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Offlinedoomy
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Registered: 04/29/20
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Help with spotty mold growth in grain jars
    #27020624 - 11/04/20 02:01 AM (3 years, 2 months ago)

Hello everyone,

Maybe you can help me with a conamination problem.

After many years i have started growing again. I used 2 different spore prints and streaked them on agar. This went very well and without signs of contamination. I then cut some slices and tranferred them to sterilized rye grain jars (80min @20psi).

First it looked good and the PC mycelium started growing. When i checked the jars after ~10 days i noticed a green spot in one jar. In the following 5-8 days more and more showed up and i had to throw out almost 90% of the jars. The mold starts showing on random spots but not around the agar(i did not shake the jars).




My first thought was insufficient sterilisation. I made another batch and this time i did ~3h @20psi. Now today i again found moldy spots in 3 of the 20 jars. Again they show up randomly and its different kinds of molds (mostly light and dark green/blue). There must be a problem somewhere in my sterile technique.

What i dont understand is that the mold shows up at random spots (not on top and not around the agar) and only after 10 or more days. So it must come from the grain or am i wrong?


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InvisibleBaba Yaga
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Re: Help with spotty mold growth in grain jars [Re: doomy]
    #27020660 - 11/04/20 03:08 AM (3 years, 2 months ago)

Mold dies way below boiling temp. What are your lids/filters like and do you take out the jars when they are still hot or do you let cool over night in you PC?

And you said you haven't shaken after inoculation but did you shake before to redistribute moisture?

Could be your technique as you said but it looks like the mold is starting to grow in the lower parts which is strange:shrug:


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Offlinedoomy
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Re: Help with spotty mold growth in grain jars [Re: Baba Yaga]
    #27020683 - 11/04/20 03:49 AM (3 years, 2 months ago)

Thank you Baba Yaga,

I use polyfill, turn it into a plug and then pull it trough the hole in the lid. Its in there tight and i have naver had any problems with it.

For the first batch i also covered every lid in aluminium foil but then read its not necessary so i kept the jars bare for the second batch.

I leave the jars to cool overnight in the pressure cooker. I shook some of the jar to redistribute moisture but not all. I also shook none of the jars from the first batch.

Its really strange that the mold growth starts in random spots and only after some time(~10days).



This arre the fully colonised jats from the first batch:



Almost fully colonised small jars from the second batch(i shook them after adding the agar):






Jar from the second batch that will probably start grow mold soon (there are some tiny spots of white growing, its hard to see):





Edited by doomy (11/04/20 03:58 AM)


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OfflineSingularFusion
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Re: Help with spotty mold growth in grain jars [Re: Baba Yaga]
    #27020686 - 11/04/20 03:51 AM (3 years, 2 months ago)

have you tried a different bag/brand of grain doomy?

I haven't had it happen to me personally yet, but there is apparently a such thing as nasty bags of grain that just contam no matter what you do

you read some horror stories, even from experienced growers

if you are sure on the rest of your sterilization and such, it couldn't hurt to use a different source for your grain and rule out another possible cause

hope you get it figured out, to my eye the grain looks prepped nicely otherwise


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Offlinedoomy
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Re: Help with spotty mold growth in grain jars [Re: SingularFusion]
    #27020693 - 11/04/20 04:02 AM (3 years, 2 months ago)

Hi Nef,

Thank you, yes thats what im going to try next. The rye berries i used are sold by the local mill for baking bread. I will try oats for horses next, its also a lot cheaper.


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InvisibleRoger Clemency
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Re: Help with spotty mold growth in grain jars [Re: doomy] * 1
    #27020809 - 11/04/20 06:13 AM (3 years, 2 months ago)

If you havenโ€™t, make sure you vent your PC for 5-10 minutes before putting the weight on and going up to pressure. Like someone said above mold does die at lower temps than the other stuff weโ€™re killing but weird stuff can happen in an un-vented pc.

Iโ€™ve never liked polyfil as a filter but if youโ€™re using it make sure itโ€™s a tight as you can get it and you should definitely keep foil on in the PC. With sfd you donโ€™t need foil but I still prefer it anyway.

