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InvisibleInfiniteDreams
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Re: Is this mold in a plate? [Re: footpath]
    #26518546 - 03/05/20 12:13 PM (3 years, 11 months ago)



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Offlinefootpath
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Re: Is this mold in a plate? [Re: InfiniteDreams]
    #26518589 - 03/05/20 12:39 PM (3 years, 11 months ago)

So this bodhiprotista guy... he like some kina Jizo or summin?
Some damn fine publications he made in back in '97. Weird that he submits to pseudonymity when he's got seasoned credentials like that.... each their own, I guess.



I'm just gonna edit in, let's see if you bite.
Quote:

InfiniteDreams said:
Quote:

InfiniteDreams said:
Jar looks way way too wet.  What is your technique there?






So cool. Let's start this dumb shit over again so I can go on to even agree that,
Quote:

footpath said:
Being wet doesn't cause contamination. Why it's wet might be the cause, however.




And then elaborate that,
Quote:

footpath said:
It was introduced somewhere. Either before sterilizing and you didn't sterilize properly. Or after sterilizing and your equipment or technique was improper... which could easily be from the original culture.
The plate is way past the point of being able to obviously identify any contaminant cultures, but it does look fine... for whatever that's worth... which is not much.
Those tiny white dots are primordium. Which will become pins. That still doesn't really give an insight as to whether or not there's a contaminant present.







Anyway, OP. Sorry to get hooked into participating in some substanceless bickering and send this thread wayward.

You'll want to sure up your technique.
If it's just the one jar, mishaps happen. If it's many, you may want to look into the integrity of your filters or lids. Because your grains are getting and staying wetter after your PC cycle, it's safe to say that there is some amount of compromise there. Whether that's for sure the catalyst to your contamination..? Couldn't say without a lot more detail, including an earlier picture of the culture that you put to those grains.</font>


Edited by footpath (03/05/20 01:02 PM)


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InvisibleInfiniteDreams
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Re: Is this mold in a plate? [Re: InfiniteDreams]
    #26518598 - 03/05/20 12:45 PM (3 years, 11 months ago)

Quote:

InfiniteDreams said:
Jar looks way way too wet.  What is your technique there?




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OfflineDr3
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Re: Is this mold in a plate? [Re: InfiniteDreams]
    #26518703 - 03/05/20 01:28 PM (3 years, 11 months ago)

Thank you for your answers. It's been very helpful. Indeed, I am capable of reading and following instructions. I'd argue my sterile technique is generally OK, since I produced those plates by cleaning MS syringues, or by picking single grains from such growth.

    I have been able to run two succesful tubes in the past, however grains were always on the wet side and full colonisation was indeed never achieved, resulting in tubes that run for 1 and 2 flushes respectively, yield being very poor, around 20/30% of the average yield you might expect.

  So the issue could very well be that grains are too wet. I only have PP5 containers, and in the past I did not unscrew them slightly before the cycle. Moreover, I can not keep them vertical inside the PC, I have to topple them to a semi-horizontal position.

Jars seem to be OK before the PC cycle, I just follow a normal TEK, throw oats while water is boiling and soak for 40 min.

My pc cycle is usually 2 hours + 10 mins, which may be overkill for plastic jars.

All in all, I reckon I have to either shorten the prep timer, shorten the PC timer, buy glass jars and/or a bigger PCooker :smile:

But it's good to spot more or less the problem

Thanks again


Edited by Dr3 (03/05/20 01:54 PM)


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InvisibleInfiniteDreams
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Re: Is this mold in a plate? [Re: Dr3]
    #26518731 - 03/05/20 01:41 PM (3 years, 11 months ago)

It's all good footpath, I'm not eating what you're cooking.

Good to hear back Dr3, I think your plates speak for themselves that you know what you are doing there.  Some things to shore up though in your grain prep.  Bod has an excellent guide on prepping oats if you want all the details.

Quote:

Jars seem to be OK before the PC cycle, I just follow a normal TEK, throw them while water is boiling and soak for 40 min.




A key step to the grain prep tek is drying the grains after cooking.  Letting them steam off can be sufficient.  A good test is to use tissue paper.  Properly dried grains won't leave behind much moisture. 

