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OfflineCliftonGK1
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Problems with syringe straight to agar
    #21709185 - 05/22/15 05:24 AM (8 years, 8 months ago)

Pretty confused about this one.

I've had really good luck starting BRF cakes from syringes.  I've even started rye berry jars right from syringes.  But every time I've tried to start syringes direct to PDA plates I run into one of two issues:  I either end up with contaminants spawning up (likely an artifact from poor technique when creating the syringe) or a clean plate that sits for weeks and nothing germinates.  I can see the outline on the agar where the inoculum sat on the plate.  I can see the heavy spore load sitting there; but nothing comes of it.

I've found myself having to take an additional step of fruiting a BRF cake and then cloning out tissue from a good looking sample when I want to start a series of agar plates to propagate and isolate.

Anyone else have issues like this when trying to start from syringe direct to agar?

For the record, I pour my own PDA plates from a purchased mix, allowing plates to set/cool for 6 hours before use, and double-wrapping edges with Parafilm to keep from drying after inoculation.  Plates are stored undisturbed in a 76 degree cabinet after inoculation.
All my tissue clones and plate-to-plate transfers look great, but this syringe-to-agar issue is just bugging the hell out of me.  :confused:


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OfflineMbrooks1024
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Re: Problems with syringe straight to agar [Re: CliftonGK1]
    #21709207 - 05/22/15 05:42 AM (8 years, 8 months ago)

A solution to the contaminated plates would be to improve your technique (duh.) or to add some antibiotic powder to your agar. This gives the spores a few more days before the contaminants start showing up.

I've personally never had any problems with agar that can't be fixed with a transfer or two.

An alternative to fruiting a BRF cake would be to inoculate grain and transfer a single grain to agar once it is colonized. This shouldn't take long if you use a smaller jar, and will start growing within a day or two.

It's also possible you are mixing your agar incorrectly, and it is too dry/wet for the spores germinate. Are you using the proper ratio on the premixed agar, as well as using a scale to determine the correct amount of powder to use?


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InvisiblePastywhyteMDiscord
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Re: Problems with syringe straight to agar [Re: Mbrooks1024]
    #21709242 - 05/22/15 06:09 AM (8 years, 8 months ago)

Spore syringes to agar is often a crapshoot. You might be making the agar to stiff or the spores may be too old. Have you tried different syringes or just one? What recipe are you using? Usually what I do is inoculate a few plates with syringes then take the one that looks best. Often some just won't germinate.


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OfflineCliftonGK1
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Re: Problems with syringe straight to agar [Re: Mbrooks1024]
    #21709273 - 05/22/15 06:29 AM (8 years, 8 months ago)

Quote:

Mbrooks1024 said:
A solution to the contaminated plates would be to improve your technique (duh.) or to add some antibiotic powder to your agar. This gives the spores a few more days before the contaminants start showing up.

I've personally never had any problems with agar that can't be fixed with a transfer or two.



Yeah, I've done cleanup on some wild harvest tissue samples which were microbial as all get out from the start, but a few transfers and they were all good.  I'm pretty sure that the bigger issue when contam has been the problem was just sloppy technique on my part.

Quote:

Mbrooks1024 said:
An alternative to fruiting a BRF cake would be to inoculate grain and transfer a single grain to agar once it is colonized. This shouldn't take long if you use a smaller jar, and will start growing within a day or two.



I'll have to give this a try.  Sometimes it's for the forest that you can't see the trees, right?  Never really thought about doing this as my mindset has been siloed with using grains for larger scale-up, and I didn't consider using them for small scale efforts.  Thanks!

Quote:

Mbrooks1024 said:
It's also possible you are mixing your agar incorrectly, and it is too dry/wet for the spores germinate. Are you using the proper ratio on the premixed agar, as well as using a scale to determine the correct amount of powder to use?



The mix I'm using says 25g to 500ml, and I'm using a 2 decimal scale to weigh out, and a 500mL +/- 5mL graduated cylinder for liquid measure.  I don't imagine that even being 1% off on the low side should cause much in the way of dehydration issues. 
I thought maybe it was that things were drying out, since I'm seeing the liquid inoculum sit on the surface for a day, and then there's an obvious ring at the edge of the natural spread across the agar as the droplet disperses across the surface to slightly larger than the initial size.  But after that first day it looks like the droplet is gone. My assumption is that the moisture of the agar and the humidity inside the sealed-edge plate would provide an environment for germination, but so far, no dice.

Might I have better luck pouring a slightly thicker plate and doing an intra-agar injection?


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OfflineCliftonGK1
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Re: Problems with syringe straight to agar [Re: Pastywhyte]
    #21709293 - 05/22/15 06:37 AM (8 years, 8 months ago)

Quote:

Pastywhyte said:
Spore syringes to agar is often a crapshoot. You might be making the agar to stiff or the spores may be too old. Have you tried different syringes or just one? What recipe are you using? Usually what I do is inoculate a few plates with syringes then take the one that looks best. Often some just won't germinate.




