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Invisibleirequirechocolate
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Agar contam?
    #23794669 - 11/02/16 01:56 PM (7 years, 2 months ago)

I've got several plates going w/ what I thought were safe transfers from a previous plate that started w/ spores from a syringe. For these newer plates, I moved over a piece of mycelium from the edge of what grew out from the spores and away from anything that appeared like a discolored contam.

(Apologies for the picture quality. I took it over a black background in an effort to show what's going on..)
Several of these new plates have an unusual center though. It is 3 dimensional, which makes some sense since a small piece of agar came with the transfer, but at the center there are small yellow beads squeezing out from the surface. Some plates have some blueing around this area as well, (not pictured).

Is this an indication of contamination, stress, quality of life???
Thanks for your opinions.


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Offlineenlightenment
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Re: Agar contam? [Re: irequirechocolate]
    #23794691 - 11/02/16 02:02 PM (7 years, 2 months ago)

Wait a few days and post this dish again.
Picture quality is bad. Looks OK to me but no guarantee.

Update this thread in two days and I'll give you feedback again.
Try to do better pictures next time.

+ post your agar recipe


Edited by enlightenment (11/02/16 02:18 PM)


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Invisibleirequirechocolate
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Re: Agar contam? [Re: enlightenment]
    #23794730 - 11/02/16 02:19 PM (7 years, 2 months ago)

Here's a better quality pic illustrating what I'm talking about. Will update later this week.

Thanks

EDIT: I'm using YPD for plates. Essentially this is the recipe:
1. To an autoclavable flask, add:

Reagent Amount to add (per 1 L final volume)
Bacto agar 24 g
Bacto peptone 20 g
Yeast extract 10 g
Water         950 mL

2. Autoclave the mixture.

3. While stirring the autoclaved mixture on a magnetic stir plate, add 50 mL of sterile 40% glucose per liter of media.

4. While still warm, pour media into 9-cm diameter plastic Petri dishes.

5. Allow agar mixture to cool and solidify.


Edited by irequirechocolate (11/02/16 02:26 PM)


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Offlineenlightenment
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Re: Agar contam? [Re: irequirechocolate]
    #23794750 - 11/02/16 02:27 PM (7 years, 2 months ago)

That's a better picture :smile: :thumbup:
Looks good but update it anyway in 2 or 3 days. I would not use the dish before it is colonized about 1/2 to 3/4. Why? Because it is easier to slice / recognize sectors...


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Invisibleirequirechocolate
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Re: Agar contam? [Re: enlightenment]
    #23794762 - 11/02/16 02:31 PM (7 years, 2 months ago)

Is there a reason only a sector is pulled out? I've seen several helpful pics where people trace/box-in specific healthy 'branches,' but why not just dump the whole or half the plate into a jar of grains?


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Offlineenlightenment
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Re: Agar contam? [Re: irequirechocolate]
    #23794767 - 11/02/16 02:34 PM (7 years, 2 months ago)

Yes there is a reason. Take a look at this video:

http://www.mushroomvideos.com/Strain-Isolation

If you are new to growing mushrooms I would recommend to just cut out a piece without caring about sectors.


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InvisibleMycolorado
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Re: Agar contam? [Re: enlightenment]
    #23794797 - 11/02/16 02:48 PM (7 years, 2 months ago)

Metabolites on the culture could be indicative of contamination.  The pics look like they have a halo of bacterial contam around the leading edge of the mycelium but it may just be the pic...you might hold it up to the light to see better.  Also, your agar recipe is very rich which can promote a more tomentose or fuzzy/fluffy mycelial growth pattern.  If you cut the nutes down a bit, you should see more rhizomorphic growth and see sectors.  You might ensure it's clean before dropping it on grain.


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Offlineenlightenment
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Re: Agar contam? [Re: Mycolorado]
    #23794805 - 11/02/16 02:50 PM (7 years, 2 months ago)

:whathesaid:

The nutrient content of the agar could be the reason for this grow pattern, yes. That's why I asked for the recipe. If the update shows more signs of bacteria you can make another transfer to clean the mycelium.


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Offlinedextr0
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Re: Agar contam? [Re: Mycolorado]
    #23794809 - 11/02/16 02:51 PM (7 years, 2 months ago)



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Invisibleirequirechocolate
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Re: Agar contam? [Re: dextr0]
    #23794825 - 11/02/16 02:58 PM (7 years, 2 months ago)

Nice, I may try this Sto's agar recipe the next time I buy some malt extract for brewing. My stuff definitely has that tomentose (cottony) rather than rhizomorphic (strandy) look to it.
Thanks a lot, you guys.


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Offlinedextr0
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Re: Agar contam? [Re: irequirechocolate]
    #23795457 - 11/02/16 06:20 PM (7 years, 2 months ago)

I've read that if using MS you will more than likely need a couple transfers to get rhizomorphic growth. So it might not necessarily be your recipe.
Something to check out.
I use the grocery tek myself. Its OK but I want to add brewers yeast. Trien figure out the numbers. Bad with math.


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OfflineAlCapone2k
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Re: Agar contam? [Re: dextr0]
    #23795537 - 11/02/16 06:43 PM (7 years, 2 months ago)

I have somethin similiar to that but only on two of round about 20 plates.

It just appeared after the the plate was almost full of mycelium, before that there wasn't anything.




