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irequirechocolate
Stranger

Registered: 10/15/16
Posts: 132
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Agar contam?
#23794669 - 11/02/16 01:56 PM (7 years, 2 months ago) |
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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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enlightenment
alchemist


Registered: 08/09/09
Posts: 1,647
Loc: Europe
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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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irequirechocolate
Stranger

Registered: 10/15/16
Posts: 132
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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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enlightenment
alchemist


Registered: 08/09/09
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That's a better picture  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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irequirechocolate
Stranger

Registered: 10/15/16
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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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enlightenment
alchemist


Registered: 08/09/09
Posts: 1,647
Loc: Europe
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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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Mycolorado
Hobbyist


Registered: 07/23/16
Posts: 8,529
Loc: Interdimensional Bootcamp
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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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enlightenment
alchemist


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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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dextr0
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irequirechocolate
Stranger

Registered: 10/15/16
Posts: 132
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Re: Agar contam? [Re: dextr0]
#23794825 - 11/02/16 02:58 PM (7 years, 2 months ago) |
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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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dextr0
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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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AlCapone2k
Beginner


Registered: 10/06/16
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Re: Agar contam? [Re: dextr0]
#23795537 - 11/02/16 06:43 PM (7 years, 2 months ago) |
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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
-------------------- Since I am from Germany, my english is not perfect but I try my best    My small Agar to Oyster Open Air Grow Trek
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irequirechocolate
Stranger

Registered: 10/15/16
Posts: 132
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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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Mad Season
hookers and blackjack



Registered: 09/16/12
Posts: 12,666
Loc: Canada
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Those don't really look like any cubes I've seen before..
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irequirechocolate
Stranger

Registered: 10/15/16
Posts: 132
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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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irequirechocolate
Stranger

Registered: 10/15/16
Posts: 132
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Updated pic from today... Not looking good, as the blue is spreading 
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dextr0
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Registered: 07/24/16
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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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mrmazdarx9
Pffffttt


Registered: 05/15/16
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Re: Agar contam? [Re: dextr0]
#23804148 - 11/05/16 05:54 PM (7 years, 2 months ago) |
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Looks a bit like mycogone/wet bubble never seen that on a plate
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Josex
#cheat_code



Registered: 11/13/15
Posts: 8,995
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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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dextr0
Stranger


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Re: Agar contam? [Re: Josex]
#23804381 - 11/05/16 06:54 PM (7 years, 2 months ago) |
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Edited by dextr0 (11/05/16 07:01 PM)
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Josex
#cheat_code



Registered: 11/13/15
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Re: Agar contam? [Re: dextr0]
#23804388 - 11/05/16 06:55 PM (7 years, 2 months ago) |
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Cocaine?
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dextr0
Stranger


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Re: Agar contam? [Re: Josex]
#23804420 - 11/05/16 07:03 PM (7 years, 2 months ago) |
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Lol wrong pic...fixed it.
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spacechildo
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Re: Agar contam? [Re: dextr0]
#23804467 - 11/05/16 07:16 PM (7 years, 2 months ago) |
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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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dextr0
Stranger


Registered: 07/24/16
Posts: 431
Last seen: 20 days, 3 hours
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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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mrmazdarx9
Pffffttt


Registered: 05/15/16
Posts: 9,796
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Re: Agar contam? [Re: dextr0]
#23804543 - 11/05/16 07:39 PM (7 years, 2 months ago) |
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Start over kill that shit dead
-------------------- COCA GROWERS come here and share your knowledge COCA GROWERS UNITE
Click here for UK trades
need some supplies in the UK check Here or PM me UK OTD uk members chat UK supplies and trade OTD place to chat shit Right Here If you use "SWIM" you should DROWN
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dextr0
Stranger


Registered: 07/24/16
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Last seen: 20 days, 3 hours
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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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Mad Season
hookers and blackjack



Registered: 09/16/12
Posts: 12,666
Loc: Canada
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Re: Agar contam? [Re: dextr0]
#23805044 - 11/05/16 10:36 PM (7 years, 2 months ago) |
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Cleaning spores is some of the funnest shit tho.. It gets you damn good at recognizing morphology
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TheEaglesGift
The Nagual


Registered: 04/10/11
Posts: 10,554
Loc: Ixtlan, Mexico
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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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