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stareatclouds
star eat clouds?



Registered: 09/29/14
Posts: 10,271
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Rate My Technique (Agar Transfer Video)
#21229496 - 02/05/15 07:40 AM (9 years, 3 months ago) |
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Hi,
I'm still new to agar and am trying to get better at my sterile technique inside my SAB. Here's a quick video of me transferring a small chunk of mycelium to a new agar plate.
Please pick out anything I'm doing wrong or sloppily. Specifically, I am curious about the following:
1. I usually put my lids bottom-down on top of an iso-towel. Should I just set them bottom-up on whatever instead?
2. My glove touched my towel on the floor of my SAB. I shouldn't do this.
3. Should I have smaller things like the tiny petri dish elevated a bit higher throughout the process?
4. How much movement is TOO much movement in a SAB. For instance, I'll often wipe my gloves again with an iso-towel inside the SAB. Will this kick up crap or is it fine?
Also, I edited out the flame sterilization for brevity.
Thanks for the help!
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Albertheisenhouer
Tryptamine doctor



Registered: 10/26/13
Posts: 734
Loc: Holland
Last seen: 22 days, 19 hours
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Re: Rate My Technique (Agar Transfer Video) [Re: stareatclouds]
#21229510 - 02/05/15 07:50 AM (9 years, 3 months ago) |
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Some things you might want to do the next time:
1) Don't use a towel, even though you used alcohol. Better work on a smooth surface, which is cleaned with a bit of alcohol.
2) You can submerge the agar tissue in a little bit of 3% H2O2 to kill any spores or bacteria that might get stuck on your agar. Mycelium is resistant to H2O2. Remember, the air is stuffed with spores and bacteria. You can also use a bit of H2O2 within the agar if doing agar to agar transfer. Spore germination should be done without any peroxide.
3) Flame sterilise the scalpel right before using it, let it get red hot, wait about 20 secs and then use it.
4) try to minimise the time that of transfer.
5) if your glove touches any surface you should use alcohol to desinfect before touching anything else
6) the size of the agar wedge is okay, but I would use the plate a bit earlier (before it is colonised 100%)
Edited by Albertheisenhouer (02/05/15 07:52 AM)
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stareatclouds
star eat clouds?



Registered: 09/29/14
Posts: 10,271
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Hi Albert,
1. My plastic floor is way too slippery without a towel. I always use freshly laundered towels to minimize gunk. Without one, the curved bottom of my SAB has mason jars and plates sliding around into the corners.
2. Shouldn't I be unconcerned with the air being dirty? It's a SAB and I'm operating on the principle that bad particles have been resting on the floor.
3. I flame sterilized the blade, although I never wait 20 seconds, I simply dip in my receiving plate to cool.
4. Valid point, will do. Still having trouble handling the blade.
5. Agreed. I only noticed I touched the bottom after reviewing the video.
6. Agreed. I received it as a trade and it was already colonized fully.
Thanks for the response!
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Albertheisenhouer
Tryptamine doctor



Registered: 10/26/13
Posts: 734
Loc: Holland
Last seen: 22 days, 19 hours
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Re: Rate My Technique (Agar Transfer Video) [Re: stareatclouds]
#21229526 - 02/05/15 08:01 AM (9 years, 3 months ago) |
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1) you can maybe use an anti-slip sheet or something what serves the same purpose.
2) I think you can better whipe all the surfaces of the SAB, then wait about half a minute and then use it. The air will always be filled with microbes when not using a HEPA filter. Waiting is not gonna do a lot about it. The power of a SAB is that there is no current of airflow, thereby minimising the risk.
3) you can also cool the blade by putting it in agar, and then wait a few moments to cool. The agar will melt but it doesn't do anything bad
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MrGiraffe

Registered: 04/04/14
Posts: 3,149
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With petri dishes I'll hold it in my left hand, use my right hand to lift the lid up and slide it just enough to gain access (usually about half way open). I'll use my thumb on my left hand to hold the half open lid against the petri. I do this with my blade already flamed and in my right hand. Use my right hand to take the wedge and then immediately use my thumb to push the cover back up and onto the dish. Sit that dish to the side and open my receiving dish lid enough to fit the blade in with out touching any of the edges slide the blade in and deposit the wedge. Of course my receiving dish is also a petri, so I can get into the middle of the plate with barely opening the dish, and the cover always stays completely over the top to help prevent anything from being able to fall in it.
--------------------
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Albertheisenhouer
Tryptamine doctor



Registered: 10/26/13
Posts: 734
Loc: Holland
Last seen: 22 days, 19 hours
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Re: Rate My Technique (Agar Transfer Video) [Re: MrGiraffe]
#21229985 - 02/05/15 10:51 AM (9 years, 3 months ago) |
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sounds like a solid technique
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taGyo
Strainiac/AMU



Registered: 10/16/14
Posts: 18,802
Loc: Journal Land
Last seen: 5 years, 7 months
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-5,
You suck Stare.
-------------------- Gyo's Better Grows TNF Q&A AMU Q&A Dominus fortunae meae sum
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stareatclouds
star eat clouds?



