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OfflineBobbit
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Re: Pastywhyte's EzLC tek. [Re: Edmunter]
    #25784457 - 02/01/19 06:36 PM (4 years, 11 months ago)

Hi all,

Made my first ezLC last night,


It seems that I have some extra water in my jars?


Should I attempt to tip it out when I have the lid off to innoc?


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OfflinePsilosopherr
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Re: Pastywhyte's EzLC tek. [Re: Bobbit]
    #25784461 - 02/01/19 06:38 PM (4 years, 11 months ago)

very common in jar agar IME. Not a problem


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OfflineBobbit
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Re: Pastywhyte's EzLC tek. [Re: Psilosopherr]
    #25787260 - 02/03/19 12:24 AM (4 years, 11 months ago)

Cheers Philo,

I did tip when dropping wedges. . .

Do you store them upside down (agar on top) also?

Didn’t think of the obvious until writing this response.


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Offlinevertygo
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Re: Pastywhyte's EzLC tek. [Re: Bobbit]
    #25789250 - 02/03/19 09:08 PM (4 years, 11 months ago)

Could you use a quart of water with a smaller round agar puck, if the water had some Karo or other additive ? In other words, can you supplement the nutrition in the water and have less agar/myc ? Would it be safe to say this would take much longer to colonize ?

I realize the actual tek calls for just sterile water by itself.

Just thought of another question, because everything seems very clean (assuming a very good sterile technique as well) could the LC be colonized at a warmer temperature to encourage rapid growth ?


Edited by vertygo (02/03/19 09:32 PM)


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OfflineBobbit
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Re: Pastywhyte's EzLC tek. [Re: vertygo]
    #25789637 - 02/04/19 03:51 AM (4 years, 11 months ago)

Quote:

vertygo said:
Could you use a quart of water with a smaller round agar puck, if the water had some Karo or other additive ? In other words, can you supplement the nutrition in the water and have less agar/myc?



I believe that the whole idea is to have the sediment trapped in the agar. Sure you could add more nutrient, though that would defeat the purpose..


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Invisible36fuckin5
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Re: Pastywhyte's EzLC tek. [Re: vertygo]
    #25789960 - 02/04/19 08:47 AM (4 years, 11 months ago)

Quote:

vertygo said:
Could you use a quart of water with a smaller round agar puck, if the water had some Karo or other additive ? In other words, can you supplement the nutrition in the water and have less agar/myc ? Would it be safe to say this would take much longer to colonize ?

I realize the actual tek calls for just sterile water by itself.

Just thought of another question, because everything seems very clean (assuming a very good sterile technique as well) could the LC be colonized at a warmer temperature to encourage rapid growth ?




You're looking for shortcuts. That never works. Just do it right.

Adding more nutes will make it slower. These things already colonize in a few days. There is no reason to use higher temps.


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i recommend common sense and figuring it out.

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Offlinevertygo
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Re: Pastywhyte's EzLC tek. [Re: 36fuckin5]
    #25791128 - 02/04/19 04:33 PM (4 years, 11 months ago)

Can I use ez felt for the GE instead of the SFD ?


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Invisiblestevo

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Re: Pastywhyte's EzLC tek. *DELETED* [Re: vertygo]
    #25794432 - 02/06/19 07:22 AM (4 years, 11 months ago)

Post deleted by stevo

Reason for deletion: .


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Offlinevertygo
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Re: Pastywhyte's EzLC tek. [Re: stevo]
    #25795694 - 02/06/19 05:26 PM (4 years, 11 months ago)

Would putting the LC jars on a orbital swirling table 24 hours a day increase the growth speed ? I'm not a scientist, so sorry if this is a dumb question.  I ask because a magnetic stirrer wouldn't apply to this situation.


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OfflineBobbit
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Re: Pastywhyte's EzLC tek. [Re: vertygo]
    #25796467 - 02/07/19 02:17 AM (4 years, 11 months ago)

I now see why you use 1 piece plastic lids.

I used band and metal lid, and had some contam problems, I think from not tightening the lids properly.

I sealed some brf lids holes with rtv, and left a few unsealed. I tested the seal, and with lids upside-down, you have to screw really tight to get an air seal.

I will try sealing lids to bands with rtv as plastic lids are hard to source here.


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OfflineBobbit
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Re: Pastywhyte's EzLC tek. [Re: Bobbit]
    #25819170 - 02/17/19 03:44 PM (4 years, 11 months ago)

Revelation, seal side down with 2 piece lids works fine if the band and seal are rtv'd together. The twist breaks the seal, just don't screw tight (just enough to bite)

I do have a question around testing LC.
What to look for on your test plate? Mold or trich, easy enough, plate covered in a day or two or green spots.

What does bacterial infection look like and should I worry?


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InvisiblePastywhyteMDiscord
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Re: Pastywhyte's EzLC tek. [Re: Bobbit] * 1
    #25819200 - 02/17/19 03:57 PM (4 years, 11 months ago)

Bacteria looks slimy and grows fast. Any colonies that grow other than the wedge you placed should be considered suspect.


