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OfflineGrambo
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frustrated
    #24238887 - 04/12/17 10:09 PM (7 years, 9 months ago)

Guys, I got this contamination that started at transfer 4 from another culture, and at transfer 8, I am still battling with it. My start with agar was very successful without many contams, since I transferred a clone from a bacterial cake I seem to be contaminating everything.

Can I get some advice on what to do please? I will do my next transfer and set aside extra wedges to do a hot pour. Am I letting the plates sit too long so that the bacteria has a firmer grip? I didn't seem to have much trouble before but now I am struggling. I am well aware of how to be sterile, I just can't seem to shake it.

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InvisibleMad Season
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Re: frustrated [Re: Grambo]
    #24238975 - 04/12/17 11:02 PM (7 years, 9 months ago)

Either try starting back up from spores to see if it's just the culture, or seriously revise your techniques. Make sure things in the SAB are nice and wet, nice massive armholes, a very still room to work in, and don't get all shaky. I'd probably do both MS and technique revisions lol


--------------------
contam and car window art
How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

AMU Q&A
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OfflineGrambo
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Re: frustrated [Re: Mad Season]
    #24240363 - 04/13/17 12:37 PM (7 years, 9 months ago)

Thank you for answering me Mad.

The armholes are six inch, the air is off and I have to transfer off to the side of centre as that is where the soap/water drips from as it collects. Both cultures were contaminant free for almost a month before this. I did transfers at my kitchen table that didn't contam in open air. At the moment I have one MS that is contam free so I will make more than two transfers. It just sucks that I can't clean this up after two months of work.

I am grateful for the advice though sir. I will try to clean this a few more times before giving up, my trays should be starting up soon and I can try again with that strain instead. Faaack I hate bacteria.

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InvisibleMad Season
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Re: frustrated [Re: Grambo]
    #24240597 - 04/13/17 02:22 PM (7 years, 9 months ago)

Well it is possible it's just been riding all along, and only been made visible recently.. Have you considered doing a couple transfers with antibacterial agents in agar like tetracycline? It really inhibits bacteria and can give you the clean transfer you needed.


--------------------
contam and car window art
How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

AMU Q&A
No trees were killed in the sending of this message. However, a large number of electrons were terribly inconvenienced.

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InvisibleReblackah
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Posts: 13,770
Re: frustrated [Re: Mad Season]
    #24240617 - 04/13/17 02:36 PM (7 years, 9 months ago)

Hey Mad, does Tetracycline survive the PC run or do you add it to the agar after the PC? And do you have any idea on measurements?

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InvisibleMad Season
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Re: frustrated [Re: Reblackah]
    #24240628 - 04/13/17 02:38 PM (7 years, 9 months ago)

I've only done pour techniques with it, but it supposedly has a melting point above 400°F, so it theoretically would survive the PC cycle.

I just toss a pinch in per plate, seems to do the job just fine.


--------------------
contam and car window art
How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

AMU Q&A
No trees were killed in the sending of this message. However, a large number of electrons were terribly inconvenienced.

Edited by Mad Season (04/14/17 12:03 PM)

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OfflineGrambo
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Re: frustrated [Re: Mad Season]
    #24241056 - 04/13/17 05:21 PM (7 years, 9 months ago)

Never heard of using tetracycline but I have some as well as minicyclin and doxycycline. I suppose I can sacrifice a tab to give it a shot if it sticks around. I just get the feeling it is flourishing with each transfer no matter how small and remote the transfer is from a contam spot.

Thanks once more.

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InvisibleReblackah
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Re: frustrated [Re: Grambo]
    #24242380 - 04/14/17 02:20 AM (7 years, 9 months ago)

it's probably riding the mycelium inside the myc strands.

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InvisiblePanda89
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Re: frustrated [Re: Reblackah]
    #24245870 - 04/15/17 07:46 AM (7 years, 8 months ago)

No dude, you act like we didnt answer you.
https://www.shroomery.org/forums/showflat.php/Number/24151528

You left out crucial info. You man handle the plates with no gloves on. That is your issue and why you have contams.

Edited by Panda89 (04/15/17 07:56 AM)

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InvisibleMad Season
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Re: frustrated [Re: Panda89]
    #24245900 - 04/15/17 08:05 AM (7 years, 8 months ago)

I don't use gloves in a SAB. Nor does bodhisatta, or Pastywhyte. Why? Because in a SAB the air flows with gravity. Thus, NEVER put anything over open media, besides the tool of choice. Even with an alcohol soaked glove, the moment you see it go over open media, consider that plate fucked. Gloves and alcohol are a safety net, and definitely more useful for people with contam issues, but it isn't the reason why it contams. It's because people's hands or a dirty handle of a tool goes over open media. At least in a SAB that is. FH would be much different, and using gloves in that is a 100% necessity.


--------------------
contam and car window art
How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

AMU Q&A
No trees were killed in the sending of this message. However, a large number of electrons were terribly inconvenienced.

