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OfflineJon
Registered: 06/28/03
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Several agar questions.
    #4297885 - 06/15/05 03:23 AM (12 years, 2 months ago)

I just started to experiment with agar, at the moment I have 2 colonized petri dishes and a mason jar full of leftover agar which was inoculated with spores as well. I was wondering if there was a specific look to the mycelium that would make it suitable to inoculate rye jars. Also I have some open ends on my knowledge of how to transfer the agar to the grain. Do I apply a segment then shake it, or is there a specific position the colonized segment has to be against the grain, or do I just leave it there. Is there also any disadvantages of having an agar layer too thick? And at last, what the hell do I do with that quart jar with colonized agar at the bottom, the jar seems to have the cleanest colonization out of the petri plate and I dont know exactly how I can put it to use. One being that I dont have to proper length in tools to reach the end of the mason jar, and also the fact that the agar layer is about a half inch thick. Any help is greatly appreciated.


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Offlineblackout
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Re: Several agar questions. [Re: Jon]
    #4297979 - 06/15/05 04:19 AM (12 years, 2 months ago)

The mycelium should look ropey, like strands. All I can see against thick agar is that it is wasteful and harder to lift. I have made long inoculation loops from coat hangers to get to the bottom of jars. You can scrape the top layer of myc off the thick agar and stick it in your jar. Just stick it in and shake it so it is covered in grain.


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InvisibleRoadkill
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Re: Several agar questions. [Re: Jon]
    #4298134 - 06/15/05 05:21 AM (12 years, 2 months ago)

you look for spots like these where I pointed in Red.
you want to transfer this agressive mycelium growth to new plates.


when you get some agressive growth...transfer again.
this might take several transfers to new plates to achieve.

so your transfer will look like this.


side view.


growing out.


growing out another view.


then you cut out some wedge sections for your jars.
tilt your jar so the substrate moves and stick a wedge down into the space you have created in the jar from tilting.
then tilt it back...the substrate will cover the agar wedge.
repeat this several times in different places inside the jar.
I really should take some pictures of this part of the procedure!~

then in about 7 days...you should have a fully colonized jar like these.


hope this was helpful to you.

tc


--------------------
Laterz, Road

Who the hell you callin crazy?
You wouldn't know what crazy was if Charles Manson was eating froot loops on your front porch!


Brainiac said:
PM the names with on there names, that means they have mushrooms for sale.



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OfflineJon
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Re: Several agar questions. [Re: Roadkill]
    #4298144 - 06/15/05 05:31 AM (12 years, 2 months ago)

more than helpful Roadkill, you kick ass.


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Offlineblackout
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Re: Several agar questions. [Re: Jon]
    #4298148 - 06/15/05 05:35 AM (12 years, 2 months ago)

Excellent pics Roadkill. They have convinced me to give agar another go instead of multispore.

If you add the final wedge to a LC instead of a jar do you find the resulting LC gives the same aggressive growth (or is it even faster?)


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Re: Several agar questions. [Re: blackout]
    #4298164 - 06/15/05 05:58 AM (12 years, 2 months ago)

thanks guys.

:smile:

good question bro!~

I have never used an agar wedge to start an LC...yet.

What I have done alot of is this...

take a scalpel and peel the mycelium off the agar in the petri dish.
take the mycelium and put it in a sterilized blender(I have a stainless steel one) with some sterile water.
turn the blender on and slice and dice it all up good.
pour the mix into a sterile jar...or whatever...I use sterilized baby food jars. :smile:
and then just make some syringes from this mixture.

bad ass mycelium syringes...with very agressive growth.

and you can do clones this way too.

tc


--------------------
Laterz, Road

Who the hell you callin crazy?
You wouldn't know what crazy was if Charles Manson was eating froot loops on your front porch!


Brainiac said:
PM the names with on there names, that means they have mushrooms for sale.



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Offlineblackout
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Re: Several agar questions. [Re: Roadkill]
    #4298192 - 06/15/05 06:41 AM (12 years, 2 months ago)

I was trying a agar experiment last night to do sort of the same thing. I have no flow hoods and work in a dirty environment.