And just as a tip you should transfer at least twice from a germ plate before considering the culture clean. I have used T1 and even germ plates to nocc some jars but always just as an impatient inoculation of a few qts. I would continue to transfer the culture at the same time to make sure.


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InvisibleFriedEggS
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Re: Help with spotty mold growth in grain jars [Re: Roger Clemency]
    #27021505 - 11/04/20 01:30 PM (3 years, 2 months ago)

try a few control jars. sterilize them and then sit them on a shelf without ever opening them. see if they contaminate. if they do, your problem is probably with your filters.


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Offlinedoomy
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Re: Help with spotty mold growth in grain jars [Re: FriedEgg]
    #27034412 - 11/12/20 01:17 AM (3 years, 2 months ago)

I now also had to throw away the last 2 glasses of the first batch (now 100% fail rate).

They had spotty white mycelium growing in them that looked very similar to PC mycel.

Only after a few weeks they started to look like this.










Here is one glass next to a contaminated one. They both have the PC mycel growing from the agar wedge.





Some more pics, it is really hard to spot without the densly growing PC mycel next to it.







Edited by doomy (11/12/20 01:19 AM)


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OfflinekunzyMAGI24
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Re: Help with spotty mold growth in grain jars [Re: doomy]
    #27034507 - 11/12/20 04:38 AM (3 years, 2 months ago)

Let it cool in the pc over night


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OfflinePBJ710
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Re: Help with spotty mold growth in grain jars [Re: FriedEgg] * 1
    #27034671 - 11/12/20 07:19 AM (3 years, 2 months ago)

Quote:

doomy said:
I used 2 different spore prints and streaked them on agar. This went very well and without signs of contamination. I then cut some slices and tranferred them to sterilized rye grain jars (80min @20psi).




I think Roger Clemency nailed it.  You need to clean up your culture by making multiple transfers from the leading growth edge before going to grain.  It's easy for other contaminations to hide on germination plates under the random mycelium growth points then show up later.  Do you have any pictures of the petris used?  Is your scalpel possibly transferring something to the jar during the A2G process?

Quote:

FriedEgg said:
try a few control jars. sterilize them and then sit them on a shelf without ever opening them. see if they contaminate. if they do, your problem is probably with your filters.




Another great idea to help isolate where the contamination is coming from.


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InvisibleBaba Yaga
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Re: Help with spotty mold growth in grain jars [Re: PBJ710]
    #27035162 - 11/12/20 12:18 PM (3 years, 2 months ago)

This strange problem of yours might be caused by mites that are coming in through your filters or with the agar wedge.

Can you post pictures of your agar plates and lid filters.

I also agree with PBJ and Roger, your agar culture is not clean.


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Offlinedoomy
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Re: Help with spotty mold growth in grain jars [Re: PBJ710]
    #27036226 - 11/13/20 01:11 AM (3 years, 2 months ago)

Thank you guys. You are right, its probably my petri dishes. I just dont understand why at the beginning the PC mycel grows normally from the agar wedge and only later mold shows up at random spots.

So far only ~40% of the second batch was contaminated but mycel growth slowed down considerably. I have now shaken the remaining jars to speed up colonisation.

I will do a 3rd Batch with all your recommendations soon and post results.

Here are some pics of fresh petri dishes i made from the one i used for batch 2. At first glance they look ok but im now worried that the fluffy white stuff is this mean doppelgaenger of the PC mycel.

The last one looks different because there is some bacteria or yeast contam around it.













Edited by doomy (11/13/20 01:26 AM)


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Offlineroarkell
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Re: Help with spotty mold growth in grain jars [Re: doomy]
    #27036417 - 11/13/20 05:47 AM (3 years, 2 months ago)

I had a similar issue a few month back... I went wayyy overkill on pc cycles, went insanely slow with my technique (even in front of a hood) and was still getting 75% contams... for me, it was my lid setup... I was only using a single layer of micropore tape... I tried using multiple layers with a bit more success but stumbled upon this little fix...

