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OfflineMych
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Registered: 03/25/05
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what could i possibly be doing wrong with my LC?
    #4271176 - 06/08/05 12:18 PM (12 years, 17 days ago)

I am trying to make some liquid cultures from my multispore syringes (bought from TLG).  I have gone through 8 4oz jars with no luck. WHAT AM I DOING WRONG????

Here is the process i have been following for 8 separate jars, and no mych has showed up after 2+ weeks EXCEPT for a single jar.. which formed mych and then the mych seems to have died and turned a tan color.  Do i need some sort of breather hole?

-100ml bottle spring water + 5ml light karo syrup
-mix into my 4oz jar
-tape over the 1 innoc hole
-PC for 20min after reaching 15psi with 2 hand towels on the bottom for insulation
-let cool for 12-24 hours and examine jars so that not carmalization occured.
-shake jars to get oxygen back into the water.
-put jars in my closed air glove box, wipe inside down with iso and let sit for a few minutes
-flame needle tip and quickly drop it inside the glove box
-wipe jar lid with iso, peel back tape, wipe jar lid again with iso
-shake needle with about .5cc of air in it (already pulled in through a glowing hot needle)
-Hold needle pointing down for 10+ seconds to let some spores settle down by the needle then wipe needle with iso.
-innoc jar with .5 to 1cc of spore sollution.
-reseal the tape
-let jar sit in my 81F incubator

... no mych forming, but you can see spore clumps :frown:


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Offlinegrow
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Re: what could i possibly be doing wrong with my LC? [Re: Mych]
    #4271200 - 06/08/05 12:26 PM (12 years, 17 days ago)

I didn't shake my jar, and I had success my first time....

What is Mych? Mycelium + Trych? by mixing words, you are misinforming cultivators who could help you. what does this mean, you are trying to grow Mycelium and green mold? well that's why you're failing, and that is the only reason I can see why you would fail from the procedures followed above...


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OfflineMych
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Re: what could i possibly be doing wrong with my LC? [Re: grow]
    #4271211 - 06/08/05 12:29 PM (12 years, 17 days ago)

mych=mycellium


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Offlinepcubmycol
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Re: what could i possibly be doing wrong with my LC? [Re: Mych]
    #4271228 - 06/08/05 12:33 PM (12 years, 17 days ago)

I see no problems with the above procedure. What temp are these jars incubating at?


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Offlinegrow
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Re: what could i possibly be doing wrong with my LC? [Re: Mych]
    #4271229 - 06/08/05 12:34 PM (12 years, 17 days ago)

I see. I have never heard of "Replacing oxygen". you might just be bringing in contaminated air, if you are truely "replacing oxygen" meaning none to begin with in the jar, it must be brought from the outside of the jar which is not sterile? ok ok I don't know just don't shake your jars. I had success my first time, I PC'd for 20 mins, I used 96+96ml + 3+3ml, So I use 3% karo, 5% may be too much for "mych"


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Offlinepcubmycol
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Re: what could i possibly be doing wrong with my LC? [Re: grow]
    #4271274 - 06/08/05 12:46 PM (12 years, 17 days ago)

I usually use 4 or 5% karo and now that i think about it, i seem to have problems with the higher nute LC. Maybe grow is right and 3% is the way to go. As a matter of fact, i noticed that 5% will only grow for me if innoced from another lc and that spores wont germinate.

5 shrooms for you grow for making me realize something that i should have before.


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OfflineMych
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Re: what could i possibly be doing wrong with my LC? [Re: pcubmycol]
    #4271293 - 06/08/05 12:50 PM (12 years, 17 days ago)

Quote:

pcubmycol said:
I see no problems with the above procedure. What temp are these jars incubating at?



a perfect 81F


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OfflineMych
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Re: what could i possibly be doing wrong with my LC? [Re: grow]
    #4271308 - 06/08/05 12:53 PM (12 years, 17 days ago)