I sealed a 35ml shot glass and some agar into a autoclave bag. Then I microwaved the whole thing. The plan was to have a small pool of sterile agar under the air filter. While the bag is still warm I could crease the bag to make a second pool (petri dish) in the bag. Then I would put a blob of silicon on the filter as a self healing port. Then inject a drop of spore solution on the agar.
After I get growth on the first "petri" I could move a piece of nice growth onto my second "petri" in the bag. This would be done by holding the shot glass through the bag and using it to scoop up a small piece of agar and dump it on the second pool of agar. (this step may no be necessary since my first pool could be made very large and the radial growth would be much greater).

When I have good growth on my agar I can scoop some up with the shot glass again. The glass can be cleaned out by manipulating it in the bag. Then I can move the shot glass under the injection site and insert a syringe with 10ml of sterile water. With the needle I should be able to cut up the agar wedge and then squirt in water and suck it up, ready to inoculate.

Anyway, I micorwaved the bag and it got a hole in it, I think the glass got very hot and melted the bag. I think the problem was that the amount of agar was tiny so while it was on low power it still got extremely hot since all the power was going into such a small mass.

I will be able to iron the part of the bag where the hole is and start again. I will have to steam it this time, or stick it in a dish of water in the microwave.

It may also be possible to work with dirty prints with this method, sucking up good growth and injecting into another similar bag.

Do you think I would need 2 "petris" or would one big one do.


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Edited by blackout (06/15/05 06:43 AM)


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InvisibleRoadkill
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Re: Several agar questions. [Re: blackout]
    #4298207 - 06/15/05 07:18 AM (12 years, 2 months ago)

I think you need to make a glove box bro!~

:wink:

but...I like the way that you think.
sounds like making pools(for a petri dish) in the autoclave bag would work to me.
my petri dishes are 4" round...so you can decide how many to make.
probably depends on the size of your bag.
your pretty smart...great idea!~

maybe try and find a small plastic container to take the place of the shot glass...the plastic would have to be the right kind of course.

----

I used to do alot of open air work with agar...and was quite successful.
I kept everything clean in my work area(kitchen) and made sure there was no air movement in the room at all.
I would spray the air with Lysol or cheaper brand from a $1 store...constantly.

I'm a firm believer that most contams are floaters riding the air currents in the room...just like mushroom spores do in a fruiting chamber.
People wonder why spores are on top of the tallest mushroom...they are riding the air movement inside the fruiting chamber.
An air draft in a house can cause all sorts of havok...spreading contams everywhere.

Do you have the large clear rubbermaid brand containers in Europe?
the 28 gal/106 L Max Latch Storage...to be exact.
I'll be posting a cheap glovebox I made from one...later this week.

Take care...I'm heading to bed!~

good luck!~


--------------------
Laterz, Road

Who the hell you callin crazy?
You wouldn't know what crazy was if Charles Manson was eating froot loops on your front porch!


Brainiac said:
PM the names with on there names, that means they have mushrooms for sale.



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Offlineblackout
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Re: Several agar questions. [Re: Roadkill]
    #4302535 - 06/16/05 04:41 AM (12 years, 2 months ago)

I look forward to seeing the 106l glove box, I won't build one but it may give me more ideas. I can stash a 106l box in my drawer!

I made my "petri bag" this morning, with a few mods.
The agar was all over the inside of the bag, it is very difficult to make a nice pool. I cut open the bag and cleaned it out. I then got a black microwaveable dish that a ready-meal came in. I cut it down so it has a 1cm lip. I then put the cap of a deodorant in the bag and about 100ml of water. The bag was resealed over the holes and microwaved for 2mins on high and then 20 onlow which kept the bag puffed up with steam.

I have a jar with sterile agar and an injection port on the cap. The plan is to suck out the excess water from the bag (some will always remain). Then I can warm my agar jar and inject some agar into my black dish (I hope the black will be a nice contrast for the white myc. Then I will work as I mentioned before, with the plastic cap replacing the shot glass. I think the shot glass would survive with 100ml of water in the bag. But when I took it out I saw it had cracked so went with your suggestion


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Edited by blackout (06/16/05 04:42 AM)


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