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OfflineRogerRabbitM
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Re: Hot Pouring Agar [Re: GungGung] * 4
    #14708588 - 07/02/11 09:15 PM (12 years, 8 months ago)

Quote:

GungGung said:

Pouring hot agar on tissue... i don't know who comes up with this shit.

One good book will save you many hours of scanning posts, and, wait, it gets better, 99% of the information will be correct, as opposed to, well, i LOVE the Shroomery, but I've seen people post some pretty uninformed things, which brings our collective average down a bit.

Also, Stamets doesn't spam, flame, or ramble...




I'll tell you who comes up with that shit.  Me.  If you don't like it, then use another method, but don't ridicule that which you do not understand.

I've never said to pour hot agar over a pin, and cloning from third or forth flush as the OP is trying to do is not a good idea.

However, pouring hot agar over an infected plate is a long-proved method of isolating mushroom mycelium from attached bacteria, and is far superior to using chemicals.

The pictures of cloned pins the OP was referring to are mine.  No hot agar was used.  If the pins are rapidly growing when picked and placed on the dish, the mycelium will outrun the contaminants if a very weak nutrient load is used in the agar.  A normal nutrient mix will favor bacteria and should not be used when cloning.
RR


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semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
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OfflineGungGung
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Re: Hot Pouring Agar [Re: RogerRabbit]
    #14711421 - 07/03/11 02:51 PM (12 years, 8 months ago)

I was referring to the pouring of hot agar over a pin. I clone whole pins in exactly the same way, based on this thread that you posted like 5 years ago, where that cool pic is from.

I have not seen a thread no read in any of my texts anything pertaining to the pouring of hot agar over a contaminated dish. I would love more information if it is a useful technique.

It appeared, to me, that the OP had been having troubles because he was using that method, on pins.

I was only recommending what i know will work, but may have been overly judgmental about a method that i was unaware of. My apologies.

-Edit-

And in regards to a low nutrient content in agar, :thumbup:

Thank you for reccomending that to me in my Penis Envy Isolation Thread. I usually use premixed antibiotic MEA from FP, especially to germinate spores, but since you told me about that, i have been mixing my own MEA with no antibiotics, and a 2/1 agar/nutrient ratio, for cloning. The results speak for themselves, i'll update that thread with new pictures soon.


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~You're already dead, but you're about to die~


Penis Envy in Monotubs      Ecuador in Monotubs

Edited by GungGung (07/03/11 03:04 PM)

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InvisibleWise Toad
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Re: Hot Pouring Agar [Re: RogerRabbit]
    #14740991 - 07/09/11 01:20 PM (12 years, 8 months ago)

Quote:

RogerRabbit said:
Quote:

GungGung said:

Pouring hot agar on tissue... i don't know who comes up with this shit.

One good book will save you many hours of scanning posts, and, wait, it gets better, 99% of the information will be correct, as opposed to, well, i LOVE the Shroomery, but I've seen people post some pretty uninformed things, which brings our collective average down a bit.

Also, Stamets doesn't spam, flame, or ramble...




I'll tell you who comes up with that shit.  Me.  If you don't like it, then use another method, but don't ridicule that which you do not understand.

I've never said to pour hot agar over a pin, and cloning from third or forth flush as the OP is trying to do is not a good idea.

However, pouring hot agar over an infected plate is a long-proved method of isolating mushroom mycelium from attached bacteria, and is far superior to using chemicals.

The pictures of cloned pins the OP was referring to are mine.  No hot agar was used.  If the pins are rapidly growing when picked and placed on the dish, the mycelium will outrun the contaminants if a very weak nutrient load is used in the agar.  A normal nutrient mix will favor bacteria and should not be used when cloning.
RR





What constitutes a weak nutrient mix? I make my MEA from 10g agar-agar, 10g LME, 500ml distilled water. Is that already weak, if not how should I mix it up to be so?