Quote:

My pc cycle is usually 2 hours + 10 mins, which may be overkill for plastic jars.




Not overkill, that is the time you need and is to sterilize the grains.  2 hours at pressure.  I assume the 10 min is the venting, but you need to bring it up to pressure after that and then start the clock once you are at pressure.

Quote:

All in all, I reckon I have to either shorten the prep timer, shorten the PC timer, buy glass jars and/or a bigger PCooker :smile:




Do not shorten the timer.  Glass jars are preferred and who doesn't love a bigger PC!


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Offlinefootpath
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Re: Is this mold in a plate? [Re: Dr3]
    #26519004 - 03/05/20 04:06 PM (3 years, 11 months ago)

The only thing your wet grains are doing is preventing your mushrooms from growing optimally.

Your grain preparation is fine, except, as noted, if you're not drying off the grains after cooking.

Many people have found success with pp5 containers - most no-pour agar is done in pp5 containers, for example.

So, the question remains as to how you introduced mold to your jar.
It is not your grain preparation.
It is not the material your containers are made of.

It may still be your sterilization process - what PSI does this pressure cooker reach?
It may still be your lids or filters - fill them with water, screw the lids tight, flip them upside down, see if they leak. Also, are they filtered?
It may still be your culture - we can't tell from your pictures how clean your culture is. If you're cleaning from MSS or grain tissue transfers, you could easily be carrying contaminants around.
It may still be your sterile technique and/or handwork - this is just a matter of practice. Work in still air or laminar flow. Sterilize any tools before contacting a nutritious media or anything else that will touch a nutritious media. Never let your hands pass over any nutritious media or anything else that will touch a nutritious media.


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OfflineDr3
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Re: Is this mold in a plate? [Re: footpath]
    #26519293 - 03/05/20 06:29 PM (3 years, 11 months ago)

Quote:

footpath said:
The only thing your wet grains are doing is preventing your mushrooms from growing optimally.

Your grain preparation is fine, except, as noted, if you're not drying off the grains after cooking.

Many people have found success with pp5 containers - most no-pour agar is done in pp5 containers, for example.

So, the question remains as to how you introduced mold to your jar.
It is not your grain preparation.
It is not the material your containers are made of.

It may still be your sterilization process - what PSI does this pressure cooker reach?
It may still be your lids or filters - fill them with water, screw the lids tight, flip them upside down, see if they leak. Also, are they filtered?
It may still be your culture - we can't tell from your pictures how clean your culture is. If you're cleaning from MSS or grain tissue transfers, you could easily be carrying contaminants around.
It may still be your sterile technique and/or handwork - this is just a matter of practice. Work in still air or laminar flow. Sterilize any tools before contacting a nutritious media or anything else that will touch a nutritious media. Never let your hands pass over any nutritious media or anything else that will touch a nutritious media.




Very informative, I'll check the lids. I use polyfill rigidly stuffed for the lids. I do not know at which PSI the PC works, it is an old one and the manual is nowhere to be found. I'd gladly buy a Presto but they are not available in my country and any PC's big enough for our purposesare really, really expensive. Say 500$ or 600$ expensive.

Indeed, I could be carrying some contaminants around. The water would then exacerbate the issue if is bacterial, if I understand correctly.

My sterile technique follows what you said mostly, I use 70% iso alcohol, torch, gloves, mask, etc. Yours is very good and succint advice anyway. I tend to struggle with spraying the SAB: the lids are still covered with foil from the PC cycle to shield lids from water droplets while spraying, but removing this foil afterwards inside the sab is awkward to say the least.

Something related:  I recently inoculated a couple of jars with a GT clone, but that original plate is now showing some growth which might be moldy. I know this shouldn't normally be done. I mostly wanted to transfer to a new plate but since I had two jars around and oats are really cheap where I leave I figured I would just go for it. I'll probably fail terribly, though. I will keep you posted. I include some pics of this project:



Blue: suspect mold
Green: Transferred to jar
Red: Transferred to plate # 2 below

2 pics, more detailed, the one from below signals the mold better I think:



plate #2, to which the small red piece was transferred



Please do not worry about the texture of the agar. It is potato flakes with honey, unfiltered. Working well thus far, since I don't have access to MLE.

