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OfflineKillerclowns87
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Agar issues
    #24008917 - 01/14/17 05:39 AM (7 years, 16 days ago)

Hello guys!! Ive been working with agar for about a month now. While ive had great success with inoculating my media (ive only used grain to noc media so far) and have gotten some really nice/clean looking cultures just from a grain, my goal of course is to make a mono. My problem lies within my transfers. For some reason every time i make a transfer to new agar in attempts to do my first isolation, the new agar plate forms a ring of liquid around the agar wedge in the middle of the new plate. this making the wedge do nothing it doesn't grow out im sure because of the stagnant water suffocating him.






Anyone have any experience with this problem? Any ideas from anyone who hasnt experienced this? Could it be my agar mix? i used a PDA premix that is consisted of 24% potatoe powder, 43%agar and 33% dextrose. It calls for 100gs premix to 2500ml water to make 90 dishes. I usually use 8g/200ml which is a little more than called for, but if i use recomendations its more like a paste than a medida.


http://
Here is a pic of agar inoculating with grain. (Just so its know mycelium will grow nice on this agar..but those transfers!!):confused:


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Offlineenlightenment
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Re: Agar issues [Re: Killerclowns87]
    #24008996 - 01/14/17 06:55 AM (7 years, 16 days ago)

How many days passed since the transfer? The mycelium needs to recover for about 2-4 days and then it starts to grow.


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OfflineKillerclowns87
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Re: Agar issues [Re: enlightenment]
    #24009413 - 01/14/17 10:25 AM (7 years, 16 days ago)

Quote:

enlightenment said:
How many days passed since the transfer? The mycelium needs to recover for about 2-4 days and then it starts to grow.





These were noc'd via agar wedge on the 8th of this month. (6 days ago)


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InvisibleIntelligentxfruit
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Re: Agar issues [Re: Killerclowns87]
    #24009490 - 01/14/17 10:51 AM (7 years, 16 days ago)

Does the puddle form immediately/after a couple hours? Or does it take 1-3days to show


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OfflineKillerclowns87
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Re: Agar issues [Re: Intelligentxfruit]
    #24012002 - 01/15/17 11:41 AM (7 years, 15 days ago)

It seems to grow in the following couple days. Not much of a puddle at all when i first noculate it.


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InvisiblemushboyMDiscord
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Re: Agar issues [Re: Killerclowns87]
    #24012015 - 01/15/17 11:45 AM (7 years, 15 days ago)

Bacteria?


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OfflineKillerclowns87
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Re: Agar issues [Re: mushboy]
    #24012130 - 01/15/17 12:36 PM (7 years, 15 days ago)

Quote:

mushboy said:
Bacteria?





Dont think so... Nothing visible atleast.. They're still in same place looking same way. The puddle stops the wedge from transfering,... like i said no problems at all noc'ing up a plate with a grain tho! This is driving me nuts!!


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Offlineenlightenment
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Re: Agar issues [Re: Killerclowns87] * 1
    #24012153 - 01/15/17 12:49 PM (7 years, 15 days ago)

You could make your own agar media. MEA worked well for a lot of people (including me).

The standard MEA recipe is:
30g malt extract
20g agar
1 L distilled water

I like to use a slightly modified MEA recipe but that doesn't matter here.

The premix you use is 17g agar/1L. It should not make a significant difference but I don't understand why you don't get growth on your dishes...
Try to raise the premix to at least 10g/200ml. I don't know if it helps but it might be worth a try. :shrug:

Do you cool the flamed scalpel blade by slicing the agar of the receiving dish?


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OfflineKillerclowns87
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Re: Agar issues [Re: enlightenment]
    #24012838 - 01/15/17 06:46 PM (7 years, 15 days ago)

Quote:

enlightenment said:


Do you cool the flamed scalpel blade by slicing the agar of the receiving dish?





Yes i do. i flame, then scrape scapel in new plate (on both sides) then make transfer. I dunno i will try uping the amount of pre-mix agar to water (10g agar to 200ml vs the 8g i usually use)


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OfflinePinPornProducer
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Re: Agar issues [Re: Killerclowns87]
    #24012858 - 01/15/17 06:55 PM (7 years, 15 days ago)

If it were just nutrients/water it wouldn't stop the mycelium from recovering or growing out. Mycelium thrives in liquid cultures. I would lean toward bacteria of some kind :shrug:


Edited by PinPornProducer (01/15/17 06:56 PM)


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InvisibleIntelligentxfruit
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Re: Agar issues [Re: Killerclowns87]
    #24012946 - 01/15/17 07:33 PM (7 years, 15 days ago)

Adding more agar gives you a firmer plate. What temps are you storing in?
Quote:

Killerclowns87 said:
It seems to grow in the following couple days. Not much of a puddle at all when i first noculate it.




Sorry man it sounds like a contaminate.
There's no way a myc wedge is generating enough heat to draw water under it(only other explanation I can think of besides soft agar), even if it did as PPP stated water wouldn't stop anything.


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OfflineOrbit
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Re: Agar issues [Re: Intelligentxfruit]
    #24013090 - 01/15/17 08:30 PM (7 years, 15 days ago)

Do you store your plates upside down? It may be condensation. Try taking a larger piece it may recover better.


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InvisiblebodhisattaMDiscordReddit
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Re: Agar issues [Re: Orbit]
    #24013106 - 01/15/17 08:38 PM (7 years, 15 days ago)

Sometimes the whole plate surface is bacteria and some people just don't notice.


