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 Dango_Bill
Seven
 
Registered: 12/01/07
Posts: 307
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 Somethin wrong with the LC? 
#7819606 - 01/01/08 10:53 AM (16 years, 3 months ago) |
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I've got six LCs of cambodian that were noc'd with spores a few weeks ago, 150ml water & 6 grams white karo in 12 ounce jars. Injection holes are sealed with silicone, the lids on 3 of the jars are upside down, the other three have them rightside up (seal ring touching glass) - wasn't sure if it made a difference with LC, so I figured only half of them would be fucked up it did.
They sat at 82 degrees in darkness, only being exposed to light when I opened the enclosure to give them a gentle swirl once a day. They've all got a nice blob of myc in them about the diameter of a quarter now.
Four days ago I cooked off twelve half-pint pf tek jars; in the ratio 2 cups verm, 1 cup brf, 1 cup water. I shook up one of the LC jars and managed to get a dense cloud of myc chunks in the syringe, each PF jar received about 1.5ml of LC total, across 4 silicone sealed injection ports.
I've yet to see any signs of growth in the pf-tek jars. I'm pretty confused as to where I'm going wrong; this would make my third try at nocking up jars with LC, and my third failure - I've tried GT strain as well, and used karo and honey, always 4% by weight. I've also nocked up other jars of LC with the LC I've made, and the LC-from-LC jars colonized nicely.
It seems to be that when I put LC into something other than LC, it doesn't grow...
Ideas, anyone?
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 MYSTIQUE
Say Hi to the elves for me.
Registered: 04/28/07
Posts: 1,764
Loc: Canada
Last seen: 5 years, 6 months
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 Re: Somethin wrong with the LC? [Re: Dango_Bill]
#7819734 - 01/01/08 11:31 AM (16 years, 3 months ago) |
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You have all the air holes sealed on your lc and the top on tight? You might be damaging your lc
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Dont know what the fuck I just said? READ THIS
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I ain't a hippy but I'm covered in dirt
Sippin lots of mushroom tea in a tye-dye shirt
Chasin' the Grateful Dead, no shoes on my feet
Beggin' in the parking lot for something to eat,
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Dango_Bill
Seven
Registered: 12/01/07
Posts: 307
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Re: Somethin wrong with the LC? [Re: MYSTIQUE]
#7820119 - 01/01/08 01:46 PM (16 years, 3 months ago) |
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Hm. I didn't know LC needed air... guess I'll go re-read some teks.
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Nibin
Getting there
Registered: 11/29/05
Posts: 4,480
Last seen: 10 years, 11 months
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Re: Somethin wrong with the LC? [Re: Dango_Bill]
#7820143 - 01/01/08 01:51 PM (16 years, 3 months ago) |
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Quote:
Dango_Bill said:
Hm. I didn't know LC needed air... guess I'll go re-read some teks.
That is one of the basics... why did you think we use tyvek, polyfill, etc?
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Newcomers guide-----> For all things shroomy
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tahoe
Noob Slayer
Registered: 11/26/03
Posts: 6,274
Loc: N38.93829W119.98108
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Re: Somethin wrong with the LC? [Re: Nibin]
#7820198 - 01/01/08 02:09 PM (16 years, 3 months ago) |
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if there was a thick cloud of mycelium then it should be fine.
4 days??? That is barely enough time. Some see growth from lc's in 24 hours some see it in a week.
Sounds like your lc was way too rich.
The best way to make a lid for a lc is like this.
I pick this up from monstermich.
here we have the lid and some oyster and shiitake floating around
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Stop experimenting half way through your first grow. Grow it to maturity, watch it, learn from it. Do this a few times then experiment with different ideas and figure out what works best for you.
