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OfflineVanboy267
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MS to agar mycelial growth?
    #26988004 - 10/16/20 09:03 AM (1 month, 12 days ago)

Hi everyone,

First ever time experimenting with MS syringe to agar using GT spores.

Before my question, wanted to give some background. Used sterile techniques to the best of my abilities, pressure cooker for agar and SAB for pouring. Also used, without prior experience, an overly nutritious agar recipe of 500mL distilled water, 10g agar, 10g LME and 1g nutritional yeast.

In one of the two plates pictured below, am I right or wrong in believing there is some form of fuzzy mycelial growth? If so, when and how should I start transferring to clean it out?

Any advice would be appreciated :laugh:

Plate 1






Plate 2






Edited by Vanboy267 (10/17/20 09:42 AM)


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OfflineArthurFungarelli
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Re: MS to agar mycelial growth? [Re: Vanboy267]
    #26988012 - 10/16/20 09:10 AM (1 month, 12 days ago)

Looks like mycellium with a cool looking contam.  In the first pic, I'd take something from the leading edge at about 4 o'clock and transfer that to a new plate, and maybe 3 o'clock from the last plate.  I will say it does look a bit different from how I'd expect mycellium to look, but I have seen that kind of clear edge around it before.  I think it'll look better once it's transferred away.  You could make those transfers now; I think you've got enough material that's far enough away from contaminants to make it worthwhile.


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OfflineVanboy267
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Re: MS to agar mycelial growth? [Re: ArthurFungarelli]
    #26988021 - 10/16/20 09:24 AM (1 month, 12 days ago)

Thanks for the advice Arthur :grin: I was really worried about opening up the agar plate if it was some form of precursor mold haha.

I read up somewhere in the forum that using overly nutritious agar possibly makes the mycelium growth fuzzy. Hence why for my next agar plates I'm only doing MEA with a slight reduction in LME.

I was thinking of cutting a rice grain sized bit of the recommended leading edge with a scalpel to transfer across. Would that be right to do?


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OfflineArthurFungarelli
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Re: MS to agar mycelial growth? [Re: Vanboy267]
    #26988028 - 10/16/20 09:32 AM (1 month, 12 days ago)

Any time friend!  There is definitely some sort of contaminant on there, so you'll want to be careful not to touch that.  I'm unsure about the overly nutritious agar, but it does ring a bell.  I just use the water from simmering my grains + agar for my plates.  It's worked alright so far, but I'm sure I'll want to branch out soon. 

Your technique sounds right; I usually cut a roughly 5mm tall triangular piece (minimize the amount of cuts) and transfer that.


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OfflineCocaineBuffet
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Re: MS to agar mycelial growth? [Re: ArthurFungarelli]
    #26989019 - 10/16/20 09:01 PM (1 month, 12 days ago)

I see nothing here except contamination.

How did you inoculate?


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OfflineVanboy267
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Re: MS to agar mycelial growth? [Re: CocaineBuffet]
    #26989278 - 10/17/20 12:52 AM (1 month, 11 days ago)

Hey CocaineBuffet, I used a spore syringe obtained from an unknown vendor. Unfortunately I've had difficulty finding fairly reputable sources for spores.

I flame sterilised, ejected a few drops to cool the tip then single drop into the middle of the plate. I did three plates like this and the other with an innoc loop with a Z streak (only bacterial contam on those three).

I had other blank agar plates closeby for the week of colonisation and those never had any contam, so I'm confident of my sterile techniques of pouring agar plates.

I was really hoping that I could do any form of cleaning from contam as I only have that one syringe :/


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OfflineArthurFungarelli
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Re: MS to agar mycelial growth? [Re: Vanboy267]
    #26989718 - 10/17/20 10:16 AM (1 month, 11 days ago)

I think you should take a couple transfers.  I could definitely be wrong, and maybe that's not just tomentose mycellium that I'm seeing, but the only thing you have to lose is time and a plate or two.


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OfflineVanboy267
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Re: MS to agar mycelial growth? [Re: ArthurFungarelli]
    #26990350 - 10/17/20 06:54 PM (1 month, 11 days ago)

Quote:

ArthurFungarelli said:
I think you should take a couple transfers.  I could definitely be wrong, and maybe that's not just tomentose mycellium that I'm seeing, but the only thing you have to lose is time and a plate or two.




Hey Arthur, that's exactly what I did. I transferred what you recommended, although I felt not too confident with my transfer technique haha. Always a good learning curve and challenge though!

I also tried a transfer of other areas I felt were decent mycelial growth so I can diversify my options. When you cut the small section Arthur, do you fully open the lid or do you angle it open so the plate is still covered?


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OfflineJohn in WI
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Re: MS to agar mycelial growth? [Re: Vanboy267]
    #26990392 - 10/17/20 07:23 PM (1 month, 11 days ago)

Just a few not so well informed opinions :-)

Pouring in a SAB might be better than trying it in open air--but I can't recall every seeing anyone do that?  I could be wrong but normally people pour in front of a laminar flow hood.  I'm not sure a SAB is good enough for that kind of work.  (If I'm wrong, someone please direct me towards a TEK.  I would LOVE to start working with actual Petris, vs. 1/2 pt jars).

