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OfflineTira
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Troubleshooting
    #23763635 - 10/23/16 09:31 AM (7 years, 4 months ago)

I recently inoculated 24 grain jars with agar wedges and today, I realized that 9 of them are contaminated.  Mostly with trich, some with black pin mold. 

I inoculated jars with agar and have only one hole on my lids for GE; which is filtered by two layers of micropore tape -one layer on the outside of the lid, and one layer on the inside. 

All of the contaminated areas are on the top of the grains, so I think there are 2 possibilities; either my sterile technique is the problem and they've got contamination during the agar-grain transfer process, or two layers of micropore tape alone is not a sufficient filter and can't keep contaminants outside.

Any ideas?


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Invisibledankington
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Re: Troubleshooting [Re: Tira]
    #23763673 - 10/23/16 09:49 AM (7 years, 4 months ago)

top of the grain in the jars, you say?
Filter fail.

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Offlineenlightenment
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Re: Troubleshooting [Re: Tira]
    #23763678 - 10/23/16 09:51 AM (7 years, 4 months ago)

:whathesaid:
I have read a lot about people failing with micropore tape.
But I also read that it works for some people.
I don't use it so I can not tell you any experience. Polyfill is a cheap way for GE holes.

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OfflineMushierage
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Re: Troubleshooting [Re: enlightenment]
    #23763709 - 10/23/16 09:59 AM (7 years, 4 months ago)

Probably already in your spawn.  Might only be showing itself on the top because the conditions are better for sporulating there.  I've had similar problems with trich, but I've only lost a couple jars so far this run, out of 20 or so.  .

What is your sterilization process and technique like?  What type of grain spawn are you using?


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Re: Troubleshooting [Re: Mushierage]
    #23763719 - 10/23/16 10:03 AM (7 years, 4 months ago)

You'd most likely see the contaminate throughout the jar if it were the grains. If it were your inoculate, you'd see the contam close to the agar wedge. If it's the filter failing, you see the contam under the filter--aka the top of the grains in the jar.
:themoreyouknow:

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OfflineTira
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Re: Troubleshooting [Re: Mushierage]
    #23764130 - 10/23/16 12:19 PM (7 years, 4 months ago)

Quote:

dankington said:
You'd most likely see the contaminate throughout the jar if it were the grains. If it were your inoculate, you'd see the contam close to the agar wedge. If it's the filter failing, you see the contam under the filter--aka the top of the grains in the jar.
:themoreyouknow:




That's what I think, but they're not right under the filter and failing with sterile technique would also cause the top of the grains to contaminate, as that would be the area exposed when I removed the lid of the jar to drop the agar wedge.

Quote:

Mushierage said:
Probably already in your spawn.  Might only be showing itself on the top because the conditions are better for sporulating there.  I've had similar problems with trich, but I've only lost a couple jars so far this run, out of 20 or so.  .

What is your sterilization process and technique like?  What type of grain spawn are you using?




I PC for 100 minutes and then let the PC cool down itself before I remove the jars.  I do not remove the foil until I get the jars in the SAB.  I did 8 WBS,  8 wheat and 8 corn jars. I inoculated  WBS jars first, and then 2 days later I inoculated wheat and corn jars.  Since WBS ones only had 1 jar contaminated, and my SAB's walls got a little dry during the inoculation of corn and wheat jars (and from those I had 8 jars contammed, 50% fail rate), I think it might be my sterile technique (maybe the SAB's walls drying?) and not the filters.

I'm positive that it's only on the top because the jars are not colonized yet.  I can see the area where I dropped the agar wedge colonizing nicely, and trich is sporulating in a seperate area.


btw I had a hard time making the agar wedge fall off from the scalpel; maybe that and SAB having dry walls together were a good chance for contaminants to get in?


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Offlinetryptkaloids
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Re: Troubleshooting [Re: Tira]
    #23764167 - 10/23/16 12:33 PM (7 years, 4 months ago)

probably a mix between sterile technique and filter fail. did you debur the holes before you put the tape on? could you have punctured the tape when you put it on? usually when you think it's your sterile technique it probably is.


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OfflineMushierage
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Re: Troubleshooting [Re: tryptkaloids]
    #23764219 - 10/23/16 12:56 PM (7 years, 4 months ago)

8 jars contaminating to me seems like it's the spawn itself but I could be wrong.    I personally don't think it's your SAB, I've been doing work in a completely dry SAB for months and only have a 10% contamination rate.  That's honestly still higher than I'd like, so I may start misting the SAB, and start getting my sterile shit together like using sfd holes instead of poly, using alcohol on gloves and Lysol on work surfaces, and allowing time to air to settle down before I start working.