Are most commercial mix agars too stiff?  I don't want to name names because I'm not sure if the company is a sponsor or not, but they're a well known supplier for commercial mycology supplies (and not prints/syringes).  Their PDA mix does seem to hold fairly firm when placing a tissue sample, and in test I could feel reasonable resistance when scoring it with a probe.  But it wasn't firm enough that it was 'crumbly' feeling (you know what I mean; when it breaks/tears rather than allowing a probe to make a clean separation/slice).

I think next round of plates (tomorrow) I'll try mixing things 20% lighter on the powder (20g PDA mix to 500ml dH20) and see if that works out better.


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InvisibleCrazycoocoo430


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Re: Problems with syringe straight to agar [Re: CliftonGK1]
    #21709308 - 05/22/15 06:39 AM (8 years, 8 months ago)

I have same problem


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OfflineMbrooks1024
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Re: Problems with syringe straight to agar [Re: CliftonGK1]
    #21709313 - 05/22/15 06:40 AM (8 years, 8 months ago)

A 1% difference is nothing. I was just wondering if you were using the tbsp=7g measurement :rolleyes:

Good luck on your future efforts! Hopefully the agar gets better, and the grains work well for you.

also, you don't mean injecting the spores into the agar, right?

Also:
Quote:

MycoAu said:
The thickness of the agar does matter.  It is usually a function of the purpose for which it is being used.  The usual thickness is (I believe) about 5 mm thick.  If you're not having problems with dehydration of the agar during the period of time in which you are using it, I wouldn't worry about it too much.




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InvisibleUncleFester
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Re: Problems with syringe straight to agar [Re: CliftonGK1]
    #21709323 - 05/22/15 06:43 AM (8 years, 8 months ago)

Quote:

CliftonGK1 said:
"I've found myself having to take an additional step of fruiting a BRF cake and then cloning out tissue from a good looking sample when I want to start a series of agar plates to propagate and isolate."

"Anyone else have issues like this when trying to start from syringe direct to agar?"

"syringe-to-agar issue is just bugging the hell out of me.  :confused:"




Dude I know that feel, my syringe to agar always ends up like shit but then again I collected my spores from a mushroom growing out of my plant soil. Either way I was wondering the same question and now I'm glad you posted this and saved me the time.

So I guess syringe to agar isn't the best way but yeah cutting a wedge out of a brf cake it's what I've had to do to get my spores from syringe to agar. Very indirect but if it works it works ya know?


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InvisiblePastywhyteMDiscord
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Re: Problems with syringe straight to agar [Re: CliftonGK1]
    #21709340 - 05/22/15 06:50 AM (8 years, 8 months ago)

I have never used commercial premixed agar (though I do have some) as I prefer to control the ratios and ingredients myself.  I have found that a slightly richer media is beneficial for spore germination. Perhaps the commercial premixed stuff was made too nutrition light. For instance if they screwed up a batch and had 1 part nutes to two parts agar instead of the 1:1 that is usual, when you mix your agar the result would be an agar that would be 3% agar and 1% nutes. This would be pretty stiff as well as thin on nutes. That could retard germination, though it would still work fine for cloning etc.

I'm just spitballing here but I would suggest making your own and see how that works. It would cost very little to pick up a packet of agar and make some up yourself.


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OfflineCliftonGK1
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Re: Problems with syringe straight to agar [Re: Mbrooks1024]
    #21709351 - 05/22/15 06:56 AM (8 years, 8 months ago)

Quote:

Mbrooks1024 said:
A 1% difference is nothing. I was just wondering if you were using the tbsp=7g measurement :rolleyes:

Good luck on your future efforts! Hopefully the agar gets better, and the grains work well for you.

also, you don't mean injecting the spores into the agar, right?



Thanks for the well wishes and the suggestions!  :smile:

And, yes, I did actually mean injecting the spores into the agar.  Just spitballing ideas to see what sticks and what stinks.  So, it sounds like that idea stinks, eh?  (you can point and laugh, it's OK.  I spent 20 years in research science, so I'm fine with people asking me, "Son, what the hell are you thinking?"  :facepalm3:

Also:
Quote:

MycoAu said:
The thickness of the agar does matter.  It is usually a function of the purpose for which it is being used.  The usual thickness is (I believe) about 5 mm thick.  If you're not having problems with dehydration of the agar during the period of time in which you are using it, I wouldn't worry about it too much.







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OfflineCliftonGK1
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Re: Problems with syringe straight to agar [Re: Pastywhyte]
    #21709354 - 05/22/15 06:59 AM (8 years, 8 months ago)

Quote:

Pastywhyte said:
I'm just spitballing here but I would suggest making your own and see how that works. It would cost very little to pick up a packet of agar and make some up yourself.




I think when I exhaust my current supply of this stuff I'll go that route.  I have this stuff, and it seems a waste just to throw it out, even though I've got enough to pour about 300 more plates from what's left.
Maybe it's project time...