It does look like the white material is mycellium.  As already stated, the strange greasy looking stuff is most certainly bacteria.  My experience with spores on agar is that the initial germination results if fluffy tomentose growth.  Apparently, with hundreds or perhaps thousands of spores germinating and fusing, you end up with a real genetic mess of compatible and incompatible strains.  I've seen it several times, where the first, second, or even third plate is fluffy, then some sectoring and rhyzomorphic growth is apparent.

First things first--select a small amount of agar from as far away from the bacteria as possible, and transfer it.  It might be folklore, but it seems like placing the transfer piece mycellium down on the new plate can favor fungal growth over bacteria.

I'm curious if your bacterial problem is from the spore syringe, or from the agar itself.  Do you have any agar plates that you have not opened?  When I do agar, I generally keep a few on the shelf for a week and examine them.  If they are contaminated, even through they were not inoculated, then you might have a problem with the agar sterilization, plates, or sterile air during the tranfer.

Anyway--you're on the right track.  Going spores to agar is really the best way to proceed.  Get nice clean mycellium, then make nice clean grain masters...

If you have any thoughts on the SAB agar pour, I'd love to hear more. I've never tried it, not saying it wouldn't work, but the idea makes me a little nervous.


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OfflineCocaineBuffet
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Re: MS to agar mycelial growth? [Re: John in WI]
    #26990493 - 10/17/20 08:48 PM (1 month, 11 days ago)



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Re: MS to agar mycelial growth? [Re: Vanboy267]
    #26990737 - 10/17/20 11:41 PM (1 month, 11 days ago)

Quote:

Vanboy267 said:

[...] do you fully open the lid or do you angle it open so the plate is still covered?




Here are two links that really helped me out. The videos are really good! I open the plate completely. Move deliberately, not in a fast or jerky motion, but do not be laissez faire either. Nothing that is not sterile over the plate!

Bod's Easy AF Video Series
BOD's Easy AF List

Good luck. I poured my first plates a month ago and everything went wrong. I just swabbed plate #250 and it is really feeling good. I still get some contams because of poor technique, but very few. It will become second nature once you do it a few times. Just keep pouring plates! Attached is one of my favorite contaminated plates so you do not feel alone!



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OfflineVanboy267
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Re: MS to agar mycelial growth? [Re: John in WI]
    #26990951 - 10/18/20 05:33 AM (1 month, 10 days ago)

Quote:

John in WI said:
Just a few not so well informed opinions :-)

Pouring in a SAB might be better than trying it in open air--but I can't recall every seeing anyone do that?  I could be wrong but normally people pour in front of a laminar flow hood.  I'm not sure a SAB is good enough for that kind of work.  (If I'm wrong, someone please direct me towards a TEK.  I would LOVE to start working with actual Petris, vs. 1/2 pt jars).






Bod's comprehensive agar tek is where I learned most of the agar tek as well as sterile techniques. In the previous post by Grenik, he has has some links to the videos I watched using the SAB. In a nutshell, SABs works via physics (i.e. any contaminants in the air settle on the bottom of the SAB) as it cannot be sterilised. So far the blank agar plates (going on 9 days) that I have poured and left besides my inoculated plates have shown no sign of contaminant growth.

Quote:

John in WI said:
Anyway--you're on the right track.  Going spores to agar is really the best way to proceed.  Get nice clean mycellium, then make nice clean grain masters...




Thanks John, that's the first hurdle that I'm going to have to tackle first before considering any grain masters. This hobby is indeed for those who are patient, but I'm so eager to see any growth everyday haha.


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OfflineVanboy267
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Re: MS to agar mycelial growth? [Re: CocaineBuffet]
    #26990966 - 10/18/20 05:51 AM (1 month, 10 days ago)

Quote:

CocaineBuffet said:
Were these plates inoculated in SAB using sterile technique? Did you flame sterilize the needle and inoculation loop? How did you make the plates (be as detailed as possible)






Hey CocaineBuffet, as for the sterile techniques and pouring of the agar plates I followed Bod's comprehensive agar tek as closely as I could. In terms of sanitisation before the inoculation of the plates, I turned on my air purifier in the room for an hour before agar work, wiped every surface down with 70% iso during the air purification. Turned off air purifier and sprayed room with Glen20 (contains 60% ethanol and the equivalent of Lysol), wiped down the outside and inside of the SAB with iso, wiped down the wire cooling rack and agar plates. Placed it all inside the SAB and had a shower. Wore nitrile gloves, sprayed my gloves and forearm with iso, and then sprayed inside of SAB again with iso and wiped it down. Sprayed a bit of soapy water on the walls and bottom of SAB. Left it for 30 minutes before I engaged in agar work.

Applying iso again over my gloves, I then proceeded to remove the parafilm from the agar plates and stacked them on top of each other. Flame sterilised the spore syringe till red hot, placed it in SAB and let a few drops off to cool the need tip, then lifted the bottom most agar plate only high enough to insert the syringe tip and placed one drop in the centre. For each plate I flame sterilised and did the same. Hopefully that's detailed enough from my recollection of that day.