Quote:

grow said:
I see. I have never heard of "Replacing oxygen". you might just be bringing in contaminated air, if you are truely "replacing oxygen" meaning none to begin with in the jar, it must be brought from the outside of the jar which is not sterile? ok ok I don't know just don't shake your jars. I had success my first time, I PC'd for 20 mins, I used 96+96ml + 3+3ml, So I use 3% karo, 5% may be too much for "mych"




you talking about the step where i shake the jars? im not introducing more air into the jar, the end up being sealed up as soon as they come out of the PC.. sometimes a bit of air gets pulled in while they are in my glove box.... but anyway the reintroduction of oxygen is simply the shaking of the water with the existing air. Aparently pressure cooking takes alot of the oxygen out of the water and shaking it will put it back in. I read that several times on this board, but i cant see how it would possibly hurt anything


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Offlinebenrules92
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Re: what could i possibly be doing wrong with my LC? [Re: Mych]
    #4271322 - 06/08/05 12:56 PM (12 years, 17 days ago)

i incubate my lc's at 87-88 ,,,,, but if your not getting anything at 81 i dont know what to say ,,, maybe try using less karo ,,, personaly i use honey and it works awsome, never had any problems with lc's


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OfflineMych
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Re: what could i possibly be doing wrong with my LC? [Re: pcubmycol]
    #4271325 - 06/08/05 12:57 PM (12 years, 17 days ago)

Quote:

pcubmycol said:
I usually use 4 or 5% karo and now that i think about it, i seem to have problems with the higher nute LC. Maybe grow is right and 3% is the way to go. As a matter of fact, i noticed that 5% will only grow for me if innoced from another lc and that spores wont germinate.

5 shrooms for you grow for making me realize something that i should have before.




yea, this was recommended to me once before and i hope to be trying that tonight. I am just trying to collect any other adjustments that i might need such as breather holes and what not.


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Offlineblackout
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Re: what could i possibly be doing wrong with my LC? [Re: Mych]
    #4271354 - 06/08/05 01:04 PM (12 years, 17 days ago)

I would let them cool down in the pc. During cooling they pull in air from the outside and this should be clean.
Shaking to aerate is good, and will mean it will work better especially if you have no filter. Are you allowing the needle to cool enough between heatings?

You could try less karo as mentioned, less will only show less eventual growth, too much will show none, but your measurements seem fine, and while in the pc it should not "boil off" (which happens in a microwave, increasing the sugar %) Does the karo have any preservatives?

If all else fails I would try a weak honey solution.

Are the syringes old, have they been subject to heat?


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OfflineMych
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Re: what could i possibly be doing wrong with my LC? [Re: blackout]
    #4271435 - 06/08/05 01:28 PM (12 years, 17 days ago)

Quote:

blackout said:
I would let them cool down in the pc. During cooling they pull in air from the outside and this should be clean.
Shaking to aerate is good, and will mean it will work better especially if you have no filter. Are you allowing the needle to cool enough between heatings?

You could try less karo as mentioned, less will only show less eventual growth, too much will show none, but your measurements seem fine, and while in the pc it should not "boil off" (which happens in a microwave, increasing the sugar %) Does the karo have any preservatives?

If all else fails I would try a weak honey solution.

Are the syringes old, have they been subject to heat?




i tried letting one batch cool for 24 hours in the PC, and the others were taken out and immediately sealed as soon as the side of the PC was cool enough to touch with my bare hand.  neither batch seemed to make a difference.

the needle tip is definately cool enough as it is kept wrapped in an alcohol soaked papertowel (after flaming) for atleast a minute prior to inoculation.

The syringes are what may be my issue.  I got them 1 month ago from thelittleguy.com  I have had ZERO germination in my BRF cakes and karo jars from 3 of the 4 syringes i have.  I have used about 6cc from each syringe.  the 4th syringe is performing nicely though (all jars have colonized, and the karo jar for that syringe actually grows.. but then turns brown)

what is the best way to determine if my syringes are viable? agar dish?  im running out of spore sollution :frown:


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InvisibleSoopaX
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Re: what could i possibly be doing wrong with my LC? [Re: Mych]
    #4271456 - 06/08/05 01:32 PM (12 years, 17 days ago)

Very weird... Try upping the karo syrup, I don't really measure mine, per se, I just use a table spoon for about 150mL of water.


