is my thinking.
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thiotimoline
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Re: Easy Antibiotic Agar Tek For The Absolute Beginner [Re: hippopotamu5]
#17608006 - 01/25/13 10:12 AM (11 years, 4 days ago) |
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What's the reason for boiling it down if you're just going to dilute it again? Does the extra cooking break it down in some way?
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piggy
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Re: Easy Antibiotic Agar Tek For The Absolute Beginner [Re: thiotimoline]
#17608066 - 01/25/13 10:28 AM (11 years, 4 days ago) |
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Very clever!
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plainoldjake



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Re: Easy Antibiotic Agar Tek For The Absolute Beginner [Re: piggy]
#17608175 - 01/25/13 11:00 AM (11 years, 4 days ago) |
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Was thinking about moving to Agar and this Tek looks "Fun"
Has anyone had any luck with it?
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The.Medicine.Man
Ethnobotanical Enthusiast


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Re: Easy Antibiotic Agar Tek For The Absolute Beginner [Re: thiotimoline]
#17608972 - 01/25/13 01:17 PM (11 years, 4 days ago) |
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Quote:
thiotimoline said: What's the reason for boiling it down if you're just going to dilute it again? Does the extra cooking break it down in some way?
I think, and I may be way off here, the boiling down is to break the starches and potato solids down and get a more homogenous mixture.
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Tipote
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Re: Easy Antibiotic Agar Tek For The Absolute Beginner [Re: teal]
#22614739 - 12/04/15 08:40 PM (8 years, 1 month ago) |
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nice guide, thanks! i was a bit intimidated by agar before but i think i'm going to try this!..
--------------------
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