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OfflineMChem
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No signs of growth in LC after 13 days
    #26643595 - 05/02/20 12:28 PM (3 years, 8 months ago)

Hi,

Recently prepared two batches of liquid culture (1g LME:250 ml water, PC 30min@15psi), using the syringe biopsy method and SAB for inoculation. The first batch was inoculated with clean B+ mycelium grown on agar, one transfer from MS. The second was inoculated with clean GT clone mycelium grown on agar. It has been 13 days since inoculation, and I am yet to see growth in either batch. There are some small specs of material that could be mycelium, but they haven't grown noticeably and may well be undissolved LME.

Should I toss these given that there is no growth after so long? The cultures are in pyrex media bottles with the lids screwed on tightly, could it possibly be the lack of gas exchange stifling growth?





Edited by MChem (05/02/20 12:51 PM)


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Offlinepoisoned
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Re: No signs of growth in LC after 13 days [Re: MChem]
    #26643808 - 05/02/20 01:58 PM (3 years, 8 months ago)

What's with the needle in that first one?

I'm guessing they both got contaminated.


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OfflineA.k.aM
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Re: No signs of growth in LC after 13 days [Re: poisoned] * 1
    #26643982 - 05/02/20 03:13 PM (3 years, 8 months ago)

It could take a while to see anything visible with that small of an inoculate.

They look clear still so that’s good but it’s really hard to tell with lc. I’d give it more time.


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Edited by A.k.a (05/02/20 03:14 PM)


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Offlinepoisoned
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Re: No signs of growth in LC after 13 days [Re: A.k.a]
    #26643994 - 05/02/20 03:15 PM (3 years, 8 months ago)

A.k.a has way more experience with LCs than I do, so he's probably right.


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OfflineA.k.aM
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Re: No signs of growth in LC after 13 days [Re: poisoned]
    #26644008 - 05/02/20 03:19 PM (3 years, 8 months ago)

I’ve only ever made them with transfer size wedges and once with spores though.

I haven’t read the poke tek but it seems like it could take awhile to expand from what could be a few cells of myc.

Could easily be wrong though, sometimes they go crazy fast.


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OfflineJustweed
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Re: No signs of growth in LC after 13 days [Re: A.k.a]
    #26645665 - 05/03/20 10:43 AM (3 years, 8 months ago)

Heh saw your name and thought you were an old friend!

Did you leave the biopsied piece in the needle and just drop the needle in? That'll be a no go, the piece is in the needle with no area to grow out to (quickly at least) and will quickly choke in the needle.
Try doing a rough shake to get some aeration in the culture, I would do that once a day until you see thorough growth, maybe even 2x a day. You could have also cooked off all your sugars by PCing for so long, but that's just a guess. I make my LC by putting ~400ml of water into a quart jar, then adding a half tablespoon of honey, then putting that in a pot of boiling water and boiling for 10-15 minutes. Then I add genetics. Works pretty well, and quick too. Maybe give that a try for your next go. I need to do a tek on it, it makes making LC too easy, I've got a whole tub full of the stuff now!


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OfflineWeavieWonder
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Re: No signs of growth in LC after 13 days [Re: Justweed]
    #26645691 - 05/03/20 10:56 AM (3 years, 8 months ago)

Speaking from experience, the mixture looks overcooked. See how the particulates at the bottom are brown and the liquid has a brownish tint to it? No bueno. Try a 20 minute pc cycle next time and don't use a lot of water in the pc. The biopsy method is my prefered method of inoculation. If you dont start to see growth 3 - 5 days after inoculation, something is fucked.


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InvisiblePiptheGreAtest
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Re: No signs of growth in LC after 13 days [Re: WeavieWonder]
    #26645761 - 05/03/20 11:23 AM (3 years, 8 months ago)

Never tried needle biopsy but when I knock up LC( All I use), I take a whole 60mm dish pizza slice it and throw it into a bottle. These two here had rapid growth colonized it about 4 days without stirring at all. I use 1g of LME to 500ml of water even.


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Invisiblemycorry
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Re: No signs of growth in LC after 13 days [Re: PiptheGreAtest]
    #26645834 - 05/03/20 12:03 PM (3 years, 8 months ago)

I have had a problem like this before. What happened was that in my focus on killing all contams for the xfer I got the blade/handle too hot and the heat killed the myc during the xfer. This leads to a crystal clear LC with a dead wedge inside.

Another cause that has happened to me many times is that some species seem to hate the temp shock of going from warm agar to cool LC. Furthermore, Some species need a large wedge to recover in an LC especially if the species is known to grow on wood.

The rhetoric around wedges is usually that you should get the smallest piece possible and that just isn't true for beginners attempting LC's, imo. It's only true if you are trying to narrow genetics which isn't a big deal anyways. Your main goal should be a clean and successful inoculation.

Things to try:
-grab a bigger wedge
-be sure to fully cool your blade(maybe cool it in different spots on the agar)
-Try to reduce the temp difference between your agar and the LC(don't keep an unused LC in the fridge, room temp is fine)
-Shotgun LC inoculations to improve success. As in, inoculate 3 LCs instead of one.
-Don't make overly nutritious LCs. 1g LME to 250ml water is perfect for most non woodlovers.
-Don't overcook LCs, 20 min @15psi is more than enough. Some people have claimed that the yellowing from overcooking can hamper inoculation success, however, I have never experienced this.

:thumbup:


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OfflineMChem
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Re: No signs of growth in LC after 13 days [Re: A.k.a]
    #26646185 - 05/03/20 02:39 PM (3 years, 8 months ago)

Thanks for the reply. I was expecting it to take longer than it would with a wedge, but I thought I'd have seen growth by this point. In the biopsy tek, it states that the LC should be ready for use in 2-3 weeks, which is not encouraging. I'll see how they progress over the coming week. Fingers crossed!


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Offlinemushpunx
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Re: No signs of growth in LC after 13 days [Re: MChem]
    #26646215 - 05/03/20 02:55 PM (3 years, 8 months ago)

Personally I wouldn't make an LC with tissue. I am very experienced
at cloning, and I still find myself having to transfer away from bacteria blobs after taking a biopsy from the virgin tissue inside.

Make LC from agar wedge.

Actually when I make LC I first blend my wedge with water in an eberbach and then pour into my LC broth + spin on a magnetic stir plate.
It's usually ready in 3-4 days.


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