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Pastywhyte
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Registered: 09/15/12
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Loc: Canada
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Re: vacutainers? [Re: D3_Myc]
#27010066 - 10/29/20 10:38 AM (3 years, 2 months ago) |
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I never run agar as short as 20 min, every time I’ve done that it usually ends with bacteria in the blank plates. The way I make LC isn’t really comparable to LI or traditional LC methods but I run it as long as I do my agar.
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D3_Myc
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Registered: 05/06/18
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Interesting, never had that issue. Always run for 20 but my pressure usually sits around 17-18psi maybe that makes up the difference. I’ve even ran as short as 15 min in the past.
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A.k.a
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Re: vacutainers? [Re: D3_Myc]
#27010096 - 10/29/20 10:52 AM (3 years, 2 months ago) |
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I’ve had the same problem with fresh plates blowing up with bacterial polka dots so I usually do 35 mins now.
You can open and pour lc even in a SAB, media bottles are great for that.
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D3_Myc
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Re: vacutainers? [Re: A.k.a]
#27010114 - 10/29/20 10:58 AM (3 years, 2 months ago) |
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Not sure where I got that 20min figure. I think Misty West, never had a plate contam on me that wasn’t streaked. Doubt distilled water makes much a difference in that aspect. I always worried about caramelizing my agar if I went too long. I do boil mine before it goes into the media bottles
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A.k.a
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Re: vacutainers? [Re: D3_Myc]
#27010120 - 10/29/20 11:00 AM (3 years, 2 months ago) |
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A lot of teks do say 20 or even 15 mins. When I did 20 it was mostly ok but maybe 10% of the time I’d lose a whole stack of plates to bacteria.
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Asura
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Re: vacutainers? [Re: D3_Myc]
#27010126 - 10/29/20 11:03 AM (3 years, 2 months ago) |
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Quote:
D3monic said: Interesting, never had that issue. Always run for 20 but my pressure usually sits around 17-18psi maybe that makes up the difference. I’ve even ran as short as 15 min in the past.
This what I do, same psi. Plus 10 minutes of venting up front. Even after flame out, it hold above 15 for awhile. Not having problems with my plates at all.
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D3_Myc
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Registered: 05/06/18
Posts: 4,399
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Re: vacutainers? [Re: A.k.a]
#27010127 - 10/29/20 11:04 AM (3 years, 2 months ago) |
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As I’m thinking out loud it’s probably the pre-boil that’s allowing me to safely cut down PC time. That and distilled vs tap or well water? Spit balling thoughts here.
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D3_Myc
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Re: vacutainers? [Re: Asura]
#27010134 - 10/29/20 11:07 AM (3 years, 2 months ago) |
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Quote:
Asura said:
Quote:
D3monic said: Interesting, never had that issue. Always run for 20 but my pressure usually sits around 17-18psi maybe that makes up the difference. I’ve even ran as short as 15 min in the past.
This what I do, same psi. Plus 10 minutes of venting up front. Even after flame out, it hold above 15 for awhile. Not having problems with my plates at all.
Yea I don’t start the timer until I’m over 15psi
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Mateja


Registered: 07/14/16
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Re: vacutainers? [Re: D3_Myc]
#27010169 - 10/29/20 11:25 AM (3 years, 2 months ago) |
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I'm pretty sure we kill off everything in a liquid media with a 15min boil, especially if you had a 10 min boil to get to pressure + 10 min venting + 5min to get up to working pressure + 15min of effective sterilization, that's 35+min of boiling a liquid media. My opinion is if you after that manage to fuck up an entire sleeve then I think the wrapping the sleeve came in was broken. I've found tiny tiny holes in some sleeves after inspecting them closer (because 10+20% of the plates were broken inside) but I threw all of those sleeves away even if they had microscopic rips in the plastic. I see this as more plausible then a liquid broth not being sterilized after 35min boil.
-------------------- Cakes inside Water Tub
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Pastywhyte
Say hello to my little friend



Registered: 09/15/12
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Re: vacutainers? [Re: Mateja]
#27010195 - 10/29/20 11:41 AM (3 years, 2 months ago) |
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Quote:
Mateah said: I'm pretty sure we kill off everything in a liquid media with a 15min boil, especially if you had a 10 min boil to get to pressure + 10 min venting + 5min to get up to working pressure + 15min of effective sterilization, that's 35+min of boiling a liquid media. My opinion is if you after that manage to fuck up an entire sleeve then I think the wrapping the sleeve came in was broken. I've found tiny tiny holes in some sleeves after inspecting them closer (because 10+20% of the plates were broken inside) but I threw all of those sleeves away even if they had microscopic rips in the plastic. I see this as more plausible then a liquid broth not being sterilized after 35min boil.
Are you calling me a liar or merely incompetent?
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Celestialexplorer1



Registered: 03/25/20
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fight fight fight FiGhT FIGHT FIGHT......jk
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 To spend just one moment in eternity
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Blue Helix
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Re: vacutainers? [Re: D3_Myc]
#27010242 - 10/29/20 12:05 PM (3 years, 2 months ago) |
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Quote:
A.k.a said: I just found and used an lc that’s a year old, made from spores but tested clean a couple months after I made it, and has been sitting at room temp the whole time which for the last four months has been about 90.

