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Offlinekpendyk
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grain spawn appears pure but doesn't grow back together.. endemic contam?
    #21439815 - 03/21/15 02:47 PM (9 years, 1 month ago)

I'm pretty new to liquid culture and have had some troubles with it. When I inoculate my grain spawn, it grows out and is a nice white brick in a little over a week, but when I shake it up it doesn't grow back together properly. It turns white and grows back again but it doesn't grow together the way it did before. When I spawn to a bulk substrate, green mold shows up after a few weeks.

I've never really had any problems with mold before, so I don't think its an environmental issue.

Here is my lc prep:

20 g lme
20 g dextrose
2 g yeast/peptone
1 g gypsum
1 l distilled water

Sterilize at 15 psi for 20 mins
Inoculate with blended agar cultures (method from ggmm)

Stirred 24/7 on a magnetic stir plate. What am I doing wrong?

Side note: I've run tests with blank bags so I don't think its sterile technique, I've pretty much narrowed it down to the liquid culture as the issue. Thanks in advance.

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Re: grain spawn appears pure but doesn't grow back together.. endemic contam? [Re: kpendyk]
    #21439824 - 03/21/15 02:48 PM (9 years, 1 month ago)

so stop with the lc and use your agar to knock it up:shrug:


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Offlinekpendyk
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Re: grain spawn appears pure but doesn't grow back together.. endemic contam? [Re: cronicr]
    #21439836 - 03/21/15 02:52 PM (9 years, 1 month ago)

I'm sick of shaking bags and jars, and would prefer to perfect lc's.

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Re: grain spawn appears pure but doesn't grow back together.. endemic contam? [Re: kpendyk]
    #21439842 - 03/21/15 02:54 PM (9 years, 1 month ago)

stop using distilled water and go through your own steps and try pin point your problem


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Re: grain spawn appears pure but doesn't grow back together.. endemic contam? [Re: kpendyk]
    #21439866 - 03/21/15 02:59 PM (9 years, 1 month ago)

Either your LC is bacterial or your grain is. If your LC tests clean then its probably your grain.  But like cron said even simpler would be to just toss a wedge in your grain and see what happens.

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Re: grain spawn appears pure but doesn't grow back together.. endemic contam? [Re: Pastywhyte]
    #21439896 - 03/21/15 03:10 PM (9 years, 1 month ago)

what you using for filters?


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Offlinekpendyk
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Re: grain spawn appears pure but doesn't grow back together.. endemic contam? [Re: cronicr]
    #21439971 - 03/21/15 03:29 PM (9 years, 1 month ago)

I've just had foil over top of an Erlenmeyer flask, and left it in front of flow hood to colonize.

I have run blank grain bags with no contamination so its gotta be the lg right? Could it be because of the addition of yeast into the mix without increasing sterilization time? Or could it be that my culture is endemically contaminated?

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Re: grain spawn appears pure but doesn't grow back together.. endemic contam? [Re: kpendyk]
    #21439976 - 03/21/15 03:31 PM (9 years, 1 month ago)

Test the LC on a plate and see what grows. You should do this before using it anyway.

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Offlinekpendyk
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Re: grain spawn appears pure but doesn't grow back together.. endemic contam? [Re: Pastywhyte]
    #21439979 - 03/21/15 03:33 PM (9 years, 1 month ago)

That's what I figured. I did that yesterday but haven't seen any growth yet. Thanks for your help I've just been driving myself crazy.

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Re: grain spawn appears pure but doesn't grow back together.. endemic contam? [Re: kpendyk]
    #21439984 - 03/21/15 03:34 PM (9 years, 1 month ago)

It will take a few days for anything to recover. Bacteria takes at least 24 hours and most myc will take at least 48 or more.

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Re: grain spawn appears pure but doesn't grow back together.. endemic contam? [Re: Pastywhyte]
    #21440002 - 03/21/15 03:42 PM (9 years, 1 month ago)

About the distilled water.. Is it actually hurting the lc? Should I use spring or tap instead?

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Re: grain spawn appears pure but doesn't grow back together.. endemic contam? [Re: kpendyk]
    #21440126 - 03/21/15 04:16 PM (9 years, 1 month ago)

distilled should only be used in spore syringes, it is toxic to mycelium:thumbup: so yes tap would be better


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I'm tired do me a favor

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Re: grain spawn appears pure but doesn't grow back together.. endemic contam? [Re: cronicr]
    #21441978 - 03/22/15 05:46 AM (9 years, 1 month ago)

Every body is so quick to blame the LC here, but nobody is trying to pin point how the LC got dirty.  LC's don't get dirty just because they are LC's.  My bet is your inoculation of the LC went wry due to dirty inoculm, or under sterilized media.  What did you inoculate you LC with, anyway?  20 minutes at 15 psi is not a long enough of a sterilization duration for MDEA.  Go with 45 minutes.