The lids of my 100mm petris fits perfectly on the plastic wide mouth lids I use... its also another way to document whats going where and all that.

I see you're using single piece lids so this may not work in your situation, but they general idea should help if you can adapt it for your setup.

Hope you get thru it!


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OfflineZsuzsi
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Re: Help with spotty mold growth in grain jars [Re: roarkell]
    #27036422 - 11/13/20 05:53 AM (3 years, 2 months ago)

Quote:

I was only using a single layer of micropore tape...




I'm a beginner but I've done lots of reading here and I believe micropore tape should never be used for GE on grain jars. Look at this thread - someone here took a microscope to it and it actually lets lots of stuff go through. One thing I'm idly wondering since we're in the middle of a pandemic is whether or not cut up surgical masks could actually be used on lids - it is supposed to stop foreign contaminants after all.

Quote:

The lids of my 100mm petris fits perfectly on the plastic wide mouth lids I use




This looks neat ! does it work ? I'd fear the lack of enough GE if they fit too tight.


Edited by Zsuzsi (11/13/20 05:57 AM)


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Offlineroarkell
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Re: Help with spotty mold growth in grain jars [Re: Zsuzsi]
    #27036465 - 11/13/20 06:49 AM (3 years, 2 months ago)

They fit perfectly... ive been doing this set up for the last few months with close to zero contams... I know MP tape isn't ideal but its the cheapest and easiest... :shrug:
In my attempts to correct all my contam issues I started doing one layer before pc and another after because I could literally see it break down after a pc cycle... that helped but did not end my contam woes... the petri lids was a happy accident that is my new standard. I also write all the details on it cuz its just gonna get shit canned when I'm done anyway... no more wiping sharpie off jars!


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OfflineZsuzsi
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Re: Help with spotty mold growth in grain jars [Re: roarkell]
    #27036509 - 11/13/20 07:37 AM (3 years, 2 months ago)

Well that's pretty cool, it looks awesome on the jars.

Meanwhile I've actually found an answer to my musing about using surgical masks: RogerRabbit says no, not enough filtration for such a purpose. Even a N95 (which would turn out more expensive than, say, polyfill anyway) wouldn't do, apparently.


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Offlineroarkell
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Re: Help with spotty mold growth in grain jars [Re: Zsuzsi]
    #27036531 - 11/13/20 07:51 AM (3 years, 2 months ago)

You also gotta check dates on stuff... that thread is over 8 yrs old... not saying it isn't still true, just a lot of folks take RR's every word as that of God himself... dude gives great advice, just some things have been updated since those posts were made.


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OfflineZsuzsi
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Re: Help with spotty mold growth in grain jars [Re: roarkell]
    #27036548 - 11/13/20 07:59 AM (3 years, 2 months ago)

Ah, good point. And one of the most frustrating things on this forum: I've read great description of ideas or approaches before realizing I'm reading a 17-year old thread and some of that stuff might've been invalidated by now. Thanks !


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OfflineCamera93
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Re: Help with spotty mold growth in grain jars [Re: Zsuzsi]
    #27036556 - 11/13/20 08:08 AM (3 years, 2 months ago)

Quote:

Zsuzsi said:
Ah, good point. And one of the most frustrating things on this forum: I've read great description of ideas or approaches before realizing I'm reading a 17-year old thread and some of that stuff might've been invalidated by now. Thanks !




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Offlinethegreatecb
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Re: Help with spotty mold growth in grain jars [Re: doomy]
    #27036663 - 11/13/20 09:18 AM (3 years, 2 months ago)

Quote:

doomy said:
I now also had to throw away the last 2 glasses of the first batch (now 100% fail rate).

They had spotty white mycelium growing in them that looked very similar to PC mycel.

Only after a few weeks they started to look like this.










Here is one glass next to a contaminated one. They both have the PC mycel growing from the agar wedge.





Some more pics, it is really hard to spot without the densly growing PC mycel next to it.











:tryingnottodie:


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