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Offlinerxb
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rxb agar jar tek - low cost low tek isolation solution * 2
    #21899443 - 07/05/15 01:31 AM (8 years, 6 months ago)

this will allow you to do isolation without a flow hood, you dont even really need a still air box, but using one wouldnt hurt anything and might help, who knows. flow hoods are great, im not knocking flow hoods. this should give you as good of results, you can even take isolated material from a mold contaminated plate without blowing mold spores all over your lab.

for most applications i prefer this method to using a flow hood, but it isnt as easy to see what you are doing, and you dont get as many style points.

you do however save hundreds of dollars :smile:

Things you need:
1) 8 oz widemouth jars with lids (they come 4 for $4 at walmart)
2) a 2+ inch needle, I use a 12 gauge 2 inch autoclavable stainless steel needle. (there are vet supply places which have them for $2, they are reusable so i dont really count this as a cost but shrug)
3) aluminum foil ($1)
4) agar (you wont even use a dollars worth per jar)
5) a straight pin, or small gauge needle for punching air holes. (free)
6) high temp silicone from an auto parts shop (optional, but i like it)
7) poly fill (free in a pillow, or 2 dollars at a fabric store)

i used a quart jar for the construction demo, but this should be done in 8oz widemouth jars, so they are short enough for this to work, all my 8 oz jars were full... you should get the idea :smile:



1) mix agar.

2) fill your jars with 50 cc of mixed agar (you can find 60cc syringes at tractor supply)

3) cut an aluminum sheet the width of the jar or more and fold it in half so that its two sheets, then do
this again, so you have 2 aluminum rectangles that are 2 layers each.



4) get your jar, and put one aluminum foil set on it, and create a little bowl in the foil and crimp along
the edge of the jar



5) take a polyfill ball about the size of a baseball and smoosh it in your hands until its very small



6) attempt to put the now tiny polyfill ball in the bowl created, and then cover with the second foil
rectangle



7) while struggling desperately to keep the polyfil ball under the second aluminum sheet put the ring on
top, stuff in any polyfill that sticks out and then push down and tighten the jar ring.



8) using a straight pen (or anything that can pierce holes in the aluminum) poke lots of holes and be sure to get through both layers of foil. this is pressure relief and air exchange without holes it will probably not survive the PC



9) repeat process until all jars are done.



10) pressure cook the jars at 15 psi for 30 minutes. Allow pressure cooker to reach 0 psi before opening
the pressure cooker.



11) allow ample time for jars to cool to room temperature.

12) apply spores or live culture to the agar with a syringe. If you have dry spores, mix with sterilized
water, then suck into syringe and drip onto agar. Use a similar process for tissue samples, live culture
and spore syringes can be used directly, be sure to flame needles.

13) after injection, if you use a large needle, its not a bad idea to put a small dot of high temp silicone
over the hole created by the needle.

14) wait until the dish is colonized to your satisfaction.

15) fit a syringe with your large guage 2 inch long or greater needle (I use 12 gauge 2 inch stainless
steel needles I get from an online vet supplier) and fill the syringe with sterilized water.

16) because we are using foil and a very short jar, a specific sector can be isolated by putting the needle
into the jar right above the sector. Make a small circle with the needle around the sector to be sampled
as a boundary layer. And scratch a grid across the top of the sector



17) be sure you are on a level work surface and put a few drops of water in the sector and stir the water
into the agar then add another drop or two. Then pull the water back into the syringe and repeat the
process until you are satisfied you have enough material.

18) remove from the jar, shake the syringe well, and flame sterilize the needle.

19) drip the contents into new agar jars.

20) repeat the process until you have an isolated mycelium colony.

21) once you have an isolated strain you can make a live culture by simply adding sterilized water and
shaking the jar and then removing all water, sometimes the mycelium will recolonize the agar after
you do this, but it usually breaks up the agar.

not the best at writing so im sure i left a few typos, thanks for reading :smile:


Edited by rxb (07/11/15 11:55 PM)


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Offlinerxb
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Re: rxb agar jar tek - low cost low tek isolation solution [Re: rxb]
    #21931186 - 07/11/15 10:25 PM (8 years, 6 months ago)

i have examples of spores in the agar jars and a transfer from multispore syringe to multispore culture and an isolation from the culture.. the problem is i have not figured out how to photograph the jars in a way that looks like ANYTHING.