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Re: grain spawn appears pure but doesn't grow back together.. endemic contam? [Re: hamloaf]
    #21442210 - 03/22/15 08:31 AM (9 years, 1 month ago)

I really dont work with LC so I dont have anything usefull to add on that.

Just wondering if have you considered LI instead, seems like a good compromise since you are having troubke with the LC but dont want to drop wedges :shrug:


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Re: grain spawn appears pure but doesn't grow back together.. endemic contam? [Re: mushpunx] * 1
    #21442230 - 03/22/15 08:37 AM (9 years, 1 month ago)

He said he made an LI Stamets-style (GGMM) which is EXACTLY the popular LI tek that is floating around here. 

Blending up agar and inoculating liquid media with it is a pretty standard operating procedure in liquid culturing techniques. 

This guy's inoculm is dirty, and or he fucked up his sterile work during either the making of his agar, the making of the LI, or the inoculation of his agar or LI.


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Re: grain spawn appears pure but doesn't grow back together.. endemic contam? [Re: hamloaf]
    #21442239 - 03/22/15 08:40 AM (9 years, 1 month ago)

:facepalm: didnt read too well I guess hahah


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Re: grain spawn appears pure but doesn't grow back together.. endemic contam? [Re: hamloaf]
    #21442525 - 03/22/15 10:27 AM (9 years, 1 month ago)

Quote:

liquidmyce said:
Every body is so quick to blame the LC here, but nobody is trying to pin point how the LC got dirty.  LC's don't get dirty just because they are LC's.  My bet is your inoculation of the LC went wry due to dirty inoculm, or under sterilized media.  What did you inoculate you LC with, anyway?  20 minutes at 15 psi is not a long enough of a sterilization duration for MDEA.  Go with 45 minutes.




Upon further research I think that may be my problem. I knew my issue was coming from my liquid culture I just didn't know what was causing it. I have admittedly messed up sterilization times because I have been adding yeast that clearly needs to be sterilized longer. Thanks for your input!

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Re: grain spawn appears pure but doesn't grow back together.. endemic contam? [Re: kpendyk]
    #21442556 - 03/22/15 10:36 AM (9 years, 1 month ago)

Quote:

kpendyk said:
Quote:

liquidmyce said:
Every body is so quick to blame the LC here, but nobody is trying to pin point how the LC got dirty.  LC's don't get dirty just because they are LC's.  My bet is your inoculation of the LC went wry due to dirty inoculm, or under sterilized media.  What did you inoculate you LC with, anyway?  20 minutes at 15 psi is not a long enough of a sterilization duration for MDEA.  Go with 45 minutes.




Upon further research I think that may be my problem. I knew my issue was coming from my liquid culture I just didn't know what was causing it. I have admittedly messed up sterilization times because I have been adding yeast that clearly needs to be sterilized longer. Thanks for your input!




20m at 15psi is more than enough for YPD(a) in a 500ml flask.
actually even that's overkill YPD broth and agar only need 15m at 15psi for 1L flasks. but I like to do overkill

liquidmyce is a source of shoddy advice though :shrug: and constantly gives advice on things of which he has no first hand experience.

I pour 100s of plates a week. and have never went longer than 20m.

Components
Ingredients (g/L)
Bacteriological peptone, 20
Yeast extract, 10
Glucose, 20
Agar, 15
Application

A solid medium for the propagation of yeast.
Quantity

65 g per liter
Reconstitution

Preparation instructions
1. Suspend 65 g in 1 L of distilled water.
2. Heat to boiling while stirring to dissolve all ingredients completely.
3. Autoclave for 15 minutes at 121 °C.

For YPD broth(LC) (-agar) it's still 15m at 15psi

Edited by Trusted cuItivator (03/22/15 10:45 AM)

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Offlinekpendyk
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Re: grain spawn appears pure but doesn't grow back together.. endemic contam? [Re: bodhisatta]
    #21442572 - 03/22/15 10:43 AM (9 years, 1 month ago)

What about a litre of liquid in a 2 l flask?

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