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Offlinemsj
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Syringe, Blood, Agar Difficulties
    #27358994 - 06/22/21 03:21 PM (2 years, 10 months ago)

First, I put this in advanced because the only things I can think of to help with this issue are maybe antibiotics or agar techniques that I don't know and I'm not sure how to search for, so feel free to move it if this is the wrong place to ask.

I had a stupid moment the other day.  I had a spore syringe with a needle capped on the luer lock tip and I, for whatever dumb reason, decided to try to open it in my SAB with two hands.  Unsurprisingly, I stabbed the shit out of myself with this hypodermic.  Not a big deal 'cause it's a sterile needle and all that so I cleaned up, bleached/iso'd/lysol'd my SAB and rack, pressure cooked my tools, and got some fresh agar plates. 

I don't have spare needles right now, so I cleaned the still-attached needle with iso to get the tiny bit of blood off the tip, then flamed the needle starting from the bit closest to the syringe and got it completely glowing red hot.  I then inoculated some plates.  Those plates changed within a day to the little bit of spore solution in the plate taking on a slimy, immovable consistency.

I'm assuming this is bacterial contamination because I stabbed myself with a needle like an idiot.  I took some more plates and after I flamed my needle again I figured that anything in it must have died by this point so I tried again.

Same result.  Did I ruin this entire syringe?  I'm going to make another attempt with a fresh needle when they come in and I'm hoping it's just the needle that's contaminated.

If I did contaminate the entire syringe, it's not the end of the world I can buy another but it's wasted money and I hate that.  I've got all this agar and plenty of time.  The other syringe I got from this vendor has been doing unexpectedly well, too, so I assume this was quality merch 'till I ruined it.

Could someone point me to some material about how to overcome bacterial contamination on agar in this circumstance?  I hate to throw away a syringe because I was dumb enough to stab myself with it.

TL;DR I stabbed myself with a spore syringe and now every plate I make with it has the weird slime that's in the picture below.  How do I grow some clean mycelium if this syringe is contaminated now?


Edited by msj (06/22/21 03:25 PM)

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OfflineSadTurkey
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Re: Syringe, Blood, Agar Difficulties [Re: msj]
    #27359112 - 06/22/21 04:51 PM (2 years, 10 months ago)

Proper flaming of the needle should sterilize it completely. I guess it might theoretically be possible some bacteria/endospores got lodged in the syringe protected by a piece of your tissue, which got pushed back into the syringe by a bit of boiling liquid in front of it while heating the needle if you were holding the syringe with the needle up or some shit. That would be some mythological bad luck, though.

Either way, assuming your sterile technique is up to scratch and you properly flamed your needle, it's likely the contamination is coming from the syringe itself, whether it was due to you stabbing yourself or just from the vendor. Remember that spore syringes are inherently not sterile, so the presence of bacteria is expected.

I've had syringes that were totally bunk, going bacterial on every plate with mycelium on only 1/10 plates. And I didn't stab myself with it lol.

Edited by SadTurkey (06/22/21 04:52 PM)

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Offlinemsj
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Re: Syringe, Blood, Agar Difficulties [Re: SadTurkey]
    #27359139 - 06/22/21 05:27 PM (2 years, 10 months ago)

This makes me feel slightly better. My luck IS mythologically awful, though. It's frustrating because I really wanted to grow that variant in particular, but I'll live. 

I'm really looking for any way to try to slow the bacteria down enough to give the myc a chance to outrun it, so to speak.  All I need is a tiny spot clear of contamination big enough to stab with a needle when mine come in (think Josex poke tek), but it's like this contam runs rampant in less than a day.  Might wind up trying antibiotics if nothing else works (they might be a handy tool in the future and I like knowing how to do stuff anyway).  You think if I let these dishes sit for a while they might grow a bit of myc and I can try to clean it up from there, or is it just a lost cause once they're this overrun?

