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bacteria from agar?


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#21 Saphroziac

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Posted 30 January 2021 - 06:58 PM

The flowhood is 1' height and 2' width. A quart jar doesn't reach above it just sitting on a table, a riser is like 6" in height or less, and the agar can sit on that. I need to do a smoke test of my own to see if I can get A2G done.


Edited by Saphroziac, 30 January 2021 - 06:58 PM.


#22 Saphroziac

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Posted 30 January 2021 - 09:14 PM

Here are the grains immediately after 3 hours of pressure cooking. I opened the PC in front of the flowhood and tightened down the jar lids. Take a look at my new kitchen rack.

3.1.jpg 3.2.jpg 3.3.jpg

 

Here are some photos of the grains immediately after 3 hours of pressure cooking at 15+ PSI I shook them up to redistribute the moisture. Some grains look more saturated than others.

3.4.jpg 3.5.jpg 3.6.jpg 3.7.jpg

 

I did my own smoke test with a smudge stick. I see what you mean about the turbulence at the bottom.

3.8.jpg 3.9.jpg 3.91.jpg 3.92.jpg


Edited by Saphroziac, 30 January 2021 - 09:14 PM.

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#23 Saphroziac

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Posted 01 February 2021 - 02:18 AM

I numbered my agar plates, and put them in my newly PC'd jars. Lets see which ones contam! I will update on how they are doing in 1-2 weeks time.

Plate 1.jpg Plate 2.jpg Plate 4.jpg Plate 5.jpg Plate 6.jpg Plate 7.jpg

 

Here are some photos I took of my agar process.

Start.jpg Step.jpg Torch.jpg X.jpg x2.jpg Cut.jpg Done.jpg How to Open Jar.jpg

 

A few more plates.

How to Open Jar.jpg APE.jpg Gallindoi.jpg GT Fell to Side.jpg Malabar 1.jpg Malabar 2.jpg Pan Cyan Aus.jpg


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#24 Saphroziac

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Posted 06 February 2021 - 05:32 PM

These have nothing to do with the above two posts, and are a completely different batch.

These are some Stargazers that have been going for a little longer than a month. Please let me know if you see any signs of contaminants bacteria or otherwise. I tried to take their photo in the right lighting this time and capture the most active face of the jar.

SG1.jpg SG2.jpg SG3.jpg SG4.jpg SG5.jpg SG6.jpg SG7.jpg SG8.jpg

 

I have a few jars that seem like they've stopped growing, or are going extremely slowly. Are there any reasons that the mycelium would have just stopped growing?  I shook one jar that seemed like it was growing well and gave it a week to recover and upon returning to it, the mycelium seemed as if it was not growing, or reaching out at all. It seemed like it was dead.

Could it have been to dry? I have a small heater in there that turns on in a 4'x4' Gorilla Tent every 30 minutes to keep it around 65-75F. Could the heated air have dried out all the grains? 

Was there an invisible contaminant that had incapacitated any mycelial growth? Was it genetics?

Some people's jars are so white and fluffy, it makes me appreciate more what it takes to grow mushrooms.


Edited by Saphroziac, 06 February 2021 - 05:34 PM.

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#25 cujoloki

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Posted 06 February 2021 - 06:03 PM

 

Some people's jars are so white and fluffy, it makes me appreciate more what it takes to grow mushrooms.

I agree totally! I too have an appreciation for the myco masters here! Jar #1 looks like it is getting yellow yeast at the top? Not very familiar with oat or rye grains but looks iffy. Wouldnt want to open that in the lab though it maybe a risk not worth it. 



#26 Saphroziac

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Posted 06 February 2021 - 06:11 PM

Here are some Agar plates I found questionable. 

In Agar 1 all my B+ plates look like that....is mycelium sometimes that thin and wispy?

Agar 1.jpg

In the rest of them, they are all the same strain PF Classic, what is that non-uniform part in all of them?

Agar 2.jpg Agar 3.jpg Agar 4.jpg Agar 5.jpg

 

 


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#27 sandman

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Posted 09 February 2021 - 04:23 PM

I suspect the non-uniform growth is nothing but how the piece was laid on the agar. A little extra schmear in a spot makes the outward growth look funny. 

