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OfflineShizzeh
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Good Agar to failed LI / LC ? Help !
    #23959771 - 12/27/16 04:11 AM (7 years, 1 month ago)

Hello,

I have cloned some mycelium on agar and isolated some onto a second plate. without any contaminants I then tried a LI tek with 4% of the water weight in raw sugar, pressure cooked for 20min then let cool for 24h before dropping a wedge of the agar plate in the LI.

Nothing happened after a week so I dropped more agar in it, now still nothing is happening.

Here are some pics of the agar plate used and then the failed LI.

Here is the first step of the cloning on agar:






Here is the one I used:


And This is the "LI / LC"







I Then want to use le LI for BRF cakes, have I failed from the start and gotten bad mycelium ?

Should I wait more for the LI ? I let it on a shelf at room temp for a week, with no direct sunlight, and the 2nd week I let it in the dark at room temp.

Thanks in advace !


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Offlineenlightenment
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Re: Good Agar to failed LI / LC ? Help ! [Re: Shizzeh]
    #23959775 - 12/27/16 04:21 AM (7 years, 1 month ago)

I use very small pieces of colonized agar to inoculate liquid media.
The dish on the first picture is contaminated BTW.



The LC should be more colonized before you use it.




It looks like your sterile technique was not good enough and the LC got contaminated.

You should make at least one transfer before you use the cloned culture.


Edited by enlightenment (12/27/16 04:30 AM)


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OfflineFerather
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Re: Good Agar to failed LI / LC ? Help ! [Re: enlightenment]
    #23959808 - 12/27/16 05:25 AM (7 years, 1 month ago)

:whathesaid:

The first plate also looks like it has malnutrition, transfers will take ages.


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OfflineShizzeh
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Re: Good Agar to failed LI / LC ? Help ! [Re: enlightenment]
    #23959948 - 12/27/16 08:20 AM (7 years, 1 month ago)

Quote:

enlightenment said:
I use very small pieces of colonized agar to inoculate liquid media.
The dish on the first picture is contaminated BTW.



The LC should be more colonized before you use it.




It looks like your sterile technique was not good enough and the LC got contaminated.

You should make at least one transfer before you use the cloned culture.





I was referring to the transfer on this picture for the clean agar, I am not of english mother language and might have said it wrong.


What makes you say the LC is contaminated? The fact that the mycelium didn't expand ?

I used rather large pieces of Agar i reckon, the ones we can see at the bottom of the LC.

Thanks !


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Offlineenlightenment
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Re: Good Agar to failed LI / LC ? Help ! [Re: Shizzeh]
    #23959973 - 12/27/16 08:43 AM (7 years, 1 month ago)

I misread your post and thought you already used it to inoculate cakes that showed contaminations.
Test your LC on one jar before you use it on more jars.


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InvisibleMycolorado
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Re: Good Agar to failed LI / LC ? Help ! [Re: Shizzeh]
    #23960015 - 12/27/16 09:08 AM (7 years, 1 month ago)

You might try for some better growth on agar and use it to noc grain or whatever before involving liquid medium.  As mentioned, the growth looks weak.  What's your agar recipe?


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OfflineKenetic
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Re: Good Agar to failed LI / LC ? Help ! [Re: Mycolorado]
    #23960158 - 12/27/16 10:25 AM (7 years, 1 month ago)

So is the second plate the one you used to inoculate your lc?  If so, it appears that you cut out a section of agar that hasnt been colonized.  Could explain it......
Im not being 100% serious, thats just what it looks like.

It could take up to a week for your culture to recover and another week at least before you see significant progress.


What type of lid are you using?


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Edited by Kenetic (12/27/16 10:38 AM)


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Offlineenlightenment
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Re: Good Agar to failed LI / LC ? Help ! [Re: Kenetic]
    #23960182 - 12/27/16 10:35 AM (7 years, 1 month ago)

Quote:

So is the second plate the one you used to inoculate your lc?  If so, it appears that you cut out a section of agar that hasnt been colonized.




I thought about this, too. But the pieces in the jar look like they do not fit in the cut spot.


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OfflineShizzeh
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Re: Good Agar to failed LI / LC ? Help ! [Re: enlightenment]
    #23960636 - 12/27/16 02:06 PM (7 years, 1 month ago)

Hey,

Quote:

Mycolorado said:
As mentioned, the growth looks weak.  What's your agar recipe?




I used stro's agar prep. It kinda failed because my pc had been shook a bit so half the jar had lost its content and was spilled inside of the PC, I only used what was left in the jar. And figured I might as well see if it would solidify and see what would be up, as I didn't have any agar left for the moment.
(i didnt use the Hefe in his recipe, only agar malt and water from tap, which is very clear and tasty where I live, let's say it comes from the mountains.)

Quote:

kenetic said:
So is the second plate the one you used to inoculate your lc?  If so, it appears that you cut out a section of agar that hasnt been colonized.  Could explain it......
Im not being 100% serious, thats just what it looks like.

It could take up to a week for your culture to recover and another week at least before you see significant progress.


What type of lid are you using?




I used wedge from the 2nd plate indeed, I had a bit that looked unsure after 2 days of transfer on that part so I cut it away because i didnt know if it was me that had touched the part with the scalpel or something that landed on the plate by mistake. I wanted to be safe.

I use lids like this :




Made with bathroom silicone, Does the LC need to breathe ? the jar is closed tightly by hand.

Quote:

enlightenment said:

I thought about this, too. But the pieces in the jar look like they do not fit in the cut spot.




Good eye !

Thanks guys i'll be going skiiing for the next two days and will let everything rest a bit more, again in the dark at room temp. If it doesn't move by the end of the week I will try the agar recipe once again and this time I will maybe put a bit more malt to see if it helps.


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Offlineenlightenment
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Re: Good Agar to failed LI / LC ? Help ! [Re: Shizzeh]
    #23960638 - 12/27/16 02:07 PM (7 years, 1 month ago)

You need gas exchange. Your lid has no filter. You can use a syringe filter for GE on LC jars.


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OfflineKenetic
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Re: Good Agar to failed LI / LC ? Help ! [Re: enlightenment]
    #23960655 - 12/27/16 02:16 PM (7 years, 1 month ago)

Yup.  I'd say that has a lot to do with it.


--------------------
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                                                :cookiemonster::elmo:



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OfflineShizzeh
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Re: Good Agar to failed LI / LC ? Help ! [Re: Kenetic]
    #23964883 - 12/29/16 08:22 AM (7 years, 1 month ago)

Thanks guys !

For the GE i just have to drill a hole and cover it with breathable tape?


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Offlineenlightenment
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Re: Good Agar to failed LI / LC ? Help ! [Re: Shizzeh]
    #23964940 - 12/29/16 09:01 AM (7 years, 1 month ago)

Filters for LC jars:
  • syringe filters
  • PTFE filter stickers
  • hydrophobic autoclavable filters



Filters for grain jars:

  • synthetic filter disks (SFDs)
  • polyfil
  • PTFE filter stickers
  • micropore tape (3 layers)



Use the search engine for your desired filter material.
I am busy ATM but if you have questions feel free to ask.
This is a great community with a lot of good cultivators around.

This is how my LC and grain jar lids look like:



Maybe this thread is also helpful.


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