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OfflineMych
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Re: what could i possibly be doing wrong with my LC? [Re: SoopaX]
    #4271501 - 06/08/05 01:42 PM (12 years, 17 days ago)

perhaps i could try honey instead as well


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OfflineHotnuts
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Re: what could i possibly be doing wrong with my LC? [Re: Mych]
    #4271569 - 06/08/05 01:57 PM (12 years, 17 days ago)

Hmmm. Your procedure sounds pretty good with one exception, cleaning the needle with alcohol and the inoculation hole after you've knocked up the jar. It only takes a minimal amount of alcohol to kill spores from the psilo species. Not to mention tape isn't exactly the cleanest way to seal up something sterile. I'd try this. Instead of using tape to cover the hole for inoculation, use silicone. Silicone also will seal back up after you knock up the jar. After you flame the needle, don't use alcohol to wipe it down. It's useless unless your sticking you needle into a nutritous substance, like brf jars. No need to do it if your using a gloove box.


Edited by hotnutz (06/08/05 01:59 PM)


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OfflineHotnuts
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Re: what could i possibly be doing wrong with my LC? [Re: Hotnuts]
    #4271582 - 06/08/05 02:00 PM (12 years, 17 days ago)

By the way! You only to pc LC jars for 20 minutes @ 5psi. That's right. 5psi.


Edited by hotnutz (06/08/05 02:03 PM)


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OfflineMych
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Re: what could i possibly be doing wrong with my LC? [Re: Hotnuts]
    #4271707 - 06/08/05 02:31 PM (12 years, 17 days ago)

Quote:

hotnutz said:
Hmmm. Your procedure sounds pretty good with one exception, cleaning the needle with alcohol and the inoculation hole after you've knocked up the jar. It only takes a minimal amount of alcohol to kill spores from the psilo species. Not to mention tape isn't exactly the cleanest way to seal up something sterile. I'd try this. Instead of using tape to cover the hole for inoculation, use silicone. Silicone also will seal back up after you knock up the jar. After you flame the needle, don't use alcohol to wipe it down. It's useless unless your sticking you needle into a nutritous substance, like brf jars. No need to do it if your using a gloove box.




on that note.. ive probably sterilized my syringes by allowing the alcohol to get into the tip of the needle and then bleed into the rest of the syringe :frown:
FUCK, no wonder the syringes seem dead


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OfflineShdwstr
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Re: what could i possibly be doing wrong with my LC? [Re: Mych]
    #4272050 - 06/08/05 03:44 PM (12 years, 17 days ago)

I've read all your comments and your proceedure seems fine.

Chances are it was your syringes.
Buying a cheap product usually gets you an inferior one.
Lesson Learned... stick with the Shroomery Vendors.

Good Luck
Shdwstr


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OfflineHotnuts
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Re: what could i possibly be doing wrong with my LC? [Re: Shdwstr]
    #4272161 - 06/08/05 04:10 PM (12 years, 17 days ago)

Dude! I don't need a link. I do shit on my own. You see the results don't ya? Higher temps caused by a pressure cooker @ 15psi can be harmful and cause carmelization if you don't hit 20 minutes on the dime. 5 psi just gives you more lead way for longer times and works just fine. Hell! You don't even have to pc a liquid culture. A 30 minute steam sterilization will do the trick also. And TLG is a fine vendor. Just ask your buddy Agar. Nocking someones buisness for an improper procedure isn't cool bro.


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OfflineMych
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Re: what could i possibly be doing wrong with my LC? [Re: Shdwstr]
    #4272211 - 06/08/05 04:20 PM (12 years, 17 days ago)

Quote:

Shdwstr said:
I've read all your comments and your proceedure seems fine.

Chances are it was your syringes.
Buying a cheap product usually gets you an inferior one.
Lesson Learned... stick with the Shroomery Vendors.

Good Luck
Shdwstr




TLG is a damn good vendor (if not one of the best) according to many on this forum. TLG also used to be a vendor here.
I will be contacting TLG to see whats up.. im not sure how to word the email though since growing = illegal.


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