Thanks.


Edited by Dr3 (03/05/20 06:40 PM)


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Offlinefootpath
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Re: Is this mold in a plate? [Re: Dr3]
    #26520241 - 03/06/20 09:03 AM (3 years, 11 months ago)

I may think that your pressure cooker could be an issue. If it does not reach 15 PSI, 2 hours will not be sufficient to sterilize and you will need to run it longer. It's okay for agar because liquids sterilize very quickly, but grains take a long time to reach a sterilizing internal temperature.
Do you have a picture of it or know the make/model?

You are correct that, if you carry along a bacterium, the wet conditions will give it better chances to outperform your mushroom mycelium.

Many people don't worry too much about spraying down their SAB. I, personally, only wipe down the floor, work surfaces, and any jars/plates going in it with 70% isopropyl before doing any work. I should note that my SAB is in a permanent place and the arm holes are covered when not in use. I will thoroughly clean it if I have to move it or if I work with a contaminant that sporulates.

You do have a good recognition of the different cultures on that plate.
What you circled in blue, I would certainly agree is a mold.
The other parts you circled, it's hard to say. It does look like mushroom mycelium is there - the only issue when it is grown out so far is that you will have a harder time knowing if you are transferring more than the intended culture.


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InvisibleInfiniteDreams
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Re: Is this mold in a plate? [Re: footpath]
    #26520262 - 03/06/20 09:20 AM (3 years, 11 months ago)

So the grain prep that definitely wasn't the issue might actually be the issue?


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Offlinefootpath
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Re: Is this mold in a plate? [Re: InfiniteDreams]
    #26520289 - 03/06/20 09:39 AM (3 years, 11 months ago)

Not at all.
It may be the sterilization process.
It may be the the lids or filters.
It may be the culture.
It now sounds unlikely to be the technique/handwork.

The (maybe) poor grain preparation process is simply providing conditions inhibitory of mycelium growth and supportive of contaminant growth that was either not eliminated by sterilization or was introduced by some other means.


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InvisibleInfiniteDreams
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Re: Is this mold in a plate? [Re: footpath]
    #26520348 - 03/06/20 10:08 AM (3 years, 11 months ago)

It may be that he angered Piltzintecuhtli.

Can you explain how sterilization of the grains in a PC is not part of the grain preparation process?

:wonka:


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Offlinefootpath
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Re: Is this mold in a plate? [Re: InfiniteDreams]
    #26520409 - 03/06/20 10:50 AM (3 years, 11 months ago)

Because it's part of the sterilization process.
As I said, the preparation is only to effect the conditions of the spawn substrate, meaning the environmental conditions. You're hydrating it to provide moisture and permeability.
The sterilization process is used to eliminate competition. It has nothing to do with how you prepared the spawn substrate.
If you prepare grains properly and sterilize improperly, you will likely get contamination.
If you prepare grains improperly and sterilize properly, you will likely not get contamination.
They're clearly two different processes by virtue of not being dependent on each other.

Once you've prepared a spawn substrate, you do not further change its properties. Rather, you eliminate an external variable.
To have an optimally viable spawn substrate, you must first prepare it in a way that provides adequate conditions; and then you must sterilize it to allow your culture exclusive access to it.

I highly recommend reading through some of those publications you linked to me earlier. That protista fella sure knows his stuff.
This is also a highly valuable resource if you need a little more clarification.


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Offlinejbgtaa
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Re: Is this mold in a plate? [Re: footpath]
    #26520444 - 03/06/20 11:15 AM (3 years, 11 months ago)

To put all this to rest I’d be willing to say that all that moisture, if it isn’t due to prep, could be from temp fluctuations due to two organisms eating and growing in the jar.

Op, you let the mold in when you opened the jar in the sab. Or the mold came in on the plate. If your filter material is fine, and the jar colonized before the mold showed up, I’d say that there was one too many mold spores floating around inside your SAB.