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OfflineSadTurkey
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Re: Syringe, Blood, Agar Difficulties [Re: msj]
    #27359608 - 06/23/21 04:21 AM (2 years, 10 months ago)

Quote:

msj said:
You think if I let these dishes sit for a while they might grow a bit of myc and I can try to clean it up from there, or is it just a lost cause once they're this overrun?




Hard to say. How long ago has this plate been inoculated? Spores take a while to germinate, so contaminants can often get a head start. It depends on the contamination and the conditions it produces whether or not the mycelium is able to grow.

If the plates keep failing and you're determined to make it work, you could try making it harder to colonize in the hopes the mycelium wins out. You could try making a plate very low in nutrients or try a plate in the fridge. I have no experience with antibiotics, but if you can get them that should help cleaning it up.

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Offlinegeat
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Re: Syringe, Blood, Agar Difficulties [Re: SadTurkey]
    #27361345 - 06/24/21 10:46 AM (2 years, 10 months ago)

When you inoculate the plates do you completely remove the lid? I would recommend holding the lid over the plate and just slight off center so as not to have your finger tips over the agar. It's like trying to place a drop inside a clam.  Place the syringe over the plate with the drop already on the tip then let it fall. Make sure NOTHING is placed over the plate that is not sterile. And spores take a while to germinate. Flaming the syring should have done the trick to clean it as long as you didnt draw blood into the spore solution. And that seems very unlikely. Notice the slime is a solid ring around the outside. Probably contam from your fingers that was carried around the plate by the condensation.

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Invisibledfwerydfhg
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Re: Syringe, Blood, Agar Difficulties [Re: geat]
    #27361439 - 06/24/21 11:57 AM (2 years, 10 months ago)

You could try putting a drop on one plate, and using a sterile swab or inoculation loop to swab a second plate from that. Benefit would be not carrying over all that water, which the bacteria uses to easily get everywhere.

This just occurred to me, but you could also try putting a couple of drops into an empty (no agar) plate, then leaving it unwrapped in your SAB until they've dried out completely. Then swab from the dry spots to a new plate. Hopefully would take out a lot of the mobile bacteria, and up your chances of separation on the new plate.

There's also Josex's BRF pucks, which some people swear by.

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Offlinemsj
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Re: Syringe, Blood, Agar Difficulties [Re: dfwerydfhg]
    #27366493 - 06/28/21 04:17 PM (2 years, 10 months ago)

Quote:

SadTurkey said:
Quote:

msj said:
You think if I let these dishes sit for a while they might grow a bit of myc and I can try to clean it up from there, or is it just a lost cause once they're this overrun?




Hard to say. How long ago has this plate been inoculated?




A while.  I gave up and threw them out.  I'm going to start over.

Quote:

geat said:
When you inoculate the plates do you completely remove the lid?




I try very hard to not open it completely but sometimes I'm clumsy.  But so far I have an excellent rate of success with the other syringes I've tried so I think it's just this one.


Quote:

dfwerydfhg said:
You could try putting a drop on one plate, and using a sterile swab or inoculation loop to swab a second plate from that. Benefit would be not carrying over all that water, which the bacteria uses to easily get everywhere.

This just occurred to me, but you could also try putting a couple of drops into an empty (no agar) plate, then leaving it unwrapped in your SAB until they've dried out completely. Then swab from the dry spots to a new plate. Hopefully would take out a lot of the mobile bacteria, and up your chances of separation on the new plate.

There's also Josex's BRF pucks, which some people swear by.




I like the drop to swab to plate idea.  The extra moisture I think is definitely a factor.  And I actually am trying BRF pucks because I read about that!!  Thanks for the heads up that I'm going in the right direction potentially.

I also found where to get some gentamicin so I think I might give that a shot for when I get really bad syringes like this in the future.  Don't think I'd make a habit of using it in every plate but that might be really handy on this one.

Edited by msj (06/28/21 04:18 PM)

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