Quote:

CocaineBuffet said:
What I would suggest if you are in the US is to PM me and buy these.




Thank you kindly for offering your assistance, unfortunately I'm located in Australia :/ I'll look into purchasing sterilised swabs after giving this current experiment of cleaning contam and practising with an innoc loop and scalpel first.


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OfflineFrrrunkis
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Re: MS to agar mycelial growth? [Re: Vanboy267]
    #26990972 - 10/18/20 06:04 AM (1 month, 10 days ago)

This might be obvious, but did you shake the syringe well before use? Basically, in my limited experience, you should get some of the black spore clumps in the liquid you use on agar.


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OfflineVanboy267
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Re: MS to agar mycelial growth? [Re: Grenik]
    #26990975 - 10/18/20 06:09 AM (1 month, 10 days ago)

Quote:

Grenik said:
Here are two links that really helped me out. The videos are really good! I open the plate completely. Move deliberately, not in a fast or jerky motion, but do not be laissez faire either. Nothing that is not sterile over the plate!

Bod's Easy AF Video Series
BOD's Easy AF List






Hey Grenik,

That's exactly where I'm learning most of my stuff for agar work as well! Glad to know that you also fully open the agar lid as I had difficulty just keeping it ajar when handling the scalpel.

Quote:

Grenik said:
Good luck. I poured my first plates a month ago and everything went wrong. I just swabbed plate #250 and it is really feeling good. I still get some contams because of poor technique, but very few. It will become second nature once you do it a few times. Just keep pouring plates! Attached is one of my favorite contaminated plates so you do not feel alone!






Really appreciate the encouragement Grenik. It's also nice to see pictures of a contaminated plate from someone in this hobby. Makes the end goal feel more feasible haha. I do feel that there could be a thread dedicated to people starting out from contaminated plates and showing their progress of cleaning it up (If there is, could someone point it out?). All I've seen mainly are contaminated jars and successful plates, so glad to know that I'm not alone :grin:


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Re: MS to agar mycelial growth? [Re: Vanboy267]
    #26991019 - 10/18/20 07:33 AM (1 month, 10 days ago)

I'll have to look into pouring in a SAB.  I use mine all the time for transfering, g2g, making liquid cultures...  It works great, it's just not commonly used for pouring plates.

In any case, germinating on agar is by far the best way.  It germinates everything in the syringe--mold, bacteria, or whatever else is in there.  But then it's not hard to transfer good growth away from shit.    It's much more likely to work than going spores to LC or spores to grain directly.

Keep us updated. It looks like that white fluffy material is mycellium.  Sometimes when I germinate spores, I come down with a jar full of cobweb mold. But that doesn't look like cobweb.


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Re: MS to agar mycelial growth? [Re: John in WI]
    #26991844 - 10/18/20 08:41 PM (1 month, 10 days ago)

Quote:

John in WI said:
I'll have to look into pouring in a SAB.  I use mine all the time for transfering, g2g, making liquid cultures...  It works great, it's just not commonly used for pouring plates.




??? It is totally used for pouring agar. I mean if it isn't in the SAB and not in front of a flowhood its being done in the wrong place (unless you are pouring into PP5/glass petris and then PCing). I have only poured agar in my SAB.

Vanboy, it sounds like your sterile procedure was done correctly (a little excessive with the air purifier IMO). Hmmm well give it another shot, a lot of learning comes from error. Look into inoculation loops to spread the drop of spore solution. You also literally only need 1 drop.


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OfflineVanboy267
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Re: MS to agar mycelial growth? [Re: John in WI]
    #26992259 - 10/19/20 06:08 AM (1 month, 9 days ago)

Quote:

John in WI said:
Keep us updated. It looks like that white fluffy material is mycellium.  Sometimes when I germinate spores, I come down with a jar full of cobweb mold. But that doesn't look like cobweb.




Will do John, I'm trying to keep a log via the Journal part of my account. Also just bought a cheap digital microscope so I can keep tabs on the growth, but also seeing things on the microscopic side of things is quite pretty.


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OfflineVanboy267
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Re: MS to agar mycelial growth? [Re: CocaineBuffet]
    #26992261 - 10/19/20 06:12 AM (1 month, 9 days ago)

Quote:

CocaineBuffet said:
Vanboy, it sounds like your sterile procedure was done correctly (a little excessive with the air purifier IMO). Hmmm well give it another shot, a lot of learning comes from error. Look into inoculation loops to spread the drop of spore solution. You also literally only need 1 drop.




Yeah, since I already have an air purifier, I'd thought it best to reduce whatever airborne contaminants there might be haha. Whatever I can do to ensure I have the best opportunity at my sterile procedures I'll employ, but yeah, it's not that time efficient on my part.

I've definitely only used 1 drop for my inoculations of the spore syringe. Learnt that the hard way with my first attempt and used 1 cc hahaha, bacterial contamination galore.


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