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OfflineBobbit
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Re: Pastywhyte's EzLC tek. [Re: Pastywhyte]
    #25819452 - 02/17/19 05:53 PM (4 years, 11 months ago)

Cheers dude.

Will be dropping LC not a wedge. I've seen slimy, waxy stuff appear around wedges before the fuzz returns, not sure how that manifests from a drop off liquid.

I guess practice will show. I've been fairly successful staying clean to date though starting with dirty wild prints has def. shown me what ugliness can look like.


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OfflineSivarted
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Re: Pastywhyte's EzLC tek. [Re: Bobbit]
    #25819940 - 02/17/19 09:26 PM (4 years, 11 months ago)

One quick question.  There are a lot of warnings here when using SFDs about being very careful not to get the filter wet while shaking/stirring.

My understanding of SFDs is that they're hydrophobic and dry very quickly though.  I have some LC jars that I've been swirling like a madman, knowing beyond any shadow of a doubt that the filter is getting wet underneath, but the top never gets even a little bit damp.

In fact, I just did some intentionally vigorous shaking on one that I have sterilized but not inoculated yet, and I couldn't even get the top of the filter to show any amount of moisture until I held it upside down for a bit.

Theoretically, any amount of LC that gets in the filter could potentially start colonizing it, but since they dry so quickly, I'm not sure that should be an issue.

Bringing a contam from outside would probably require moisture getting through to the surface of the filter, which I haven't had happen with any amount of vigorous swirling, even with about 3/4 full jars.  (quarts, pints, and even a couple half gallons)  And even then if it dries fast enough there shouldn't be an opportunity for the contam to work its way through the filter.

So, am I way off or should SFDs be safer on lids than indicated here?

Thoughts?


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OfflineBobbit
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Re: Pastywhyte's EzLC tek. [Re: Sivarted]
    #25833858 - 02/24/19 02:46 AM (4 years, 10 months ago)

So I tipped water off the top of my puck after transferring a wedge in the SAB. Some must have touched the water as a few days later I got a dozen white spots appear and turn into fuzz balls.

Me of little faith was waiting for them to turn green, yet no, they all joined up happily and started climbing the glass . . . This is where the problems arise.

I sterilized a few half pints of water last night and aspirated onto this covered puck. Now it seems that the fuzz is waterproof and none of the agar surface is available to leach anything into the water. . .

Any ideas?


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InvisibleStoneJackBaller
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Re: Pastywhyte's EzLC tek. [Re: Bobbit]
    #25838833 - 02/26/19 10:57 AM (4 years, 10 months ago)

Thank you for another helpful tek.  I am mostly newb with some successful tub grows in the past.  I do ok with transfers for the most part but I am colorblind and do not trust myself with identifying contaminants on agar 100%.  Would there be any downside to adding H2O2 to the agar when making it up to mitigate mold and bacteria possibilities? 

If so, would it make sense to add it to the agar mix alone, or to also add after colonization when adding sterile H2O?

Much Gratitude


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Re: Pastywhyte's EzLC tek. [Re: StoneJackBaller]
    #25838879 - 02/26/19 11:18 AM (4 years, 10 months ago)

Adding H2O2 is pointless. It becomes water in about 2 min during sterilization


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Invisiblesh4d0ws
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Re: Pastywhyte's EzLC tek. [Re: Sivarted]
    #25838887 - 02/26/19 11:23 AM (4 years, 10 months ago)

I thought SFD's could get wet? I still use tinfoil but I remember reading about people who use SFD's eliminating tinfoil.


Maybe a solution of LC hitting it as opposed to steam water while sterilizing is a little different but the pores should be the same no matter what :shrug:


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Offlinevertygo
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Re: Pastywhyte's EzLC tek. [Re: StoneJackBaller]
    #25839214 - 02/26/19 02:23 PM (4 years, 10 months ago)

Quote:

StoneJackBaller said:
Thank you for another helpful tek.  I am mostly newb with some successful tub grows in the past.  I do ok with transfers for the most part but I am colorblind and do not trust myself with identifying contaminants on agar 100%. 




I'm color blind too, I find it helps to just dye the agar. Few drops of blue food coloring (or whatever works for you) and it's good to go.  Also, a magnifying glass/lens can sometimes help. Cheers.


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Offlinebulbstoo
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Re: Pastywhyte's EzLC tek. [Re: vertygo]
    #25885638 - 03/20/19 11:51 AM (4 years, 10 months ago)

Hey Pasty - love the idea of growing out agar in a no pour style chamber, then injecting water with no vectors as long as your SHIP/needle/whatman are well set up. IMO, it removes a lot of risk associated with dropping a puck into the LC. I imagine this is when most contams occur.

  • Why not inject sugar+water mixture into the agar plate? Simply becuase the plate already has all the nutes we need?
  • Why not use a stirrer bar in the container + magnetic stirrer to speed up time/add the ability to really break up the agar well early on, which I imagine promotes growth moreso over the long-term


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