I will say you can load wet grain and it’ll colonize fine with the proper filter material, given you work with it in a sterile environment such as flow hood. Contaminated have to be introduced with anything you’ve PCd. The only exception is if you don’t know how to prorperly use a PC for sterilization, but even then that’s the user problem and learning curve.
Calm down everyone


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Offlinefootpath
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Re: Is this mold in a plate? [Re: jbgtaa]
    #26520456 - 03/06/20 11:24 AM (3 years, 11 months ago)

I've given OP that advice and more and am attempting to help them troubleshoot their actual problem.
I'm open to receiving these challenges to my comprehension - it helps to really draw out unnecessarily detailed explanations for unrelated processes that some may not understand.


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InvisibleInfiniteDreams
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Re: Is this mold in a plate? [Re: footpath]
    #26520492 - 03/06/20 11:53 AM (3 years, 11 months ago)

I find your way with words fascinating.  No accountability and lots of virtue signaling.  Might work better for OP to stick to trusted methods instead of distracting with your personal philosophy. 

Now you are making up your own definitions and distinctions, which adds to the confusion. 

Grain prep has always included sterilization.


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Offlinemimir
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Re: Is this mold in a plate? [Re: Dr3]
    #26520509 - 03/06/20 12:08 PM (3 years, 11 months ago)

I didn't read all the other answers, so maybe someone already said this. The plate looks fine to me, a bit overgrown compared to what I'm used to but not a problem imho. The jar doesn't look too good, but the white stuff I can see behind the condensation is probably mycelium, the green is obviously contamination but given that it's not over the whole jars I don't think the inoculant was the problem. Here's three pics from when I didn't know better, you can see the agar plate covered with a white mold that was then duly inoculated on grains before realising that's not how mycelium looks like :lol:


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OfflineDr3
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Re: Is this mold in a plate? [Re: InfiniteDreams]
    #26520534 - 03/06/20 12:20 PM (3 years, 11 months ago)

I find all the info in this post very useful, I  can now draw a clear picture of how to move forward.

pd: I tried for leaks on the lids and water does not get out, however the polyfil does drip water when it is soaked.Sadly, the jars go half horizontally inside the PC which definitely isn't good.

Moreover, the PC doesn't specify model, as I said, it is kinda old. I can certainly try a longer PC cycle, if needed, just to remove that variable next time. I'll do the paper check also before a PC cycle to try and address the issue of humidity inside the jar, just to remove the posibility that I am not drying and straining grains properly before going into the PC.


Cheers


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Offlinefootpath
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Re: Is this mold in a plate? [Re: Dr3]
    #26520552 - 03/06/20 12:34 PM (3 years, 11 months ago)

Cool. That minimizes the source of the problem a good bit.
We now have,
- pressure cooker PSI
- possible contaminated culture
- introducing something while transferring

Polyfil will not be totally waterproof - that's okay as far as contamination goes so long as they don't get wet.

In regard to your wet grain,
Does your foil dip into the water at all?
If it does, this can essentially create a straw that will draw water into your jars through the polyfil.


Edited by footpath (03/06/20 01:02 PM)


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InvisibleInfiniteDreams
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Re: Is this mold in a plate? [Re: Dr3]
    #26520696 - 03/06/20 02:08 PM (3 years, 11 months ago)

The PC sounds quite suspect, I advise investing in a new one if possible. Presto 23qt is good value if you can find one.

If you need to raise the jars use a trivet.  You can use balls of foil to raise the level of the trivet.

IF everything is working correctly with the PC the sterilization process should not effect the water content of the jars.


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OfflineDr3
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Re: Is this mold in a plate? [Re: InfiniteDreams]
    #26523950 - 03/08/20 01:26 PM (3 years, 11 months ago)

Yes, the foil is in contact with water. I will fix this the next time I prepare jars. I'll raise the trivet and decrease the amount of water.

It's going to be very difficult to find a Presto cooker in this part of Europe, sadly.

I'll also increase the PC cycle to 2 and a half hours next time, see how that goes.

I'll keep you posted the next time I prepare jars, I reckon it will be around week or so.


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