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randy420rhoads


Registered: 02/24/07
Posts: 535
Last seen: 11 years, 5 months
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WTF contams
#14361943 - 04/27/11 04:04 PM (12 years, 9 months ago) |
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I'm using a glovebox doing agar to grain. I flame sterilize between transfers and spray down my box with lysol. Last time I lost all but one to bacterial contam, and I gave that one a shake because it was 90% done and now it looks like it's not recovering. Now my new batch I used tyvek on and this popped up on all but 2 but i'm sure they're next. It looks like neon green in the center of good oyster mycellium? Can anyone ID the contams and give me some tips on what the fuck i'm doign wrong now...
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Perun
Mahapralaya...



Registered: 11/11/10
Posts: 585
Last seen: 2 years, 2 months
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How sure r u about your agar plates? R u 100% they r clean? how long r u PCing your jars? 15psi?
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randy420rhoads


Registered: 02/24/07
Posts: 535
Last seen: 11 years, 5 months
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Re: WTF contams [Re: Perun]
#14362032 - 04/27/11 04:19 PM (12 years, 9 months ago) |
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My agar is in 1/2pint wide mouths. They look great to me 150% colonized( the mycellium started up the sides of the jar and off the agar) and are all fluffy white with no signs of contams.
I pc'd all jars for at least 90 mins.
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Ganjabonga
Chief



Registered: 10/07/08
Posts: 543
Loc: Washington
Last seen: 10 years, 3 months
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Is all of this coming from the same syringe? If everything is being done right, than that would most likely be the culprit.
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steelmonkey
Homejigger



Registered: 02/22/11
Posts: 1,822
Loc: Nova Scotia,Canada
Last seen: 2 years, 4 months
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Try isolating on more agar plates to be sure
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randy420rhoads


Registered: 02/24/07
Posts: 535
Last seen: 11 years, 5 months
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Quote:
Ganjabonga said: Is all of this coming from the same syringe? If everything is being done right, than that would most likely be the culprit.
Syringe? I said agar to grain. I cloned oysters from the PF tek.
What would isolating to more agar plates do? The first batch was bacteria and had none of this. I used multple plates 2 quart jars per plate, and they all have the same contams, So wouldn't that mean the contams are from something else? Or I just got unlucky and all my plates appeared fine but had the same contams.
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aqh
pothead



Registered: 04/10/11
Posts: 181
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Quote:
randy420rhoads said:
Quote:
Ganjabonga said: Is all of this coming from the same syringe? If everything is being done right, than that would most likely be the culprit.
Syringe? I said agar to grain. I cloned oysters from the PF tek.
What would isolating to more agar plates do? The first batch was bacteria and had none of this. I used multple plates 2 quart jars per plate, and they all have the same contams, So wouldn't that mean the contams are from something else? Or I just got unlucky and all my plates appeared fine but had the same contams.
Sounds like you already know everything.
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Ectius
Werecat Ninjabeast



Registered: 02/24/10
Posts: 36
Last seen: 12 years, 8 months
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If the culture was clean and your transfer flawless, then it'd have to be the grains. Making a proofing jar with your next batch can give you that answer.
Looking at the pics, though, the contams seem localized to your mycelium. Which suggests that it was either on the agar culture, or came in during the transfer.
Outbreaks suck, dude. Especially when you're lost as to the source. 
Suggestions: - Make a proofing jar of grain during your next batches. - Mist the interior of your GB? (If you aren't already) - Make another isolate, like aqh suggested. It's worth the work. - Try, try again.
Oh, nice snake.
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3n1gm4
3N!9M4T!C



Registered: 01/13/11
Posts: 2,727
Loc: The Downside of Up
Last seen: 12 years, 7 months
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Re: WTF contams [Re: Ectius]
#14362694 - 04/27/11 06:05 PM (12 years, 9 months ago) |
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Your lids suck, sorry but they do. Tyvek pores can spread open and let in contams, you need some red RTV and some SFDs. http://www.shroomery.org/forums/showflat.php/Number/14186384/page/1
-------------------- http://www.shroomery.org/6257/Magic-Mushroom-Dosage-CalculatorLOL when you zoom in to try to read my sig pics you will get lost in the crystal forrest of ghanni!
   
