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maelstrominabox
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Registered: 02/17/15
Posts: 2
Last seen: 8 years, 2 months
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First time working with agar. Tips/critiques/flames appreciated
#22367156 - 10/12/15 09:01 AM (8 years, 3 months ago) |
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Note: Currently at work, don't have pics to show. Will update later with pictures.
I had a spore syringe and a couple of liquid culture syringes that needed to be processed this weekend and after a few back-and-forth's I decided to go with Pastywhyte's Easy Agar Tek. I figured it would be simple and fairly foolproof, little did I know. I wasn't able to get the round containers that he showed, so I went with the square ones figuring it shouldn't be too big of a deal. I prepped the agar as described in the instructions (hoping that the preservatives in the potato flakes won't have too much of an effect on early growth) and spooned into the containers. I may have ended up putting in too much agar due to the container having a raised center and wanting a thin level layer. (possible 1st mistake)
Folded a half-piece of paper towel on top of the containers like I thought it showed in the directions and then covered with aluminum foil. (possible human error here)
Put all 32 containers into the pressure cooker, they fit fairly well, although may have been a bit off balanced. Should have been a little more patient with this, as well as maybe less water in the pot. (this was most likely a big mistake in hind-sight)
Thought I had brought up the agar to temperature evenly per the TEK, but after seeing all the condensation in my containers I'm not quite certain.
Pressure cooked and let sit overnight to cool. Didn't get a chance to actually use them until late last night, but figured that since they were remaining in the unopened PC until then it shouldn't be too big of a deal. I put them in my SAB after wiping the outside with 10% bleach solution. This is when I noticed that there was a fairly large amount of condensation on the the sides of the containers. Left covered until I was fully ready to work.
When I went to go work inside my SAB, I removed the aluminum foil and paper towels, and even through my gloves I could tell that a few of the paper towels were drenched and my micropore tape looked wet as well. At this point, my heart sank because I know that is the easiest way for contamination to enter into the culture.
I decided that at this point I might as well test my luck and continue forward. I wiped everything down as best I could with my paper towel soaked in alcohol, and proceeded to inoculate with everything I have. This is when I noticed that a few of the containers had been warped during PC (yes, I did make sure they were all PP5) and knew that was the "popping" sound I heard in the PC as it was cooling down. So, they are all sitting on my shelves in my room now, "incubating" and I'm hoping that I can manage to pull something out of this total mess up. I woke up this morning late, but could still see water on the sides of the containers. When I get home, I'll check all the containers and take pictures.
I'm trying to fully figure out where I went wrong and how to fix those errors next time so that I don't waste precious materials and time. Plus, this whole thing was really disheartening, but I'm not going to let that have an effect on continuing. Did I do the right thing by continuing to inoculate even though I knew that most weren't as sterile as they should have been? What can I do to reduce all the extra water I had going on?
TLDR: Followed a no-pour agar TEK for my first agar work. PP5 containers full of condensation on the inside. Will this screw things up? And how do I make sure that it doesn't happen again?
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Fantastic Mr. Fox
Grass Seed Connoisseur


Registered: 02/12/15
Posts: 3,225
Loc:
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Re: First time working with agar. Tips/critiques/flames appreciated [Re: maelstrominabox]
#22367198 - 10/12/15 09:09 AM (8 years, 3 months ago) |
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(Im probably going to get flamed for saying this, but oh well)
I suggest using small glass half pint jars (4oz) for "no-pour"
I also used to use the 5 PPE containers, but found tht they warp after use.
Now I use glass "no-pour" The filter still gets wet sometimes, but when I unload into my SAB I just pour off the excess water onto the floor of the SAB. All in all I'd say the glass is superior to the 5 PPE plastic, just more costly
Good luck,
-------------------- Giving is all we have, for we're just grateful to be alive

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filthyknees
no coincidence


Registered: 03/08/13
Posts: 6,283
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Re: First time working with agar. Tips/critiques/flames appreciated [Re: Fantastic Mr. Fox]
#22368536 - 10/12/15 02:40 PM (8 years, 3 months ago) |
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Get a bulk case of Petri dishes and premix agar and you will have a good laugh at how you just skipped out on a lot of bullshit
You won't really be able to see through the glad plastic and fiddling with them in an sab is nightmarish... so just get the right tools if you're serious about getting into it.
-------------------- But if you're in a hurry, and really got to go If you're in a hurry, might have to find out slow That it's one thing to try and another to fly You get there quicker just a step at a time It's one thing to bark, another to bite The show ain't over till you pack up at night
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Fantastic Mr. Fox
Grass Seed Connoisseur


Registered: 02/12/15
Posts: 3,225
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Re: First time working with agar. Tips/critiques/flames appreciated [Re: filthyknees]
#22368597 - 10/12/15 02:54 PM (8 years, 3 months ago) |
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Petri's suck IMO. lol Why pour when you can "no-pour"?! I'd understand if you making petri's up to send cultures, but besides that it's just another step(s) (having to pour, stack, then wait to cool, followed by inoculating, and parafilm/plastic wrapping them)
I know I'm lazy.... But it's the truth. lol
-------------------- Giving is all we have, for we're just grateful to be alive