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OfflineRogerRabbitM
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Re: Hot Pouring Agar [Re: Wise Toad]
    #14742530 - 07/09/11 07:49 PM (12 years, 8 months ago)

Cut the malt extract in half.
RR


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semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
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OfflineGungGung
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Re: Hot Pouring Agar [Re: RogerRabbit]
    #14748651 - 07/10/11 11:09 PM (12 years, 8 months ago)

Quote:

RogerRabbit said:
Cut the malt extract in half.
RR




:whathesaid:

2:1 agar : nutrients should be good. for 1 sleeve of (20) dishes that works out to~

10.5 g agar agar
5 g Pure light malt extract
500 ml H20

The extra .5g agar is to absorb the H20 that the other 5g of malt would have been responsible for. I have poured with 5g nutrients and 10g agar and seen condensation on top of the agar. I feel that the extra .5g helps the agar solidify to the proper degree, without the full nutrient load.

Am i out of my mind here or does this sound legit?


--------------------
~You're already dead, but you're about to die~


Penis Envy in Monotubs      Ecuador in Monotubs

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InvisibleWise Toad
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Re: Hot Pouring Agar [Re: GungGung]
    #14749442 - 07/11/11 02:29 AM (12 years, 8 months ago)

Thanks for the advice RR

The weaker nutrients are the more rhizomorphic mycelium will be due to adaptation correct?

So then would it be better to adjust my standard MEA mix, that is agar 10/LME 10/water 500, to a lower nutrient ratio from now on?

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OfflineRogerRabbitM
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Re: Hot Pouring Agar [Re: Wise Toad]
    #14758599 - 07/12/11 06:40 PM (12 years, 8 months ago)

A weaker solution will favor faster growth of the mushroom mycelium as it seeks a food source.  It's the best way to outrun molds, which tend to favor a richer mix.  Just experiment to find what works best.

Whatever the mix, rapid transfers and hot pouring will work wonders, but you have to be right there when it's time to make a transfer or the molds will break through within hours of the mushroom mycelium.
RR


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semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
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OfflineNoFaceToSave
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Re: Hot Pouring Agar [Re: RogerRabbit]
    #14770971 - 07/15/11 03:42 AM (12 years, 8 months ago)

Alright, here's where we are at, last run of petri dishes we hot poured over live tissue when the agar was cooled down enough to handle the wiskey bottle with out burning my hands in rubber gloves. Finally had sucessful growth without bacterial problems. None of the myc turned black and died from being scorched alive. Still having condensation issues on the lid of the petri dishes so maybe I should let the agar get just slightly cooler, like possibly on the verge of setting (general concensus of agar setting at approx 95 degrees).

I would say 95-100  was roughly about what that agar was at. I think this time im going to set the agar outside of the 921 and let it cool infront of the laminar flow hood (with snythetic filter disc cap on covered in foil during pc) until its fine to pick up, but i'll leave the petri dishes in the 921 with the hot water so they stay warm, this way when i introduce the agar into the petri dish their will be less of a temperature dish creating condensation.

I've picked up some rather interesting things i've been looking for from this thread, The 10% bleach soak for 15 mins from RR's notes and when Mycoelf mentioned that he has seen where people soak the mushie in 5 % bleach for 10 min. I'm not entirely entrhalled about the thought of h202 on pins, I understand that it works well for you but in my person struggles with live tissue I haven't had much luck with it for anything other than cobweb. Last time i tried 10% for 15 my pins turned completely white, but perhaps 10% just dunked for a minute would hit it or 5% at a few mins.

This weak nutrient load RogerRabbit is talking about has peaked my interest. As previously mentioned in my posts the agar recepie I've been using is:

150 grams of potatoe boiled 500ml for 30 mins which is then filtered through a tshirt cloth, creating a potato water solution. I then replace any water that was boiled off to have 500ml of potato water solution. I add 9 grams of agar and melt that down. I then add 1 tablespoon honey

I dont remeber the specifics on why i add honey because its been years since i've looked up the specifics on why but im really thinking about cutting the amount of potato wayyy down and the amount of honey way down. Perhaps just a splash of honey and 4 grams of potato boiled or even less potato like maybe a gram?

Any suggestions of a good mix with the potato, honey, agar mix for this particular transfering live tissue scenerio? For this morning im experimenting with 75 grams of potato and with about half a tablespoon. Perhaps less of a mix would be ideal but any information on how long into the growth cycle the myc goes before it starts using any substantial amount of nutrients would be handy for keeping in mind.

I really apprecieate everyone chiming in, sometimes my research leads to some rather akward dead ends or confusion.