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Ectius
Werecat Ninjabeast



Registered: 02/24/10
Posts: 36
Last seen: 12 years, 8 months
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Re: WTF contams [Re: 3n1gm4]
#14362768 - 04/27/11 06:18 PM (12 years, 9 months ago) |
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Quote:
3n1gm4 said: Your lids suck, sorry but they do. Tyvek pores can spread open and let in contams, you need some red RTV and some SFDs. http://www.shroomery.org/forums/showflat.php/Number/14186384/page/1

Implying that synthetic discs are immune to material degradation, and that one-shot tyvek will experience more wear and tear than discs glued to lids.
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anonjon
Partially Right

Registered: 11/03/08
Posts: 6,322
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Re: WTF contams [Re: Ectius]
#14362797 - 04/27/11 06:25 PM (12 years, 9 months ago) |
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sorry for your loss.
-------------------- The above post is fictional, hypothetical, or downright nonsensical.
Edited by anonjon (04/27/11 06:26 PM)
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randy420rhoads


Registered: 02/24/07
Posts: 535
Last seen: 11 years, 5 months
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Re: WTF contams [Re: anonjon]
#14362828 - 04/27/11 06:33 PM (12 years, 9 months ago) |
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Can you explain what a proofing jar is please?
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3n1gm4
3N!9M4T!C



Registered: 01/13/11
Posts: 2,727
Loc: The Downside of Up
Last seen: 12 years, 7 months
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Re: WTF contams [Re: anonjon]
#14362832 - 04/27/11 06:34 PM (12 years, 9 months ago) |
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That is envelope tyvek on those jars, implying that envelope tyvek will block .2 micron and envelope tyvek is better than SFDs?
I'm not saying it WAS the tyvek, I was just saying it could have been. SFDs aren't immune but they are better than tyvek
-------------------- http://www.shroomery.org/6257/Magic-Mushroom-Dosage-CalculatorLOL when you zoom in to try to read my sig pics you will get lost in the crystal forrest of ghanni!
   
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randy420rhoads


Registered: 02/24/07
Posts: 535
Last seen: 11 years, 5 months
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Re: WTF contams [Re: 3n1gm4]
#14362885 - 04/27/11 06:44 PM (12 years, 9 months ago) |
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AH HA. Figured out the problem thanks to the suggestion of the contams being localized to the agar wedges in the grain I went and checked something. 4 out of 6 jars are bad. The 4 bad ones were labeled "good strain 1" and the 2 good jars labelled " shit strain." I used one plate with my good strain and those all went bad. I used a whole plate of my less than desirable cloned agar plate on the 2 other jars and they are ok. Meaning it must have been the plate.
Sucks that my good strain was bad and bad strain was viable. I'm wondering how a 100% colonized healthy agar plate can be contaminated if it hadn't been opened since the cloning. If they were introduced then why didn't the spores germinate then?
uuughh I know it takes time but each time I go to the next step I fail and repeat. At first my jars needed GE so I put the tyvek lids on as someone suggested. Now they are not something with good success. What is the correct or "best way" to make the lid? The same way but with more tyvek layers? Stuffed pollyfill between the tvek and lid? BTW where do you aquire pollyfill? Never heard of it.If envelop tyvek is .2 microns isn't that sufficiant? What is SFD ?
Edited by randy420rhoads (04/27/11 06:46 PM)
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anonjon
Partially Right

Registered: 11/03/08
Posts: 6,322
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I've tried every kind including fancy ones with whatman filters and a rubber injection port.
i decided I like a simple plastic lid with a single quarter inch hole stuffed with polyfill. Don't really need the injection ports.
-------------------- The above post is fictional, hypothetical, or downright nonsensical.
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steelmonkey
Homejigger



Registered: 02/22/11
Posts: 1,822
Loc: Nova Scotia,Canada
Last seen: 2 years, 4 months
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Thats why I suggested isolating it out on more agar plates sometimes it takes awhile for certain molds to get a foot hold and incubate growing them out and further isolating the substrain results in stronger healthier more robust mycelium thats more able to fight off molds
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randy420rhoads


Registered: 02/24/07
Posts: 535
Last seen: 11 years, 5 months
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Re: WTF contams [Re: anonjon]
#14362938 - 04/27/11 06:53 PM (12 years, 9 months ago) |
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Quote:
anonjon said: I've tried every kind including fancy ones with whatman filters and a rubber injection port.
i decided I like a simple plastic lid with a single quarter inch hole stuffed with polyfill. Don't really need the injection ports.
How did you drill the hole every time I did the platic lids cracked. Had to use metal.
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FooMan



Registered: 02/02/05
Posts: 8,957
Loc: Earth
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You don't want your plates to colonize 100%. You want to grab the newest myc from the outer edges of the growth early on. You may have had some unseen contaminant on the plate. Green mold usually grows white at first and then turns green once it begins to sporulate.
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Quick WBS Prep
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anonjon
Partially Right

Registered: 11/03/08
Posts: 6,322
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Quote:
randy420rhoads said: How did you drill the hole every time I did the platic lids cracked. Had to use metal.
set it upside down on a piece of wood as you're drilling. It'll give u a clean hole.
-------------------- The above post is fictional, hypothetical, or downright nonsensical.
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randy420rhoads


Registered: 02/24/07
Posts: 535
Last seen: 11 years, 5 months
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Re: WTF contams [Re: FooMan]
#14362985 - 04/27/11 07:00 PM (12 years, 9 months ago) |
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SO when transfering agar to grain you want to make the transfer before 100% and ONLY use the outside edges of the agar?
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