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Invisiblechampinhom
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can't seem to get a clean culture
    #22321089 - 10/01/15 08:39 PM (8 years, 3 months ago)

I have been trying to isolate a clean culture of a mutant cube mushroom. I took a piece of the stem of the mushroom in question, dipped it in H2O2 and put it in an agar jar. a few weeks later the piece had grown out but their were numerous circles of green mold in the jar as well. So I did a transfer to another jar. Same thing occurred. So I made 3 transfers to three jars--and damned if the exact same pattern didn't repeat. Below is a pic of one of the jars. All the others were like it:


Today, not wanting to give up (but just about to) I did two more transfers. This time I did something a little different. I took as tiny a piece of the mycelium as I could get on the end of my transfer blade. My rationale here was this: In all the prior transfers, even though I have taken only what looked like clean mycelium, somehow I have also taken mold spores or mold mycelium along each time. It must be therefore that the mold is somewhere mixed with the cube mycelium. when I turn the agar jars over and look at the bottom, I see green spots beneath what looks like a white expanse from above. So assuming that it is the case that the mycelium is contaminated through and through, I tried to get a shred of it that might have escaped the mold plague.

Has anyone experience with this sort of situation? If so, how to proceed?


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My father used to say: I don't care what else you do in life, just don't be an asshole. People, forgive me when I forget what my daddy said.

Cut back the proliferating list of people whose opinions can hurt you. Unless they have done or want to do you some good, their views are just not worth tracking.
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Those whom the gods would save, they dower with compassion. Mr. P.  Silocybin


Edited by champinhom (10/01/15 08:42 PM)


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Invisiblemrbart4444
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Registered: 09/13/14
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Re: can't seem to get a clean culture [Re: champinhom]
    #22321126 - 10/01/15 08:45 PM (8 years, 3 months ago)

Your process isnt clean enough. I dont know where youre going wrong but try changing agar or just drop that culture .


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Offlinethebitterbuffalo26
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Re: can't seem to get a clean culture [Re: champinhom]
    #22321127 - 10/01/15 08:46 PM (8 years, 3 months ago)

It might take a few transfers done pretty quickly to isolate the fungus. Each one of those mold circles is where a mold spore landed.  If the jar was clean before you opened it, then those came from when you transferred into it.  gotta clean up that part of your technique maybe?


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OfflineBoomertown
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Re: can't seem to get a clean culture [Re: champinhom]
    #22321135 - 10/01/15 08:46 PM (8 years, 3 months ago)

bump


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Invisiblemrbart4444
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Re: can't seem to get a clean culture [Re: champinhom]
    #22321144 - 10/01/15 08:49 PM (8 years, 3 months ago)

Also what do you use a SAB or flow hood?


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InvisiblePastywhyteMDiscord
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Re: can't seem to get a clean culture [Re: mrbart4444]
    #22321260 - 10/01/15 09:14 PM (8 years, 3 months ago)

Satellites like that are indicative of sloppy technique. How long are you waiting for the air to settle before opening the plate after you bring your flamed tools back in the box? Ensure you never reach over a open plate. Try to use smooth movement but at the same time never have a plate open for more than 5-7 seconds. Be sure the walls of the sab are always wet.

I have never seen satellites that bad before.  Even open air usually looks better. Your agar looks odd as well. Review all steps of tek. Dipping in h2o2 is completely unnecessary for transfering away from mold (I dislike doing it ever).


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OfflineSillyPsy
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Re: can't seem to get a clean culture [Re: Pastywhyte]
    #22321327 - 10/01/15 09:32 PM (8 years, 3 months ago)

I thought the agar looked funny too. Have you tried leaving a jar of prepared agar out as a control? Not sure if you're using a no-pour tek, but that might help eliminate some variables in your technique.


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Invisiblechampinhom
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Re: can't seem to get a clean culture [Re: mrbart4444]
    #22321557 - 10/01/15 10:45 PM (8 years, 3 months ago)

Quote:

mrbart4444 said:
Also what do you use a SAB or flow hood?



I use a SAB. I have done many many agar jars of all types in my time.  Never had a problem like this before.


--------------------
My father used to say: I don't care what else you do in life, just don't be an asshole. People, forgive me when I forget what my daddy said.

Cut back the proliferating list of people whose opinions can hurt you. Unless they have done or want to do you some good, their views are just not worth tracking.
Saul Bellow

“People are just cannibals unless they leave each other alone.” Doris Lessing

Those whom the gods would save, they dower with compassion. Mr. P.  Silocybin


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Invisiblemrbart4444
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Re: can't seem to get a clean culture [Re: champinhom]
    #22321560 - 10/01/15 10:47 PM (8 years, 3 months ago)

Quote:

champinhom said:
Quote:

mrbart4444 said:
Also what do you use a SAB or flow hood?



I use a SAB. I have done many many agar jars of all types in my time.  Never had a problem like this before.



could be the mutant culture but still that mold is from spores in your sab


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Invisiblechampinhom
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Re: can't seem to get a clean culture [Re: Pastywhyte]
    #22321578 - 10/01/15 10:54 PM (8 years, 3 months ago)

Quote:

Pastywhyte said:
Satellites like that are indicative of sloppy technique. How long are you waiting for the air to settle before opening the plate after you bring your flamed tools back in the box? Ensure you never reach over a open plate. Try to use smooth movement but at the same time never have a plate open for more than 5-7 seconds. Be sure the walls of the sab are always wet.