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LazyMonkey
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Registered: 08/16/15
Posts: 53
Last seen: 7 years, 10 months
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Re: First time working with agar. Tips/critiques/flames appreciated [Re: Fantastic Mr. Fox]
#22368865 - 10/12/15 03:43 PM (8 years, 3 months ago) |
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The condensation can even out some if they sit in the SAB for a few days. My pasties had a good bit, but now they're looking a lot better when it comes to condensation.
As to ease of use, I have found that I can hold two in one hand. For when making a transfer, I can have them upside down, loosen the lids and then pick up two, holding them at an angle, but more inverted, and make a quick transfer from one to the other without having to take a sample off of one and then put it down, pick up the other, or have my hands hovering over open containers sitting upright.
So far it's worked. YMMV.
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maelstrominabox
Stranger


Registered: 02/17/15
Posts: 2
Last seen: 8 years, 2 months
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Re: First time working with agar. Tips/critiques/flames appreciated [Re: Fantastic Mr. Fox]
#22368867 - 10/12/15 03:43 PM (8 years, 3 months ago) |
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Yeah, I think if I'm going to have to go through all the trouble I already have, that I might as well get the right tools for the job. I should just order the pre-made ones and just call it a day. At least until I can get my feet under me and wouldn't worry about losing pretty much everything. I tried doing it on a whim, and I learned a few things that will definitely help me going forward. Plus it's not like I really wasted much of anything (hopefully). Thanks for the tips, back to the drawing board for me and trying to do another round this weekend. *fingers crossed*
p.s. I'm also going to have pictures up later tonight of my Gallindoi that I have growing in jars. I think that there might be something a bit "off" with a few of them.
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DrCrumbs
Alpha Blue


Registered: 10/25/11
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Last seen: 4 years, 2 months
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Re: First time working with agar. Tips/critiques/flames appreciated [Re: maelstrominabox]
#22369715 - 10/12/15 06:14 PM (8 years, 3 months ago) |
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I know ordering stuff can look like an easy way out.
With agar your probably ok.
But stuff like pasteurized substrate, etc. can be a nightmare. You can end up with gnats and all sorts of stuff.
Now I have pre-sterilized grains and such that have been sitting for over 5 years with no signs of contams.
Everyone can have issues.
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filthyknees
no coincidence


Registered: 03/08/13
Posts: 6,283
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Re: First time working with agar. Tips/critiques/flames appreciated [Re: Fantastic Mr. Fox]
#22371981 - 10/13/15 07:15 AM (8 years, 3 months ago) |
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Quote:
Fantastic Mr. Fox said: Petri's suck IMO. lol
I know I'm lazy.... But it's the truth. lol
I'll just leave this here.
More likely small plastic tubes are used to send cultures because they fit in an envelope.
One thing I think fox & I would agree on though, and tell me if I'm wrong is to avoid buying prestrrilized stuff. Diy. Like the poster above me said. Better to learn how to do the whole operation am I right?
-------------------- But if you're in a hurry, and really got to go If you're in a hurry, might have to find out slow That it's one thing to try and another to fly You get there quicker just a step at a time It's one thing to bark, another to bite The show ain't over till you pack up at night
Edited by filthyknees (10/13/15 07:24 AM)
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Fantastic Mr. Fox
Grass Seed Connoisseur


Registered: 02/12/15
Posts: 3,225
Loc:
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Re: First time working with agar. Tips/critiques/flames appreciated [Re: filthyknees]
#22373156 - 10/13/15 12:34 PM (8 years, 3 months ago) |
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Speak for yourself, I receive full plates. Not everybody's cheap..
But your right about using "presterilized" equipment/supplies. Unless you have your own "UV sterilizer" to ensure sterility.. But even then it'll be pointless 'cause you could save some money and sterilize your own dishes.
-------------------- Giving is all we have, for we're just grateful to be alive

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Sivarted
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Registered: 08/22/15
Posts: 1,084
Last seen: 2 months, 10 days
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Re: First time working with agar. Tips/critiques/flames appreciated [Re: Fantastic Mr. Fox]
#22373185 - 10/13/15 12:43 PM (8 years, 3 months ago) |
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I started with pasty plates too, they'really working *great* for what I'm doing, but they seem like they'd be a pain for strain isolation due to the sheer numbers and space that would be required, so I ordered a bunch of 4-section plates too.
I had pretty significant condensation issues too btw, it cleared up in a few days though.
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filthyknees
no coincidence


Registered: 03/08/13
Posts: 6,283
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Re: First time working with agar. Tips/critiques/flames appreciated [Re: Fantastic Mr. Fox]
#22373627 - 10/13/15 02:15 PM (8 years, 3 months ago) |
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-------------------- But if you're in a hurry, and really got to go If you're in a hurry, might have to find out slow That it's one thing to try and another to fly You get there quicker just a step at a time It's one thing to bark, another to bite The show ain't over till you pack up at night
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