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OfflineFreeWorldOrder
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Re: Problems with syringe straight to agar [Re: CliftonGK1]
    #21709478 - 05/22/15 07:58 AM (8 years, 8 months ago)

I've ran into similar issues. Now I usually either drop on a colonized grain, a drop of GLC  or the tissue culture route. Seems even "swabbing" with a clay sculpting tool workd better than spores in water. Don't really know why spore syringes can be finicky, most likely since they are not truly "clean" and contams set in before the spores have a chance to germinate. And the present water makes it all the easier. Just a thought. I'm no expert by any means, but have pretty good success after a couple years of "studying".

Also like Pasty said... clean it up with a few transfers. He knows his shit...

Agar work is pretty easy once you get the hang of it.

I've transfer a white leading edge of agar away from a plate full of trich & black mold (more as an experiment) and actually got away with it. Albeit, it took numerous transfers to get a clean plate.


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Edited by FreeWorldOrder (05/22/15 08:13 AM)


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Invisibletomentose
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Re: Problems with syringe straight to agar [Re: FreeWorldOrder]
    #21709881 - 05/22/15 09:57 AM (8 years, 8 months ago)

1. My success went way up after using the needle tip to scratch a small trench into the agar, and then a drop or two of spore solution into the gouged agar. As a side note: this produces very cottony mycelia which is not a problem.

2. Your particular spores may need a higher temperature for germination. Plus that tends to evaporate the agar, keeping the spores nice and moist.


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InvisiblePastywhyteMDiscord
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Re: Problems with syringe straight to agar [Re: tomentose]
    #21710009 - 05/22/15 10:40 AM (8 years, 8 months ago)

Quote:

tomentose said:
1. My success went way up after using the needle tip to scratch a small trench into the agar, and then a drop or two of spore solution into the gouged agar. As a side note: this produces very cottony mycelia which is not a problem.

2. Your particular spores may need a higher temperature for germination. Plus that tends to evaporate the agar, keeping the spores nice and moist.




:smbfacepalm:


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OfflineFreeWorldOrder
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Re: Problems with syringe straight to agar [Re: tomentose]
    #21710246 - 05/22/15 11:57 AM (8 years, 8 months ago)

Never heard of these "concepts" before.... it will benefit you tremendously to do some serious research friend...:thumbup:


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Invisibletomentose
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Re: Problems with syringe straight to agar [Re: FreeWorldOrder]
    #21710823 - 05/22/15 02:38 PM (8 years, 8 months ago)

Quote:

Pastywhyte said:


:smbfacepalm:





Quote:

Pastywhyte said:


:smbfacepalm:



Quote:

FreeWorldOrder said:
Never heard of these "concepts" before.... it will benefit you tremendously to do some serious research friend...:thumbup:




Do what works or STFU. Using dry spores and an inoculation loop does the same thing. The spores are effectively in a pool of nutritious fluid and breeding. enfolded within the agar. Do it my way and have transferable mycelia in 6 days. Too many retards on this site.


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Stametsian


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InvisibleFriedEggS
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Re: Problems with syringe straight to agar [Re: tomentose]
    #21710935 - 05/22/15 03:10 PM (8 years, 8 months ago)

Quote:

tomentose said:
Do what works or STFU. Using dry spores and an inoculation loop does the same thing. The spores are effectively in a pool of nutritious fluid and breeding. enfolded within the agar. Do it my way and have transferable mycelia in 6 days. Too many retards on this site.



:goodluckwiththat2:

i'm pretty sure all p. cubensis spores have the same germination requirements





OP: I know you're using premixed agar, but try adding some more nutes and more water to your agar.  I had problems germinating spores on agar for a while.  Making my agar softer worked.


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Edited by FriedEgg (05/22/15 03:16 PM)


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InvisiblePastywhyteMDiscord
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Re: Problems with syringe straight to agar [Re: tomentose]
    #21710976 - 05/22/15 03:23 PM (8 years, 8 months ago)

I wasn't facepalming the suggestion to scratch the agar to hold the solution. That's a shitty way to do it but it should work. No I was facepalming the other 4 completely wrong statements you made in that post. I'm on my phone so I never bothered to remove the one element that wasn't completely wrong. My apologies.


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Offlinemushpunx
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Re: Problems with syringe straight to agar [Re: Pastywhyte]
    #21711072 - 05/22/15 03:49 PM (8 years, 8 months ago)

Sometimes when I cant get spores in a solution to germinate on agar I inoculate like a half pint of rye and let it colonize, take a colonize grain out with my flame sterilized scalpel and drop it on my plate, then go from there :shrug:


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InvisiblePastywhyteMDiscord
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Re: Problems with syringe straight to agar [Re: mushpunx]
    #21711090 - 05/22/15 03:52 PM (8 years, 8 months ago)

Quote:

mushpunx said:
Sometimes when I cant get spores in a solution to germinate on agar I inoculate like a half pint of rye and let it colonize, take a colonize grain out with my flame sterilized scalpel and drop it on my plate, then go from there :shrug:




That is a fine way, also cuts the genetics down real fast :thumbup:


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