--------------------
Don't like researching posts? Read this! . Also, if you're new and your posts contain the words: Humidifer, incubator, air-stone, or heater, then you need to read and UTFSE before asking people to review your setup.  OR... You should be cultivating reptiles and fish, not mushrooms.


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InvisibleComebackKid
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Re: Troubleshooting [Re: Mushierage]
    #23764228 - 10/23/16 01:00 PM (7 years, 4 months ago)

I use double microporous tape with success...
I also secure a coffee filter atop my lids with an elastic band and let my jars colonize in a SAB

Call me paranoid but it works so :shrug:

Also corn sucks


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Invisibledesr
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Re: Troubleshooting [Re: ComebackKid]
    #23764244 - 10/23/16 01:06 PM (7 years, 4 months ago)

yeah totally filter fail. what may work for one, may not work for another, and you shouldnt push cut-corner methods.  micropore tape has "pore sizes" way bigger than tyvek or a sfd...like trying to catch mosquitoes with a volleyball net.

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OfflineTira
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Re: Troubleshooting [Re: tryptkaloids]
    #23764257 - 10/23/16 01:12 PM (7 years, 4 months ago)

Quote:

Mushierage said:
8 jars contaminating to me seems like it's the spawn itself but I could be wrong.




You mean the agar?  But wouldn't I see the contaminants sporulating in the plate too?  I still have the plate and it looks nice and clean.

Quote:

ComebackKid said:
I also secure a coffee filter atop my lids with an elastic band...


Also corn sucks




That sounds like a good idea, I'll try that.  Extra protection couldn't hurt.

I've also read that corn sucks but wanted to give it a try myself .)

Quote:

tryptkaloids said:
probably a mix between sterile technique and filter fail. did you debur the holes before you put the tape on? could you have punctured the tape when you put it on?




No I make sure the are around the holes is nice and flat using a hammer.


--------------------
Useful Links for Beginners

The Basics              AMU Teks

Frank''s Teks          Agar

Noob Forum Reccomended Teks

Agar for guaranteed spawn,
Proper pasteurization for guaranteed substrate.

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Invisiblecrackbaby
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Re: Troubleshooting [Re: Tira]
    #23764295 - 10/23/16 01:32 PM (7 years, 4 months ago)

i would say polyfill stuffed tightly into 1/2 inch holes drilled into center of the lid is the way to go...

Also, if you live in an area where it's still warm, instead of throwing out the bad jars you can find a shady spot in the backyard and bury em (2-3 inches deep, with some added moist vermiculite).  I've done this with jars that were totally fucked up and gotten nice flushes (which surprised the shit out of me at first :smile:)


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Offlineomgwtfstfu
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Re: Troubleshooting [Re: Tira]
    #23764316 - 10/23/16 01:45 PM (7 years, 4 months ago)

Betting it's a filter issue. SFD, tyvek or very tight polyfill would be best for grains.

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OfflineTira
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Re: Troubleshooting [Re: omgwtfstfu]
    #23764328 - 10/23/16 01:49 PM (7 years, 4 months ago)

It's hard to find tyvek where I live.  You think it would be ok to buy and cut some tyvek sleeves and use them as filters?


--------------------
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The Basics              AMU Teks

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Agar for guaranteed spawn,
Proper pasteurization for guaranteed substrate.

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Offlinemrmazdarx9
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Re: Troubleshooting [Re: Tira]
    #23764552 - 10/23/16 03:32 PM (7 years, 4 months ago)

The overall type yeah good way to go.


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Invisibledankington
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Re: Troubleshooting [Re: mrmazdarx9]
    #23764627 - 10/23/16 04:01 PM (7 years, 4 months ago)

coffee filters do nothing, CBK.

But try and find SFDs if you can. I have seen someone use E-Z felt with success too.



That dude is legit, so though I haven't tried it, I don't doubt it works.

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Offlinecr1s1s
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Re: Troubleshooting [Re: dankington]
    #23764641 - 10/23/16 04:05 PM (7 years, 4 months ago)

I would try to only put your micropore tape on the outside of the jar lid. If the tape on the inside is getting wet that could also be your problem. Or I would just move to SFDs.

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