My Legacy
https://www.shroomery.org/forums/showflat.php/Number/22140987#22140987
Teh=The
I need to proofread
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2newtz
Melungeonoid
Registered: 01/04/07
Posts: 847
Loc: Cumberland Gapt
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Re: Somethin wrong with the LC? [Re: tahoe]
#7820217 - 01/01/08 02:16 PM (16 years, 3 months ago) |
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didnt read the wholrpst... butmy LC's were very slow yo start... but now the shits so white
-thedudenj
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~I'm not In a Position to Properly run Anything?~
~Twice the Fun in Half the Time!~
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Dango_Bill
Seven
Registered: 12/01/07
Posts: 307
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Re: Somethin wrong with the LC? [Re: Nibin]
#7820736 - 01/01/08 04:54 PM (16 years, 3 months ago) |
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Quote:
Nibin said:
Quote:
Dango_Bill said:
Hm. I didn't know LC needed air... guess I'll go re-read some teks.
That is one of the basics... why did you think we use tyvek, polyfill, etc?
Well; the LC that i used to knock up the new round of PF jars was one that had the lid on upside down - so there was some ventilation, at least.
I knocked up another liquid culture with that same jar of LC about a week ago, and the new LC spawned from it is definitely growing. I don't think the myc in the original LC is dead.
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tahoe
Noob Slayer
Registered: 11/26/03
Posts: 6,274
Loc: N38.93829W119.98108
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Re: Somethin wrong with the LC? [Re: Dango_Bill]
#7820850 - 01/01/08 05:27 PM (16 years, 3 months ago) |
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upside down lid=contams. cant trust that set up with lc's just grain and its an oldschool way of doing things
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Stop experimenting half way through your first grow. Grow it to maturity, watch it, learn from it. Do this a few times then experiment with different ideas and figure out what works best for you.
My Legacy
https://www.shroomery.org/forums/showflat.php/Number/22140987#22140987
Teh=The
I need to proofread
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fastfred
Old Hand
Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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Re: Somethin wrong with the LC? [Re: Nibin]
#7822398 - 01/02/08 01:14 AM (16 years, 3 months ago) |
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Quote:
Nibin said:
Quote:
Dango_Bill said:
Hm. I didn't know LC needed air... guess I'll go re-read some teks.
That is one of the basics... why did you think we use tyvek, polyfill, etc?
LC doesn't need any air. The only reason to have a port in them is to equalize the pressure so that contams don't get sucked in.
Don't tell someone that their question is one of the basics and then give a wrong answer, LOL.
-FF
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deucedbi9
Stranger
Registered: 10/24/06
Posts: 4,613
Loc: UK
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Re: Somethin wrong with the LC? [Re: fastfred]
#7822496 - 01/02/08 02:36 AM (16 years, 3 months ago) |
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>each PF jar received about 1.5ml of LC total, across 4 silicone sealed injection ports...
looks like this could be your problem.
you have sealed holes and therefore no provision for GE (gas exchange) the myc is suffocating.
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whether low pressure sucks or high pressure blows...
it's a bugger to cycle in.
even though I'm feeling good
Something tells me I'd better activate my prayer capsule
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fastfred
Old Hand
Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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Re: Somethin wrong with the LC? [Re: deucedbi9]
#7822505 - 01/02/08 02:44 AM (16 years, 3 months ago) |
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Quote:
deucedbi9 said:
>each PF jar received about 1.5ml of LC total, across 4 silicone sealed injection ports...
looks like this could be your problem.
you have sealed holes and therefore no provision for GE (gas exchange) the myc is suffocating.
You should have open holes on your substrate jars. But this shouldn't cause it to fail initially. Sealed jars do just fine until they are about 70%+ colonized. If they're quick colonizers started with LC they will often colonize just fine. But any delay in colonization will cause them to stall before completion when the oxygen is consumed.
Anyways, why does everyone post these questions on day 3 or 4? WTF? It's called patience people!
-FF
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Nibin
Getting there
Registered: 11/29/05
Posts: 4,480
Last seen: 10 years, 11 months
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Re: Somethin wrong with the LC? [Re: fastfred]
#7824089 - 01/02/08 03:06 PM (16 years, 3 months ago) |
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Quote:
fastfred said:
Quote:
Nibin said:
Quote:
Dango_Bill said:
Hm. I didn't know LC needed air... guess I'll go re-read some teks.