Still looks good but we’ll see.
Wow! I'm surprised it kept that long.Quote:
D3monic said: If you’re doing LC I take it injection ports and syringe is a must? Or if you’re working in front of a hood is tip and pour acceptable similar to LI?
Well, I use an piece of gaffers tape on my bag and inject through it. Once I'm done I hot glue over it to seal and sterilize the hole. I'm not a big fan of working larger things in my still-air box and I don't have a flow hood, so I try to avoid all that through liquid cultures. For me, one of the beauties of liquid culture is that clean technique is far less critical when doing injections. You might still flame the needle and spritz some alcohol on where you are injecting, but that's about it.
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Blue Helix
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Registered: 02/02/03
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Quote:
trippleblack said: I want to store cultures 10+ years.. I go back to slants twice a year to regrow them on agar -almost lost cultures waiting a full year. I'm going to try your vacutainer lc tek, maybe using a centrifuge to seperate the mycellium if i can figure that out.
When you spin the LCs, the mycelium should compact on the bottom of the vacutainer. You can then very slowly invert the vacutainer (septum down) and draw off all the sugar water (or nearly all). Then reinject sterile water, give it a shake, and you are good to go.
Edited by Blue Helix (10/29/20 01:31 PM)
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Mateja


Registered: 07/14/16
Posts: 7,948
Loc: Here
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Quote:
Pastywhyte said:
Quote:
Mateah said: I'm pretty sure we kill off everything in a liquid media with a 15min boil, especially if you had a 10 min boil to get to pressure + 10 min venting + 5min to get up to working pressure + 15min of effective sterilization, that's 35+min of boiling a liquid media. My opinion is if you after that manage to fuck up an entire sleeve then I think the wrapping the sleeve came in was broken. I've found tiny tiny holes in some sleeves after inspecting them closer (because 10+20% of the plates were broken inside) but I threw all of those sleeves away even if they had microscopic rips in the plastic. I see this as more plausible then a liquid broth not being sterilized after 35min boil.
Are you calling me a liar or merely incompetent?
I wasn't trying to call anyone out about "lying" I have no reason to believe anyone is lying in this thread about having a whole sleeve contaminate I was just offering my perspective on it.
Quote:
Celestialexplorer1 said: fight fight fight FiGhT FIGHT FIGHT......jk
You're suppose to go: "JERRY! JERRY! JERRY!"
Also my LC's pin after full colonization
 
-------------------- Cakes inside Water Tub
Edited by Mateja (10/29/20 12:15 PM)
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A.k.a
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Re: vacutainers? [Re: Mateja]
#27010283 - 10/29/20 12:33 PM (3 years, 2 months ago) |
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Do you guys have centrifuges?
I’d have to strap it to a bike tire and ride as fast as I can.
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Pastywhyte
Say hello to my little friend



Registered: 09/15/12
Posts: 37,809
Loc: Canada
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Re: vacutainers? [Re: Mateja] 1
#27010287 - 10/29/20 12:34 PM (3 years, 2 months ago) |
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Quote:
Mateah said:
Quote:
Pastywhyte said:
Quote:
Mateah said: I'm pretty sure we kill off everything in a liquid media with a 15min boil, especially if you had a 10 min boil to get to pressure + 10 min venting + 5min to get up to working pressure + 15min of effective sterilization, that's 35+min of boiling a liquid media. My opinion is if you after that manage to fuck up an entire sleeve then I think the wrapping the sleeve came in was broken. I've found tiny tiny holes in some sleeves after inspecting them closer (because 10+20% of the plates were broken inside) but I threw all of those sleeves away even if they had microscopic rips in the plastic. I see this as more plausible then a liquid broth not being sterilized after 35min boil.
Are you calling me a liar or merely incompetent?
I wasn't trying to call anyone out about "lying" I have no reason to believe anyone is lying in this thread about having a whole sleeve contaminate I was just offering my perspective on it.
Quote:
Celestialexplorer1 said: fight fight fight FiGhT FIGHT FIGHT......jk
You're suppose to go: "JERRY! JERRY! JERRY!"
Also my LC's pin after full colonization
  