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InvisiblePanda89
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Re: frustrated [Re: Mad Season]
    #24245931 - 04/15/17 08:19 AM (7 years, 8 months ago)

My thought process was, since he has been having contams take that extra precaution to try and clean up the sample. I mean if what you are doing now isnt working, what is the harm of trying it a different way? That's how I grew with the hobby.

Edited by Panda89 (04/15/17 08:20 AM)

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InvisibleMad Season
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Re: frustrated [Re: Panda89]
    #24245967 - 04/15/17 08:34 AM (7 years, 8 months ago)

Definitely agree extra precaution is good, but I don't think it is why stuff contaminates. Its all in the techniques, rather than the chemicals. I've had no gloves and no alcohol for at least a year now, and that's because I ensure the techniques are flawless. A Sab is amazing, since the air is still, everything flows downwards. Thus your hella dirty hands should always be as far away as possible.

Y'all should read my improve your sterile techniques link in my SIG


--------------------
contam and car window art
How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

AMU Q&A
No trees were killed in the sending of this message. However, a large number of electrons were terribly inconvenienced.

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InvisiblePanda89
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Re: frustrated [Re: Mad Season]
    #24246003 - 04/15/17 08:50 AM (7 years, 8 months ago)

:rockon: thanks bro, going to check it out now. Grambo best of luck getting your agar cleaned up :goodluck:

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OfflineGrambo
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Re: frustrated [Re: Panda89]
    #24250497 - 04/16/17 11:33 PM (7 years, 8 months ago)

Thank you fellas.

Panda, "Follow sterile technique" didn't really constitute as advice or a suggestion to the question of "what steps do you guys think I should take from here?", so I thought I might find a more appropriate response on this contamination forum. I wasn't doing it to be a turd or anything, just looking for some alternate avenues to explore. Thank you very much for sending me the good luck brother!

Mad, I don't know man. I am going to try being more diligent with the transfers, I tilt my minis to transfer to lessen surface area, maybe that is the vector? I don't wipe them with iso either, never have and didn't have a problem for the first month. Hit me with any input brother, I'm just going to try a few experiments to see if they work but if you or Panda have any idea's I'll gldly add them to the list.

And... good luck to you guys too

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InvisibleMad Season
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Re: frustrated [Re: Grambo]
    #24250501 - 04/16/17 11:37 PM (7 years, 8 months ago)

Contams are a bitch, but once you've pinned the source, it's such a cinch to get past. That's why all you can really do is be diligent in every aspect until you've figured out what the issues are.

Get new cultures, double check your techniques (making sure that only sterile things go over open media), and try out antibacterial agar.


--------------------
contam and car window art
How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

AMU Q&A
No trees were killed in the sending of this message. However, a large number of electrons were terribly inconvenienced.

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InvisiblePanda89
Kung fu panda
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Re: frustrated [Re: Mad Season]
    #24251657 - 04/17/17 01:29 PM (7 years, 8 months ago)

I'm just curious looking at the first pics you are using pasty plates, what are you using as a filter on the containers? I'm just thinking about what it could be and wicking in through filter came to mind.

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OfflineGrambo
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Re: frustrated [Re: Panda89]
    #24256061 - 04/19/17 02:27 AM (7 years, 8 months ago)

Sorry, I was away.

I use two layers of micropore. I had a look at my new plates. I made 3 transfers from the single plate that didn't contam and only 1 came out contam free. I fumbled like crazy as with each transfer the wedge would keep sticking higher and higher on the blade and I just kept carving up the agar until I could get something to drop. I would have thought all three would have gone bad.

The other two are extremely peppered with blobs, like 10, 15 spots. The 2 clones are also contamed again. The clones were from a cake with a grey mold but didn't contaminate until a month later.  My original transfers were in open air but I only had contamination problems a few times and they were black spots. Those plates were tossed though.

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InvisibleReblackah
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Re: frustrated *DELETED* [Re: Grambo]
    #24256114 - 04/19/17 03:26 AM (7 years, 8 months ago)

Post deleted by LizardWizard

Reason for deletion: ok

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InvisibleReblackah
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Re: frustrated [Re: Reblackah]
    #24256115 - 04/19/17 03:28 AM (7 years, 8 months ago)

Oh, and if your first plates were open-air, and didn't have contams, could it be that it had hidden contams? I think I have had some, the mycelium shouldn't "bubble up" or lay a thicker layer in spots, if I understand correctly.

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OfflineGrambo
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Re: frustrated [Re: Reblackah]
    #24256125 - 04/19/17 03:37 AM (7 years, 8 months ago)

That is interesting and I have seen people here do that before but I had forgotten.  I appreciate the suggestion and it is definitely worth trying.  If I can only get two clean transfers I would like to try a sandwich or a hot pour. My four trays of golden teacher should be ready to fruit soon so I can try new clones. I just have a bad feeling that I will be transferring contamination from my B+ clones now that the bacteria seems to have a toehold.

Even if I destroy all my contaminated culture now I'm not sure that that will prevent contamination from reappearing on future cultures. My brain is getting sore.

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