In another thread they told me it could be mycogone


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Invisibleirequirechocolate
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Re: Agar contam? [Re: enlightenment]
    #23798413 - 11/03/16 05:23 PM (7 years, 2 months ago)

Here's an early reply/photo of that same plate. I'm even more concerned now that it's blueing.


Below is another one that is 2 days older (I transferred it 48hrs sooner). It has the same droplets but no other major discoloration.


And here is a transfer I made 2 days ago cuz I was paranoid about the others. I took a pretty big chuck of the agar along w/ it.


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InvisibleMad Season
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Re: Agar contam? [Re: irequirechocolate]
    #23798435 - 11/03/16 05:31 PM (7 years, 2 months ago)

Those don't really look like any cubes I've seen before.. :shrug:


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Invisibleirequirechocolate
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Re: Agar contam? [Re: Mad Season]
    #23798448 - 11/03/16 05:34 PM (7 years, 2 months ago)

Well shucks.
They seem to be quite invasive too, as when the plate is flipped over I can see that it is cracking the agar at the center underneath.

I have plenty more spores left, so perhaps I'll try again and w/ that malt extract recipe.


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Invisibleirequirechocolate
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Re: Agar contam? [Re: enlightenment]
    #23803899 - 11/05/16 04:41 PM (7 years, 2 months ago)

Updated pic from today... Not looking good, as the blue is spreading  :frown:


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Offlinedextr0
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Re: Agar contam? [Re: irequirechocolate]
    #23804071 - 11/05/16 05:31 PM (7 years, 2 months ago)

Were the bubbles always yellowish?? I have two jars that have clear bubbles. I thought it was condensation at first.


Edited by dextr0 (11/05/16 05:32 PM)


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Offlinemrmazdarx9
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Re: Agar contam? [Re: dextr0]
    #23804148 - 11/05/16 05:54 PM (7 years, 2 months ago)

Looks a bit like mycogone/wet bubble never seen that on a plate


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InvisibleJosex
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Re: Agar contam? [Re: mrmazdarx9]
    #23804255 - 11/05/16 06:21 PM (7 years, 2 months ago)

Keep the agar recipe as simple as posible op, and with as few steps as possible. PDA or LME agar are fine, but don't make them far more complicated than they should be, cos shit like this happens and throws all that work out of the window.

I'll hazard a guess and say that you introduced bacteria into the plates with an improperly flame-sterilized scalpel.


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Offlinedextr0
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Re: Agar contam? [Re: Josex]
    #23804381 - 11/05/16 06:54 PM (7 years, 2 months ago)



Edited by dextr0 (11/05/16 07:01 PM)


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InvisibleJosex
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Re: Agar contam? [Re: dextr0]
    #23804388 - 11/05/16 06:55 PM (7 years, 2 months ago)

Cocaine?:rail2:


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Offlinedextr0
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Re: Agar contam? [Re: Josex]
    #23804420 - 11/05/16 07:03 PM (7 years, 2 months ago)

Lol wrong pic...fixed it.


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Offlinespacechildo
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Re: Agar contam? [Re: dextr0]
    #23804467 - 11/05/16 07:16 PM (7 years, 2 months ago)

that's no good.
take transfers as soon as you see some healthy growth, no point in using agar if you're gonna let everything from the print sporulate anyway.
start over.


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Offlinedextr0
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Re: Agar contam? [Re: spacechildo]
    #23804531 - 11/05/16 07:33 PM (7 years, 2 months ago)

Space if your talking to me no way I'm messing with that one its trash. I'm super tired of trying to isolate a clean culture. I opened tthe jar to take picture for the fuck of it. I have a new plate that I was hoping was gonna be my clean culture but two bubbles showed up on it. I might make a few transfers off that one since its only two bubbles. Idk tho.
Seems safer and smarter to move onto some cleaner plates. Or just start over from new spores.


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Offlinemrmazdarx9
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Re: Agar contam? [Re: dextr0]
    #23804543 - 11/05/16 07:39 PM (7 years, 2 months ago)

Start over kill that shit dead


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Offlinedextr0
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Re: Agar contam? [Re: mrmazdarx9]
    #23804623 - 11/05/16 08:08 PM (7 years, 2 months ago)

Hey Mr. I know man; its just a PITA. I've been working with agar almost two months now. Not one clean plate. Between the trich and milk bacteria I been getting hammered by both. (Wow never thought I'd say no shit like that...lol) Now this shit comes up. I'm dedicated but its a lil aggravating. I actually just threw my big jar of spawn that I'm sure was pretty bacterial. Stalled at 70% colonization. I threw this into a small tub with some unpasteurised straw. Surprisingly its colonized 80% of the sub no contams. I had little hope for it but now I'm just like go mushies!!! All I want is a fruit or clone to work with anyway because the above plate was the last of its variety.


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InvisibleMad Season
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Re: Agar contam? [Re: dextr0]
    #23805044 - 11/05/16 10:36 PM (7 years, 2 months ago)

Cleaning spores is some of the funnest shit tho.. It gets you damn good at recognizing morphology


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InvisibleTheEaglesGift
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Re: Agar contam? [Re: Mad Season]
    #23805363 - 11/06/16 03:48 AM (7 years, 2 months ago)

Mold.

Don't get discouraged, keep trying. Learning the morphology of mold vs the myc you want is important. Pics help, but then real time practice is where the real learning happens.


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