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“By calling attention to ‘a well regulated militia’, the ’security’ of the nation, and the right of each citizen ‘to keep and bear arms’, our founding fathers recognized the essentially civilian nature of our economy. Although it is extremely unlikely that the fears of governmental tyranny which gave rise to the Second Amendment will ever be a major danger to our nation, the Amendment still remains an important declaration of our basic civilian-military relationships, in which every citizen must be ready to participate in the defense of his country. For that reason, I believe the Second Amendment will always be important.”
John F. Kennedy, April 1960

Edited by NoFaceToSave (07/15/11 03:55 AM)

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OfflineRogerRabbitM
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Re: Hot Pouring Agar [Re: NoFaceToSave]
    #14771337 - 07/15/11 07:44 AM (12 years, 8 months ago)

If you're doing a hot agar pour over mycelium, pour at around 140F.  The bottle will burn you hands so you'll need protection.
RR


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semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
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Offlineiforgot
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Re: Hot Pouring Agar [Re: NoFaceToSave]
    #14789826 - 07/18/11 11:05 PM (12 years, 8 months ago)

As far as condensation, the only way I've ever been able to avoid lots of it is to allow the plates to sit overnight under a laminar flow hood after pouring; I've also gone so far as to replace the lids with sterile lids, granted this is wasteful - but hey, I'm used to working with bacteria, and not so much fungi. 

A tip for preventing burns (as I've gotten my fair share from the thousands of liters of molten media I've poured) is to simply wrap a paper towel around the part where you grasp the bottle/flask from which you are pouring.  Its simple, yet effective, and also prevents hot agar from dripping across your fingers and into the petri dish.

Like RR said, the key is rapid transfers, and using a weaker nutrient concentration.  Since you are making your media using potatoes, I would be more apt to halve the amount of liquid from the potato liquid; in other words, if you boil 150 grams of potatoes in 500 ml of water, and then add water to make up that lost in evaporation, I would add another 500 ml of water to the strained potato-water to actually cut the concentration in half.  Then add 20 grams of agar, heat until the agar is dissolved completely, and autoclave (or PC), then allow to cool to about 55 - 60 degrees C (or 140 F), then pour. 

The easiest thing to do as far as perfecting a recipe would be to order the dehydrated powder online, or even order the pre-made plates... although, that is an expensive alternative. 

Of course, I'm probably just telling you a bunch of stuff you already know, and if so, I humbly apologize - I am just a noobie after all!!!

Cheers!

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OfflineNoFaceToSave
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Re: Hot Pouring Agar [Re: iforgot]
    #14809203 - 07/22/11 02:41 PM (12 years, 8 months ago)

Its all good stuff, last time I was in my flow hood room, we ran out of filter masks and needless to say we got bacterial contams on all the dishes, not making that mistake again.

Gloves, Masks, And re-arranging the work area so I can work faster, and keeping the work RIGHT infront of the flow hood is the strategy. Probably doing another run in the next 3 days or so. I tried a pour at 140 and the myc turned blue like it was bruised an died but who knows what the accuracy of my meat thermometer is, if its like +- 10 degrees thats a hugggge deal.


--------------------
“By calling attention to ‘a well regulated militia’, the ’security’ of the nation, and the right of each citizen ‘to keep and bear arms’, our founding fathers recognized the essentially civilian nature of our economy. Although it is extremely unlikely that the fears of governmental tyranny which gave rise to the Second Amendment will ever be a major danger to our nation, the Amendment still remains an important declaration of our basic civilian-military relationships, in which every citizen must be ready to participate in the defense of his country. For that reason, I believe the Second Amendment will always be important.”
John F. Kennedy, April 1960

Edited by NoFaceToSave (07/22/11 02:58 PM)

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Offlineiforgot
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Re: Hot Pouring Agar [Re: NoFaceToSave]
    #14833263 - 07/27/11 01:44 PM (12 years, 7 months ago)

I hope that you didn't put the meat thermometer into the agar to test the temp?! If so, that's where the contamination is coming from.  Also, when you are letting the agar cool to a temp so that you can pour it, leave it capped or the foil over the lid - whatever was on the lid when pressure cooking or autoclaving it - as it cools.  Another error I'm quite familiar with - once you open a sleeve of petri dishes, any that are not used within 24 hours may as well be tossed in the trash, as they are no longer sterile.  For example, if you have a fresh package of 25 plates, but you only pour 10 plates, you really can't count on the other 15 plates to remain sterile for a few days until you pour more plates.

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