I was just busting your chops a bit but, I do find it frustrating that people preach things that have caused me nothing but failure. I’ve never had a LC caramelize from over sterilization but I’ve had lots go bacterial from too short. Maybe it’s the media I’m using or something else but personally I can’t risk it.
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D3_Myc
Weeb Trash



Registered: 05/06/18
Posts: 4,399
Loc: Year Zero
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Re: vacutainers? [Re: A.k.a]
#27010308 - 10/29/20 12:47 PM (3 years, 2 months ago) |
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Quote:
A.k.a said: Do you guys have centrifuges?
I’d have to strap it to a bike tire and ride as fast as I can.
Lmfao 🤣 , I have one at work but it’s too big to sneak home.
When I do my LC i’ll let it go for 30 min with a batch of agar and what ever else I can pack in for a short run. Not hurting anything. Right now RW the only culture I plan on keeping in constant rotation. That’s why I was considering LC. Everything else is a first time run or cross.
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Blue Helix
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Registered: 02/02/03
Posts: 1,565
Last seen: 6 months, 18 days
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Quote:
Pastywhyte said:
Quote:
Mateah said:
Quote:
Pastywhyte said:
Quote:
Mateah said: I'm pretty sure we kill off everything in a liquid media with a 15min boil, especially if you had a 10 min boil to get to pressure + 10 min venting + 5min to get up to working pressure + 15min of effective sterilization, that's 35+min of boiling a liquid media. My opinion is if you after that manage to fuck up an entire sleeve then I think the wrapping the sleeve came in was broken. I've found tiny tiny holes in some sleeves after inspecting them closer (because 10+20% of the plates were broken inside) but I threw all of those sleeves away even if they had microscopic rips in the plastic. I see this as more plausible then a liquid broth not being sterilized after 35min boil.
Are you calling me a liar or merely incompetent?
I wasn't trying to call anyone out about "lying" I have no reason to believe anyone is lying in this thread about having a whole sleeve contaminate I was just offering my perspective on it.
Quote:
Celestialexplorer1 said: fight fight fight FiGhT FIGHT FIGHT......jk
You're suppose to go: "JERRY! JERRY! JERRY!"
Also my LC's pin after full colonization
  
I was just busting your chops a bit but, I do find it frustrating that people preach things that have caused me nothing but failure. I’ve never had a LC caramelize from over sterilization but I’ve had lots go bacterial from too short. Maybe it’s the media I’m using or something else but personally I can’t risk it.
I've forgotten about my LC for about four hours, and although it was slightly darker compared to another I had that went only 30 minute, it worked fine. I use 10g light malt extract, 10g dextrose (corn sugar), and 500ml water. I think the caramelization issue is more a theoretical concern than a reality for the length of times we are talking about, and while I'm sure exposing the LC for very long period of times might cause some caramelization, the mycelium doesn't seem to mind a tiny bit of caramelization too much in my experience.
I pressure cook my LCs for about 30 minutes, timer starting around half pressure (7 psi); however, before I put the LC in the pressure cooker and cook, I microwave it to just under boiling (5 minutes for 500 ml). I've never had contamination using this procedure.
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Blue Helix
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Re: vacutainers? [Re: D3_Myc] 1
#27010425 - 10/29/20 01:44 PM (3 years, 2 months ago) |
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Quote:
D3monic said:
Quote:
A.k.a said: Do you guys have centrifuges?
I’d have to strap it to a bike tire and ride as fast as I can.
Lmfao 🤣 , I have one at work but it’s too big to sneak home.
When I do my LC i’ll let it go for 30 min with a batch of agar and what ever else I can pack in for a short run. Not hurting anything. Right now RW the only culture I plan on keeping in constant rotation. That’s why I was considering LC. Everything else is a first time run or cross.
One might also be able to get away with using a hydrophilic syringe filter too. I've not tried it, but such a filter would catch the mycelium and probably could handle 10ml of LC. After sucking the LC through the filter with a syringe, you'd just push 10ml of sterile water into the vacutainer which should dislodge the mycelium that was captured by the filter. Like I said, though, I've never tried this. It's possible that the filter would just clog up too fast or that the mycelium would not dislodge when pushing in the water. I might try it--because I do have some hydrophilic syringe filters--and report back on Shroomery as to what I find.
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D3_Myc
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Registered: 05/06/18
Posts: 4,399
Loc: Year Zero
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Fwiw I have an experiment thread going under mushcult. You’re welcome to post it or any other tests/experiment you feel like. Wasn’t expecting the thread to be as dead as it is... 
I intentionally left it out advanced forum so it wouldn’t be intimidating for the general public to participate.
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