I have never seen satellites that bad before.  Even open air usually looks better. Your agar looks odd as well. Review all steps of tek. Dipping in h2o2 is completely unnecessary for transfering away from mold (I dislike doing it ever).




The agar I used in that jar is the BRF agar tek. But the agar I was using before that were Pasty dishes and the agar I used for my latest transfer is Pasty too.

I have never had a problem like this. It baffles me. I have the sides of the SAB sprayed down with bleach water. I flame my knife, bring it into the SAB and while it cools I loosen the jar lids. Then I open the jar to be transfered, get my sample, close that jar and open the other and tip the lid just enough to have room to stick my transfer in.

I think my technique is pretty sound. I get the feeling that I have been transfering mold mycelium along with the cube myc.


--------------------
My father used to say: I don't care what else you do in life, just don't be an asshole. People, forgive me when I forget what my daddy said.

Cut back the proliferating list of people whose opinions can hurt you. Unless they have done or want to do you some good, their views are just not worth tracking.
Saul Bellow

“People are just cannibals unless they leave each other alone.” Doris Lessing

Those whom the gods would save, they dower with compassion. Mr. P.  Silocybin


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InvisibleLocN9ne
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Re: can't seem to get a clean culture [Re: champinhom]
    #22321584 - 10/01/15 10:57 PM (8 years, 3 months ago)

I don't think you are transfering that mold via mycelium wedge...they are originating from different places than your wedge...they are falling (most likely off your hand or scalpel) into your dishes...


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InvisibleToadstool5
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Re: can't seem to get a clean culture [Re: LocN9ne]
    #22321745 - 10/01/15 11:42 PM (8 years, 3 months ago)

Have you tried using bleach on the transfered mycelium? Bleach works wonders for mold spores and is not as damaging to the mycelium as peroxid is


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Invisiblechampinhom
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Re: can't seem to get a clean culture [Re: LocN9ne]
    #22321762 - 10/01/15 11:48 PM (8 years, 3 months ago)

Quote:

LocN9ne said:
I don't think you are transfering that mold via mycelium wedge...they are originating from different places than your wedge...they are falling (most likely off your hand or scalpel) into your dishes...




I wear alcohol wiped gloves.


--------------------
My father used to say: I don't care what else you do in life, just don't be an asshole. People, forgive me when I forget what my daddy said.

Cut back the proliferating list of people whose opinions can hurt you. Unless they have done or want to do you some good, their views are just not worth tracking.
Saul Bellow

“People are just cannibals unless they leave each other alone.” Doris Lessing

Those whom the gods would save, they dower with compassion. Mr. P.  Silocybin


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OfflinecronicrFacebook
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Re: can't seem to get a clean culture [Re: champinhom]
    #22321811 - 10/02/15 12:20 AM (8 years, 3 months ago)

Quote:

champinhom said:
I have been trying to isolate a clean culture of a mutant cube mushroom. I took a piece of the stem of the mushroom in question, dipped it in H2O2 and put it in an agar jar. a few weeks later .....



that's far too much time to let it go, get your first transfers out asap and avoid this in the future


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Invisiblechampinhom
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Re: can't seem to get a clean culture [Re: cronicr]
    #22323443 - 10/02/15 12:22 PM (8 years, 3 months ago)

Quote:

cronicr said:
that's far too much time to let it go, get your first transfers out asap and avoid this in the future




Yes, that is something that has been floating around in my mind for a while now--"Maybe I shouldn't be waiting around until these mold spores germinate and do a number on me.


--------------------
My father used to say: I don't care what else you do in life, just don't be an asshole. People, forgive me when I forget what my daddy said.

Cut back the proliferating list of people whose opinions can hurt you. Unless they have done or want to do you some good, their views are just not worth tracking.
Saul Bellow

“People are just cannibals unless they leave each other alone.” Doris Lessing

Those whom the gods would save, they dower with compassion. Mr. P.  Silocybin


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Offlinetripdawg420
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Re: can't seem to get a clean culture [Re: champinhom]
    #22323543 - 10/02/15 12:41 PM (8 years, 3 months ago)

is that red agar i woud say change your agar i use mea 


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Invisiblechampinhom
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Re: can't seem to get a clean culture [Re: tripdawg420]
    #22325330 - 10/02/15 07:19 PM (8 years, 3 months ago)

Quote:

tripdawg420 said:
is that red agar i woud say change your agar i use mea 





actually it is brown rice flour and water with a drop of food dye so that myc will show up better. I like this method in that I don't have to fool around witha lot of preliminaries.

I simply put 5 grams of flour, and 16-18 ml of food dyed water in each of 8 four oz mason jars, or Glad Mini-Rounds, cover with foil and load into the PC. 15 psi for 45 mins and you've got a bunch of very capable agar dishes. They are particularly useful in that if you leave them unopened long enough pins will start forming and you can make some really fast colonizing clone cultures from such pins.


--------------------
My father used to say: I don't care what else you do in life, just don't be an asshole. People, forgive me when I forget what my daddy said.

Cut back the proliferating list of people whose opinions can hurt you. Unless they have done or want to do you some good, their views are just not worth tracking.
Saul Bellow

“People are just cannibals unless they leave each other alone.” Doris Lessing

Those whom the gods would save, they dower with compassion. Mr. P.  Silocybin


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