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Killerclowns87
Homie



Registered: 06/11/16
Posts: 161
Loc: Florida
Last seen: 1 year, 5 months
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Agar issues
#24008917 - 01/14/17 05:39 AM (7 years, 16 days ago) |
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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!!)
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enlightenment
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Registered: 08/09/09
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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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Killerclowns87
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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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Intelligentxfruit
Earth Hippy


Registered: 01/06/13
Posts: 1,545
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Does the puddle form immediately/after a couple hours? Or does it take 1-3days to show
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Killerclowns87
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Registered: 06/11/16
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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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mushboy
modboy



Registered: 04/24/05
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Bacteria?
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Killerclowns87
Homie



Registered: 06/11/16
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Re: Agar issues [Re: mushboy]
#24012130 - 01/15/17 12:36 PM (7 years, 15 days ago) |
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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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enlightenment
alchemist


Registered: 08/09/09
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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. 
Do you cool the flamed scalpel blade by slicing the agar of the receiving dish?
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Killerclowns87
Homie



Registered: 06/11/16
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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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PinPornProducer
Buy the ticket, take the ride



Registered: 08/23/14
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Loc: Rocky Point R.I
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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
Edited by PinPornProducer (01/15/17 06:56 PM)
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Intelligentxfruit
Earth Hippy


Registered: 01/06/13
Posts: 1,545
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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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Orbit
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Registered: 12/06/16
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Do you store your plates upside down? It may be condensation. Try taking a larger piece it may recover better.
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bodhisatta 
Smurf real estate agent


Registered: 04/30/13
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Re: Agar issues [Re: Orbit]
#24013106 - 01/15/17 08:38 PM (7 years, 15 days ago) |
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Sometimes the whole plate surface is bacteria and some people just don't notice.
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Pastywhyte
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Registered: 09/15/12
Posts: 37,810
Loc: Canada
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I'm gonna just call that bacteria.
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Killerclowns87
Homie



Registered: 06/11/16
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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!
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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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Orbit
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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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bodhisatta 
Smurf real estate agent


Registered: 04/30/13
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Re: Agar issues [Re: Orbit]
#24015727 - 01/16/17 07:54 PM (7 years, 14 days ago) |
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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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PinPornProducer
Buy the ticket, take the ride



Registered: 08/23/14
Posts: 9,981
Loc: Rocky Point R.I
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Dump it out before transferring
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Orbit
Stranger


Registered: 12/06/16
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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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Killerclowns87
Homie



Registered: 06/11/16
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Loc: Florida
Last seen: 1 year, 5 months
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Re: Agar issues [Re: Orbit]
#24018282 - 01/17/17 07:27 PM (7 years, 13 days ago) |
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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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