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InvisibleGhatti
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Bacterial lc's
    #22181215 - 09/02/15 11:34 PM (8 years, 4 months ago)

So I make my lc's with a ship and a whatman filters but they all turn bacterial when I get them on agar.

It's kinda a bitch getting them open to drop in a clean wedge since I use metal lids and I'm thinking that's how bacteria is getting in.

I've tried flaming my scalpel and prying the lid open, I've done it by hand, I've done it with an alcohol soaked paper towel and they're all bacterial.

I even upped my pc times to 45mins for pints.

I'm out of ideas. The agar wedges are clean, all my transfers are clean so its gotta be something with the jar and how I'm doing things.

Anyone got any ideas?


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OfflineMoldy thumb
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Re: Bacterial lc's [Re: Ghatti]
    #22181281 - 09/02/15 11:53 PM (8 years, 4 months ago)

Are you using a flow hood or glove box?


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OfflinePussyFart
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Re: Bacterial lc's [Re: Ghatti]
    #22181299 - 09/02/15 11:59 PM (8 years, 4 months ago)

Quote:

Ghatti said:
So I make my lc's with a ship and a whatman filters but they all turn bacterial when I get them on agar.

It's kinda a bitch getting them open to drop in a clean wedge since I use metal lids and I'm thinking that's how bacteria is getting in.

I've tried flaming my scalpel and prying the lid open, I've done it by hand, I've done it with an alcohol soaked paper towel and they're all bacterial.

I even upped my pc times to 45mins for pints.

I'm out of ideas. The agar wedges are clean, all my transfers are clean so its gotta be something with the jar and how I'm doing things.

Anyone got any ideas?



Skip LCs and join the rest of us.....agar and g2g transfers FTW!


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InvisibleGhatti
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Re: Bacterial lc's [Re: Moldy thumb]
    #22181304 - 09/03/15 12:01 AM (8 years, 4 months ago)

A hood. I don't believe that to be the issue since all agar work done in front of it is clean.


I already use agar but liquid is so much faster. Even if I decide to never use an lc I'd still like to be able to make a clean one.


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OfflinePussyFart
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Re: Bacterial lc's [Re: Ghatti]
    #22181313 - 09/03/15 12:05 AM (8 years, 4 months ago)

Quote:

Ghatti said:
I already use agar but liquid is so much faster. Even if I decide to never use an lc I'd still like to be able to make a clean one.



by a few days at the most....you would still have to shoot up and grow out test jars/plates to make sure it's viable....in the meantime you could have had 100 grain jars ready to spawn....the headache of LCs is enough to drive a crazy person sane.....I would just G2G....


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InvisibleMad Season
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Re: Bacterial lc's [Re: Ghatti]
    #22181320 - 09/03/15 12:07 AM (8 years, 4 months ago)

Quote:

Ghatti said:
I already use agar but liquid is so much faster. Even if I decide to never use an lc I'd still like to be able to make a clean one.



Just take the rubber seal off the lid of the LC jar (assuming you're using metal lids). Makes taking the lid off real nice. I do that with my grain jars too. Plastic lids are boss too. Plus they don't rust.


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Edited by Mad Season (09/03/15 12:09 AM)


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OfflinecronicrFacebook
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Re: Bacterial lc's [Re: Ghatti]
    #22181530 - 09/03/15 01:37 AM (8 years, 4 months ago)

Quote:

Ghatti said:



I already use agar but liquid is so much faster.



Quote:

Ghatti said:
So I make my lc's with a ship and a whatman filters but they all turn bacterial when I get them on agar.





https://www.shroomery.org/forums/showflat.php/Number/21337668#21337668
seems this has been an issue for you for a long time, i don't see how that's "faster":shrug:


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Invisibleblindingleaf
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Re: Bacterial lc's [Re: cronicr]
    #22181618 - 09/03/15 02:53 AM (8 years, 4 months ago)

liquids are a tight rope walk.

some dudes do it really well.

but most dudes are too fast, too juiced off junk tree.

make smaller masters>pints.

go to G3 ALWAYS.  u won't run out of spawn.

or check eat u alives stuff.  he is liquid king IMO.


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Offlinehamloaf
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Re: Bacterial lc's [Re: Ghatti]
    #22181658 - 09/03/15 03:40 AM (8 years, 4 months ago)

Quote:

Ghatti said:
So I make my lc's with a ship and a whatman filters but they all turn bacterial when I get them on agar.

It's kinda a bitch getting them open to drop in a clean wedge since I use metal lids and I'm thinking that's how bacteria is getting in.

I've tried flaming my scalpel and prying the lid open, I've done it by hand, I've done it with an alcohol soaked paper towel and they're all bacterial.

I even upped my pc times to 45mins for pints.

I'm out of ideas. The agar wedges are clean, all my transfers are clean so its gotta be something with the jar and how I'm doing things.

Anyone got any ideas?



Good job, man, sticking out your trials and tribulations with LC, as well as, in the face of persecution from your peers.  Sounds like you just about have the art figured out as you have got your issue pretty much isolated down to a single vector.  It takes time and patience to make it to that point in the liquid culture career.  Not talking slurries either.  We are talking the art of colonization of a nutrient rich liquid media by mushroom mycelium. 

Yea man switch to plastic lids.  You want smooth motion when removing the vessel closure.  Not to be fighting to get the lid off.  Also, instead of placing metal lids rubber side down place metal lids facing rubber side up before PC'ing and that seal from lid to jar won't be formed after the PC cycle.  Just be sure to remodel new lids with your ship and filtering material on the "bottom" of the lids before loading with liquid media and pressure sterilizing.  Furthermore, do not forget to tighten those jar lid rings down tight on those lids before sterilization.

Edit to add ~ Are you running your flowhood for at least an hour, showering, surgical mask and wearing freshly laundered clothes before entering the lab?  Also, are you wiping all tools, media vessels, and work surface area of the flowhood before placing any of the aforementioned items into the direct sterile downstream of the flowhood??


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Edited by hamloaf (09/03/15 03:51 AM)


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InvisibleGhatti
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Re: Bacterial lc's [Re: hamloaf]
    #22181889 - 09/03/15 07:10 AM (8 years, 4 months ago)

Lately my hood has stayed running. I didn't like the plastic lids I used once because they leaked.

And when I say its faster I don't so much mean on a single grain jar. For larger bags its no contest.

I'm seriously being as clean as possible wipe, flame, flame pf.


It's more of a personal vendetta for me to conquer lc.


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