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Re: Agar issues [Re: bodhisatta]
    #24013154 - 01/15/17 08:57 PM (7 years, 15 days ago)

I'm gonna just call that bacteria.


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OfflineKillerclowns87
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Re: Agar issues [Re: Pastywhyte]
    #24015614 - 01/16/17 07:20 PM (7 years, 14 days ago)

Quote:

Orbit said:
Do you store your plates upside down? It may be condensation. Try taking a larger piece it may recover better.




Deff do not store them upside down!

Quote:

Pastywhyte said:
I'm gonna just call that bacteria.





What makes you say that? Out of all my 60+ plates poured, ive only seen one visible bacteria in plate. What are some signs i have bacteria in my plates? I want to mention again that i've never had a problem getting a colonized grain to transfer to agar.... im so confused!


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OfflineOrbit
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Re: Agar issues [Re: Killerclowns87]
    #24015713 - 01/16/17 07:50 PM (7 years, 14 days ago)

Really? I thought it was common practice to store agar upside down. No condensation running around that can spread contaminants. Why do you say def not Killer?


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InvisiblebodhisattaMDiscordReddit
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Re: Agar issues [Re: Orbit]
    #24015727 - 01/16/17 07:54 PM (7 years, 14 days ago)

For bacteriological work yes. The lid gets discarded. But for us it causes more problems than good when condensation drops back on the agar


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OfflinePinPornProducer
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Re: Agar issues [Re: bodhisatta]
    #24015731 - 01/16/17 07:57 PM (7 years, 14 days ago)

Dump it out before transferring


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OfflineOrbit
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Re: Agar issues [Re: PinPornProducer]
    #24015775 - 01/16/17 08:16 PM (7 years, 14 days ago)

Maybe its just my cold climate/incubator. If I leave my dishes right side up, the top gets condensation on it. If you tilt it, it will go to one side and run down onto the agar. If I keep dishes upside down i get next to no condensation.


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OfflineKillerclowns87
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Re: Agar issues [Re: Orbit]
    #24018282 - 01/17/17 07:27 PM (7 years, 13 days ago)

Quote:

Orbit said:
Really? I thought it was common practice to store agar upside down. No condensation running around that can spread contaminants. Why do you say def not Killer?





Do you not get condensation on the lid when you do store them upside down? i do!
I've done a lot of reading on the matter and IME (and ive tried both) storeing upside down is a no-no. RR seems to think the same thing

Quote:

RogerRabbit said:
I've read of people turning them upside down for thirty years. They were wrong then and they're still wrong.  Laying them on their side does the same thing.  The fact is, cold material can hold less moisture than warm material.  As the refrigerator goes through its cycles, the temperature changes a few degrees each time.  With these cycles, if your dishes are upside down, more condensation drips to the lid.  It can't re-absorb into the agar on the up cycle, because it's now on the lid which is the bottom.  Within a month or two, you have a dried out agar disk on top, and a pool of water on bottom.

Leave jars right side up and you have none of these problems as long as you have the dishes wrapped up.  Even unwrapped, store right side up.  I knew someone would come along to repeat the store upside down mistake, so I got the correct method in first.  Mushworld is wrong with that advice.  I should shoot a picture of my lab refrigerator.  There's nearly 100 petri dishes in there, all right side up, and not a bit of condensation buildup on any of them.  Most are unwrapped.
RR







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Re: Agar issues [Re: Killerclowns87]
    #24018498 - 01/17/17 08:41 PM (7 years, 13 days ago)

I think it depends what side is warmer. I do alot of yeast culturing and (storage )in the fridge they are real bad for moisture on the lid if right side up, beer fridge so cooling is comming from above. My incubator has heat at the bottom. So moisture acts the same moving upward.

Try both ways and see witch works better for you. My current conditions upsidedown works best.


Not sure what RR is talking about hear. "cold material can hold less moisture than warm material"?

Its opposite cold material attracts moisture and warm releases it.
Boiling pot vs ice cold window where is the moisture going?



Wikipedia

Petri plates are incubated upside-down to lessen the risk of contamination from airborne particles settling on them and to prevent the accumulation of any water condensation that may otherwise disturb or compromise a culture.


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Re: Agar issues [Re: Orbit]
    #24018569 - 01/17/17 09:19 PM (7 years, 13 days ago)



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Offlinestromam
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Re: Agar issues [Re: Intelligentxfruit]
    #27580406 - 12/13/21 07:13 PM (2 years, 1 month ago)

Quote:

Intelligentxfruit said:
Does the puddle form immediately/after a couple hours? Or does it take 1-3days to show




I'm sorry to revive a fairly old post, but I've been having this exact problem with a large number of my plates. I think I might have an idea.  Could it be cold agar/tissue creating condensation on itself?  I've seen this problem from both agar to agar and tissue cultures.  I didn't take notes on the temperatures of the samples I was transferring from, but to the best of my recollection all the plates that have puddled like this came from wedges or mushrooms that had recently been refrigerated.  Here are some pictures.





This last one is the only one of about 10 different plates I made/transferred yesterday that didn't puddle overnight. It came from a pretty old (1 year+) and pretty dry looking plate, so perhaps the condensation it collected immediately got resorbed into the dry wedge.



My "puddle wedges" have always grown out just fine with no obvious contamination, other than the mycelium seems to follow the drop.  By that I mean if the drop doesn't stay put when I pick up a dish, but slides around on it plate it leaves a "snail trail" of mycelium spawn points.

If that's the cause I guess I need to let my samples come to room temp over a couple hours before making my transfers.  Maybe the more experienced are doing this already.


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