Registered: 09/29/14
Posts: 10,271
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Re: Rate My Technique (Agar Transfer Video) [Re: taGyo]
#21231041 - 02/05/15 03:08 PM (9 years, 3 months ago) |
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Thanks, Giraffe. I'll try and shield the dishes with the lids next time.
Tag,
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Psilicon
Really Nice Guy


Registered: 08/26/12
Posts: 7,057
Last seen: 3 years, 4 months
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Re: Rate My Technique (Agar Transfer Video) [Re: stareatclouds]
#21231111 - 02/05/15 03:25 PM (9 years, 3 months ago) |
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I do my work elevated, and I never put my lids down. With pasty plates I loosen the lid, and then with my left hand I'll grab the dish and raise it up. With my middle finger and thumb I'll crack it open, but my palm keeps the side of the lid opposite the tab pressed firmly against the dish so it opens like a clam. I do my work at about a 45° angle, with the lid acting as a shield to keep any falling spores from falling into the dish. Does that make sense? I can take a picture if you want.
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stareatclouds
star eat clouds?



Registered: 09/29/14
Posts: 10,271
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Re: Rate My Technique (Agar Transfer Video) [Re: Psilicon]
#21231224 - 02/05/15 03:45 PM (9 years, 3 months ago) |
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I believe that makes sense, but a picture would be awesome! In some applications I'll need to set lids down though so I'm trying to find the best place for them.
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Psilicon
Really Nice Guy


Registered: 08/26/12
Posts: 7,057
Last seen: 3 years, 4 months
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Re: Rate My Technique (Agar Transfer Video) [Re: stareatclouds]
#21231273 - 02/05/15 03:52 PM (9 years, 3 months ago) |
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stareatclouds
star eat clouds?



Registered: 09/29/14
Posts: 10,271
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Re: Rate My Technique (Agar Transfer Video) [Re: Psilicon]
#21231286 - 02/05/15 03:54 PM (9 years, 3 months ago) |
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Thanks a lot, van. That's what I imagined, but wasn't sure. I'll definitely try that technique next time. The added protection should drop my contam rate.
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TheEaglesGift
The Nagual


Registered: 04/10/11
Posts: 10,554
Loc: Ixtlan, Mexico
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Re: Rate My Technique (Agar Transfer Video) [Re: stareatclouds]
#21231367 - 02/05/15 04:09 PM (9 years, 3 months ago) |
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Your dexterity while gloved up in a SAB will get better with time.
Pretty good. Everyone else has already said anything that I'd have suggested to you.
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Psilicon
Really Nice Guy


Registered: 08/26/12
Posts: 7,057
Last seen: 3 years, 4 months
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Re: Rate My Technique (Agar Transfer Video) [Re: TheEaglesGift]
#21231389 - 02/05/15 04:13 PM (9 years, 3 months ago) |
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Haha, so true, TEG. I found it difficult to do that without gloves. Usually I rely on the latex to grip the lid for me.
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stareatclouds
star eat clouds?



Registered: 09/29/14
Posts: 10,271
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Re: Rate My Technique (Agar Transfer Video) [Re: Psilicon]
#21231397 - 02/05/15 04:15 PM (9 years, 3 months ago) |
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Thanks, Eagle. I need to design a new SAB. My holes are not shoulder length and it being so long, but not tall enough makes it really hard to maneuver in there, especially without crossing hands and hovering over important stuff.
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Psilicon
Really Nice Guy


Registered: 08/26/12
Posts: 7,057
Last seen: 3 years, 4 months
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Re: Rate My Technique (Agar Transfer Video) [Re: stareatclouds]
#21231407 - 02/05/15 04:17 PM (9 years, 3 months ago) |
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Consider mounting your dominant arm's hole a bit higher than the other. It makes it easier to transfer, pour grain in G2Gs, clone and so on. I think it was PW who gave me that idea.
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tripdawg420
low life with no life



Registered: 02/02/09
Posts: 7,480
Loc: illinois
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Re: Rate My Technique (Agar Transfer Video) [Re: Psilicon]
#21231433 - 02/05/15 04:22 PM (9 years, 3 months ago) |
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like a boss im digging these vid teks
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stareatclouds
star eat clouds?



Registered: 09/29/14
Posts: 10,271
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Re: Rate My Technique (Agar Transfer Video) [Re: tripdawg420]
#21231461 - 02/05/15 04:26 PM (9 years, 3 months ago) |
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van,
I actually did that when I first made this one, but didn't have the proper utensils to cut them out properly. When I was able to fix it, they wound up being level. I'll definitely do that next with with a taller box.
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TheEaglesGift
The Nagual


Registered: 04/10/11
Posts: 10,554
Loc: Ixtlan, Mexico
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Re: Rate My Technique (Agar Transfer Video) [Re: Psilicon]
#21231472 - 02/05/15 04:28 PM (9 years, 3 months ago) |
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Quote:
van der griegen said: Consider mounting your dominant arm's hole a bit higher than the other. It makes it easier to transfer, pour grain in G2Gs, clone and so on. I think it was PW who gave me that idea.
Awesome idea.
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Psilicon
Really Nice Guy


Registered: 08/26/12
Posts: 7,057
Last seen: 3 years, 4 months
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Re: Rate My Technique (Agar Transfer Video) [Re: stareatclouds]
#21231497 - 02/05/15 04:33 PM (9 years, 3 months ago) |
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Quote:
stareatclouds said: van,
I actually did that when I first made this one, but didn't have the proper utensils to cut them out properly. When I was able to fix it, they wound up being level. I'll definitely do that next with with a taller box.
When I made the one in the picture that you can kind of see, I didn't have the right tools, either. So I traced the holes I needed and cut them out with my X-acto, heating it up with my torch as needed. It took about five minutes.
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