--------------------
->$10 FLOW HOOD ALTERNATIVE <-

. i cleaned a mold contaminated live culture and saved it. (might have useful applications)

[quote]Enlil said:
I'd be the guy with thousands of minions doing my bidding and all of the hot women locked in a cage for my use.[/quote]


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Offlinerxb
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Re: rxb agar jar tek - low cost low tek isolation solution [Re: rxb]
    #21931189 - 07/11/15 10:27 PM (8 years, 6 months ago)

i suppose i'll eventually just do some jars i dont care about and open them up and photograph them.

at the moment im just enjoying that this is easier than my flow hood, and seems to be working.


--------------------
->$10 FLOW HOOD ALTERNATIVE <-

. i cleaned a mold contaminated live culture and saved it. (might have useful applications)

[quote]Enlil said:
I'd be the guy with thousands of minions doing my bidding and all of the hot women locked in a cage for my use.[/quote]


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Invisibleazur
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Re: rxb agar jar tek - low cost low tek isolation solution [Re: rxb]
    #21931385 - 07/11/15 11:27 PM (8 years, 6 months ago)

Easier than your flow hood?  I missed something. I didn't think if could get easier than a flow hood.
It is a promising tek though. Well thought out and performed. Good job


--------------------


A cube is NOT a cube.

FALL IN LOVE WITH LC
FOTTSE!!!
ALL NOOBS READ THIS!!!



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Offlinerxb
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Re: rxb agar jar tek - low cost low tek isolation solution [Re: azur]
    #21931391 - 07/11/15 11:28 PM (8 years, 6 months ago)

i cant use the flow hood on my couch.

can you?


--------------------
->$10 FLOW HOOD ALTERNATIVE <-

. i cleaned a mold contaminated live culture and saved it. (might have useful applications)

[quote]Enlil said:
I'd be the guy with thousands of minions doing my bidding and all of the hot women locked in a cage for my use.[/quote]


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Invisibleazur
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Re: rxb agar jar tek - low cost low tek isolation solution [Re: rxb]
    #21931404 - 07/11/15 11:30 PM (8 years, 6 months ago)

Not quite;)


--------------------


A cube is NOT a cube.

FALL IN LOVE WITH LC
FOTTSE!!!
ALL NOOBS READ THIS!!!



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Offlinerxb
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Re: rxb agar jar tek - low cost low tek isolation solution [Re: rxb]
    #21931406 - 07/11/15 11:31 PM (8 years, 6 months ago)

i guess if i moved the flow hood beside the couch... they would be equally easy :smile:


--------------------
->$10 FLOW HOOD ALTERNATIVE <-

. i cleaned a mold contaminated live culture and saved it. (might have useful applications)

[quote]Enlil said:
I'd be the guy with thousands of minions doing my bidding and all of the hot women locked in a cage for my use.[/quote]


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Invisibleazur
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Re: rxb agar jar tek - low cost low tek isolation solution [Re: rxb]
    #21931416 - 07/11/15 11:33 PM (8 years, 6 months ago)

Either way, though I'll never use this tek, it's great for beginners that find sab's cumbersome and don't have loot for a hood.
You should have it moved to cult. Probably do better there


--------------------


A cube is NOT a cube.

FALL IN LOVE WITH LC
FOTTSE!!!
ALL NOOBS READ THIS!!!



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Offlinerxb
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Re: rxb agar jar tek - low cost low tek isolation solution [Re: azur]
    #21931439 - 07/11/15 11:38 PM (8 years, 6 months ago)

you might try it one day just cuz its great for long term storage as little jars of colonized agar are like huge slants :smile:


--------------------
->$10 FLOW HOOD ALTERNATIVE <-

. i cleaned a mold contaminated live culture and saved it. (might have useful applications)

[quote]Enlil said:
I'd be the guy with thousands of minions doing my bidding and all of the hot women locked in a cage for my use.[/quote]


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Invisibleazur
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Re: rxb agar jar tek - low cost low tek isolation solution [Re: rxb]
    #21931450 - 07/11/15 11:40 PM (8 years, 6 months ago)

I may make some in the little jars


--------------------


A cube is NOT a cube.

FALL IN LOVE WITH LC
FOTTSE!!!
ALL NOOBS READ THIS!!!



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Offlinerxb
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Re: rxb agar jar tek - low cost low tek isolation solution [Re: azur]
    #21931457 - 07/11/15 11:43 PM (8 years, 6 months ago)

yeah the little jars are kind of the selling point... little jar + big needle = isolation without $$


--------------------
->$10 FLOW HOOD ALTERNATIVE <-

. i cleaned a mold contaminated live culture and saved it. (might have useful applications)

[quote]Enlil said:
I'd be the guy with thousands of minions doing my bidding and all of the hot women locked in a cage for my use.[/quote]


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Invisibleazur
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Re: rxb agar jar tek - low cost low tek isolation solution [Re: rxb]
    #21931474 - 07/11/15 11:46 PM (8 years, 6 months ago)



--------------------


A cube is NOT a cube.