That is one of the basics... why did you think we use tyvek, polyfill, etc?
LC doesn't need any air. The only reason to have a port in them is to equalize the pressure so that contams don't get sucked in.
Don't tell someone that their question is one of the basics and then give a wrong answer, LOL.
-FF
Of course an LC needs air. Growth in an LC with no gas exchange will be poor compared to one with gas exchange. Once the oxygen inside the container is used up, the mycelium will use it's anaerobic metabolism exclusively, obtaining only two molecules of ATP per glucose molecule instead of the possible 36/38 molecules of ATP it would get by using aerobic metabolism.
Given the limited nutrient supply of an LC, that makes a lot of difference.
A stirrer and Gas exchange are crucial to a top notch LC.
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Newcomers guide-----> For all things shroomy
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fastfred
Old Hand
Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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Re: Somethin wrong with the LC? [Re: Nibin]
#7825072 - 01/02/08 07:49 PM (16 years, 3 months ago) |
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LC's work just fine sealed up. The myc is limited to using dissolved oxygen in the liquid. Circulating gases above the liquid has little effect on the dissolved gas concentration.
When sterilizing you are going to drive out almost all of the dissolved gasses, so it's a good idea to give them a good shake once they're cool. Ports, polyfill, and filters limit one's ability to shake up the LC, thus resulting in lower dissolved gas concentrations.
Stirring and air exchange are not required for LCs. They will do just fine without them. That said, I used to use mag stirring and a polyfilled hole on mine most of the time. The stirring can help a bit to break up the myc and speed colonization, but the pollyfilled hole is just for pressure equalization and because you have to have some sort of hole for deposits and withdrawls.
Give it a try sometime. The LC stage benefits little to none from optimization, since it always works if you use good sterile technique and it is never a bottleneck in the process. I'm sure you could use a micropore filter, aquarium pump, and an air stone if you really wanted to oxygenate your LC, but that's a good deal of work and supplies for no real benefit in the overall scheme of things.
-FF
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Dango_Bill
Seven
Registered: 12/01/07
Posts: 307
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Re: Somethin wrong with the LC? [Re: fastfred]
#7836650 - 01/05/08 05:50 PM (16 years, 3 months ago) |
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I'm pretty sure my LC have enough air; they're each only using 150ml of liquid and they're in 12 ounce jars - 150ml = 5 ounces; so half of the space in the jars is air. I have been swirling them daily & whenever i open my incubator, and they were shaken before i inoculated them. The pressure in the LC jars should be equal to atmospheric; I remembered to use a filter syringe to equalize it before innoculating the ones with the lids on seals-down.
I threw away two of the LCs with the lids on upside down yesterday; they had formed a few little black spots in them which i assumed were contams, no big loss, i still have a few of them, and luckily neither of those contamed jars were used to noc up any PF jars.
I still don't quite understand why I can't get any liquid culture to even begin to colonize a PF jar; as far as I can tell I'm doing everything correctly. I suck up pretty decent sized chunks of myc when I fill syringes with LC, so I know that there is myc going into the PF jars. I've checked to make sure that my needles are penetrating the straight verm layer on the top of the PF jars, and they are.
Any more ideas, guys?
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Nibin
Getting there
Registered: 11/29/05
Posts: 4,480
Last seen: 10 years, 11 months
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Re: Somethin wrong with the LC? [Re: fastfred]
#7838188 - 01/06/08 03:52 AM (16 years, 3 months ago) |
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Quote:
fastfred said:
LC's work just fine sealed up. The myc is limited to using dissolved oxygen in the liquid. Circulating gases above the liquid has little effect on the dissolved gas concentration.
When sterilizing you are going to drive out almost all of the dissolved gasses, so it's a good idea to give them a good shake once they're cool. Ports, polyfill, and filters limit one's ability to shake up the LC, thus resulting in lower dissolved gas concentrations.