 

I don't know exactly why the faint growth happens but I consider it dudded out. Sometimes old plates that looked like that will even have pins on them but I dont use them.


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#28 sandman

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Posted 09 February 2021 - 04:28 PM

You can get really clean circular growth with really clean circular transfers.

20200722_191033.jpg

IMG_0083.jpg

Not a particularly impressive agar plate just one I had around.  But the little tool I made from a piece of 1/4" stainless tube I ordered on ebay. One end does the punch and one end is cut to a scalpel type end to transfer.


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#29 cujoloki

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Posted 09 February 2021 - 08:18 PM

Thats cool!



#30 Microbe

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Posted 10 February 2021 - 06:22 AM

@JRH

Sorry, they're not very close up photos. The grain is Rye Berries.

How can these jars sit there for a month or two and have nothing show up and then right when I put an agar plate in they get bacteria and other random contams. :(
I am transferring them with gloves, shit loads of alcohol 70% and even Hydrogen Peroxide on the outside of the jar, face mask, torch sterilized scalpel, cut it and transfer it as fast as possible, open the jars 1-3 times to get the wedges in in front of a flippin flowhood.

I didn't shake them this time because I wanted to see if bacteria would form where I put the agar plate, and not in some random spot in the jar....I wanted to try and confirm it was coming from the plate.

Have you conducted a plate test on your flow hood? Take two plates that have been incubated after pouring and are determined to be clean, take one plate and lay flat on your working surface and remove the cover and wait a few seconds and then replace the cover. Take the second one and after removing the cover, turn the plate on its side so that the agar surface is facing the filter and after a few seconds place the cover back on. Wrap in parafilm or whatever you use to protect your plates and label them as filter face and working surface and incubate for a week.

Note you can increase the amount of plates you use to test so you so you can get filter face left, filter face center, and etc.

If you determine that the flow hood is clean, then you need to start looking at your cultures and or technique. Always incubate your plates for a week until the issue is resolved. Mycelium can easily colonize a plate before the bacteria becomes visible but if its there its there even if the plate looks good.

As far as sterilizing grains, i do quarts at 15 psi for 1 hour and without issue. So if your running at least an hour and at 15 psi, then your grains are being sterilized.
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#31 Saphroziac

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Posted 17 February 2021 - 10:52 PM

It turned out that of the 7 plates, 2 contaminated.  1,2,5,6,7 seem all to be good. Maybe a little on the slow side because they were transferred 2 weeks ago, but one of them seems to be going pretty strong. I don't see any wet spots anywhere.

 

Jar 1

1.jpg

 

Jar 2 

SG2.jpg

 

3-4 Contaminated with bacteria so I assume it was from the bad plates that I  previously worked directly on the table in front of turbulence.

 

Jar 5

gt5.jpg

 

Jar 6

GT 6.jpg

 

Jar 7

7.jpg

 

and here are some near fully colonized Stargazer jars that I put into bulk sub today, I shook them up a couple weeks ago because they seemed like they were growing strong. I looked at them with a light and it didnt seem like there was any mold.

Colo 1.jpg Colo 2.jpg Colo 3.jpg

 

A couple notes. I did some BRF to agar transfers the other day and it seems like BRF transfers are more likely to contaminate. I also did a grain transfer to agar from a GWM jar and all 3 plates got super gross bacteria. Which is weird because GWM Quarts looked like they were going fine, then I shook one  up and it seems like it just stopped growing.  Let me know what you think of my jars!


Edited by Saphroziac, 17 February 2021 - 10:56 PM.


#32 FunG

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Posted 18 February 2021 - 06:44 AM

Hey saph.

Sorry things are going so buggy for you.

Its extrodinarly hard to say where things are going wrong at this point. Your plates all look good, your technique is good as well but that leaves it to the flowhood.

Try doing some A2G transfers in a glovebox to try a alternative approach to transfers is the only recommendation I can offer.
I'm pretty stumped

And the jars you over killed (lol) they look normal, rye always has a few kernels that look wet after pc'ing.

Hopefully someone more experienced with the methods your using can point out where the flaw is since I'm strictly spores to grain.




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