FALL IN LOVE WITH LC
FOTTSE!!!
ALL NOOBS READ THIS!!!



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Offlinerxb
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Re: rxb agar jar tek - low cost low tek isolation solution [Re: azur]
    #21931483 - 07/11/15 11:48 PM (8 years, 6 months ago)

ps thanks for bumping the shit out of my tek :smile:


--------------------
->$10 FLOW HOOD ALTERNATIVE <-

. i cleaned a mold contaminated live culture and saved it. (might have useful applications)

[quote]Enlil said:
I'd be the guy with thousands of minions doing my bidding and all of the hot women locked in a cage for my use.[/quote]


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InvisiblePrisoner#1
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Re: rxb agar jar tek - low cost low tek isolation solution (moved) [Re: rxb]
    #21931500 - 07/11/15 11:50 PM (8 years, 6 months ago)

This thread was moved from Advanced Mycology.

Reason:
moved at user request


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Invisibleazur
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Re: rxb agar jar tek - low cost low tek isolation solution (moved) [Re: Prisoner#1]
    #21931514 - 07/11/15 11:52 PM (8 years, 6 months ago)

We're gonna be tired in the morning pris.


--------------------


A cube is NOT a cube.

FALL IN LOVE WITH LC
FOTTSE!!!
ALL NOOBS READ THIS!!!



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Invisibleazur
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Re: rxb agar jar tek - low cost low tek isolation solution (moved) [Re: azur]
    #21935266 - 07/12/15 09:44 PM (8 years, 6 months ago)

Bumping to share


--------------------


A cube is NOT a cube.

FALL IN LOVE WITH LC
FOTTSE!!!
ALL NOOBS READ THIS!!!



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InvisibleMr.GuessWork
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Re: rxb agar jar tek - low cost low tek isolation solution (moved) [Re: azur]
    #21936511 - 07/13/15 09:15 AM (8 years, 6 months ago)

The use of the syringe to pick up the isolate is a pretty cool idea. Isn't it kind of hard to do a streak to get good separation between colonies with all that stuff in the way though?


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Re: rxb agar jar tek - low cost low tek isolation solution (moved) [Re: Mr.GuessWork]
    #21936674 - 07/13/15 10:14 AM (8 years, 6 months ago)

While this could work okay for transfers & myc. for inoculations, it seems it may be tough to isolate sectors like this... :shrug:

SAB still seems much easier than going through all the process of making up the jars and trying to isolate with a syringe needle through the 2 layers of foil and polyfil.

Not saying it can't work, just seems like a lot of effort & time when one can just use a $7 SAB and do the same thing in just minutes or even seconds...


--------------------
"They who can give up essential liberty, to obtain a little temporary safety, deserve neither liberty nor safety." - Benjamin Franklin

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Edited by FreeWorldOrder (07/13/15 10:21 AM)


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Offlinerxb
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Re: rxb agar jar tek - low cost low tek isolation solution (moved) [Re: FreeWorldOrder]
    #21937254 - 07/13/15 01:04 PM (8 years, 6 months ago)

Quote:

FreeWorldOrder said:
While this could work okay for transfers & myc. for inoculations, it seems it may be tough to isolate sectors like this... :shrug:

SAB still seems much easier than going through all the process of making up the jars and trying to isolate with a syringe needle through the 2 layers of foil and polyfil.

Not saying it can't work, just seems like a lot of effort & time when one can just use a $7 SAB and do the same thing in just minutes or even seconds...




it works PERFECTLY for isolating sectors.

because you can put the needle anywhere on the lid, you can get material from very very specific locations. thats really the POINT of the tek.

i have a number of SAB and a flow hood, and a posative pressure box. putting the needle through 3 layers is effortless. isolation is effortless. WAAAAAAY easier than a SAB in my opinion.


--------------------
->$10 FLOW HOOD ALTERNATIVE <-

. i cleaned a mold contaminated live culture and saved it. (might have useful applications)

[quote]Enlil said:
I'd be the guy with thousands of minions doing my bidding and all of the hot women locked in a cage for my use.[/quote]


Edited by rxb (07/13/15 01:06 PM)


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InvisibleMr.GuessWork
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Re: rxb agar jar tek - low cost low tek isolation solution (moved) [Re: rxb]
    #21937299 - 07/13/15 01:16 PM (8 years, 6 months ago)

How do you inoculate the plate with the needle? Do you only do liquid inoculation? It seems's like it'd be hard to get a good streak, especially if you wanted to do a couple streaks to get really good separation.


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