Stirring and air exchange are not required for LCs. They will do just fine without them. That said, I used to use mag stirring and a polyfilled hole on mine most of the time. The stirring can help a bit to break up the myc and speed colonization, but the pollyfilled hole is just for pressure equalization and because you have to have some sort of hole for deposits and withdrawls.
Give it a try sometime. The LC stage benefits little to none from optimization, since it always works if you use good sterile technique and it is never a bottleneck in the process. I'm sure you could use a micropore filter, aquarium pump, and an air stone if you really wanted to oxygenate your LC, but that's a good deal of work and supplies for no real benefit in the overall scheme of things.
-FF
All I know is that when in the past I have made LC in a sealed container, introducing air (through an empty syringe without the plunger filled with polyfill and closed off with micropore) only to equalize pressure and not exchanging air after that, the size of the mycelium cloud was smaller and stopped growing sooner than in the containers with a permanent air exchange filter.
Same mix of nutrients (just under 4% honey) and same volume in the different jars, all given a good swirl once a day and had shards of glass in them.
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Newcomers guide-----> For all things shroomy
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fastfred
Old Hand
Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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Re: Somethin wrong with the LC? [Re: Nibin]
#7838930 - 01/06/08 11:29 AM (16 years, 3 months ago) |
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> Any more ideas, guys?
If your jars are prepared right, and you're sucking up myc, and it's getting to the substrate I don't see what could be going wrong. It should colonize very quickly compared to spores.
Nibin, LCs don't need to grow all that large. It is preferable to limit the myc growth by way of limiting nutrients and reducing respiration rather than having the culture die in it's own waste products.
There are all kinds of culture techniques used in science. Continuous flow reactors, bioreactors, fixed substrate matrices in liquid, etc.. So you definitely can do different things for different results. Some systems use continuous liquid flow, aeration, and stirring. But to simply germinate some spores and propagate the myc for use in starting cultures you don't need any of that. Sealed jars with no shaking will still work just fine.
-FF
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Nibin
Getting there
Registered: 11/29/05
Posts: 4,480
Last seen: 10 years, 11 months
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Re: Somethin wrong with the LC? [Re: fastfred]
#7838939 - 01/06/08 11:32 AM (16 years, 3 months ago) |
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Quote:
fastfred said:
There are all kinds of culture techniques used in science. Continuous flow reactors, bioreactors, fixed substrate matrices in liquid, etc.. So you definitely can do different things for different results. Some systems use continuous liquid flow, aeration, and stirring. But to simply germinate some spores and propagate the myc for use in starting cultures you don't need any of that. Sealed jars with no shaking will still work just fine.
-FF
No need to tell me about it  I will be taking a subject related to this next year. I'll be spending 6 months in the lab preparing cultures. I'm looking forward to it.
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Newcomers guide-----> For all things shroomy
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fastfred
Old Hand
Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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Re: Somethin wrong with the LC? [Re: Nibin]
#7839041 - 01/06/08 12:08 PM (16 years, 3 months ago) |
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Good luck. I suggest getting an mp3 player as preparing hundreds or thousands of cultures quickly gets tedious.
-FF
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Nibin
Getting there
Registered: 11/29/05
Posts: 4,480
Last seen: 10 years, 11 months
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Re: Somethin wrong with the LC? [Re: fastfred]
#7839168 - 01/06/08 12:50 PM (16 years, 3 months ago) |
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Quote:
fastfred said:
Good luck. I suggest getting an mp3 player as preparing hundreds or thousands of cultures quickly gets tedious.
-FF
A lot of them will be faeces cultures so yes, I will need to find something to distract me.
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Newcomers guide-----> For all things shroomy
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fastfred
Old Hand
Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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Re: Somethin wrong with the LC? [Re: Nibin]
#7839198 - 01/06/08 12:53 PM (16 years, 3 months ago) |
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We once had a liquid fecal sample in micro labeled "Watery Walter". LOL
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