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huey.bluey


Registered: 01/16/19 
Posts: 2,042
Loc: Mississippi River
Last seen: 1 day, 18 hours
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What are these contaminants and is any of this salvagable
#28469820 - 09/15/23 09:36 AM (1 year, 3 months ago) |
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Hello I have 6 distinct plates that show random spherical abnormalities away from what Ive transferred. These are all T1's from spore plates.
I notice these white rings appear around the edges and away from the transferred material. Can someone identify what these contaminants are called? Also could I try transferring away from most of these contaminants or should I just compost the agar within them and try again from T0?
Thank you for the help
1) 2) 3) 4) 5) 6)
Edited by huey.bluey (09/15/23 11:06 AM)
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huey.bluey


Registered: 01/16/19 
Posts: 2,042
Loc: Mississippi River
Last seen: 1 day, 18 hours
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Re: What are these contaminants and is any of this salvagable [Re: huey.bluey]
#28469912 - 09/15/23 11:37 AM (1 year, 3 months ago) |
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Here is my agar process:
1) The 64q SAB
 Towel underneath cookie rack, secondary rack on top. I do transfers on that top rack. Sometimes I dont switch out a new towel, usually I do. I spray ISO on the walls and wipe it down every other use and before I get the agar out of the PC. 2) Agar plates I usually PC 500 ml of MPG agar for 1 hour in my instant pot ~13psi not quite 15.
Quote:
(germ plates) ------- - MEA - ------- 500 ml water 10 grams agar 10 grams malt extract
Quote:
(colony plates) ------- - MPA - < mainly do this one, have not tried PDY yet. ------- 500 ml water 10 grams agar 10 grams malt extract 2.5 grams ground rye 2.5 grams soy peptone 1 gram yeast
Quote:
(colony plates) ------- - PDY - ------- The filtered, extracted broth from boiling 150 grams of sliced potatoes in 500 ml of water for 1 hour. 10 grams agar 5 grams dextrose sugar 1 gram yeast
I leave the lid on the agar bottle slightly loose, then when done I never release pressure until 30-40 minutes later and I drop the remaining 1-2psi to 0 and tighten the cap. No pours I just pour cover and PC. I let the agar cool 90 minutes until lukewarm to the touch, shaking the bottle every 20 minutes. Then I pour in my SAB. Mask and gloves on, 10% bleach solution rubbed on the gloves. I usually shower before but sometimes not. I also use a 70% iso hand sanitizer on my forearms. Then I pour to petris and let sit for 4-24 hours.
3) Transferring I wipe the SAB with iso again, or just spray with 10% bleach. Let sit for 15 minutes. Put my transfer plate on the high rack. Put more bleach on my hands and in the sab. Wipe my scalpel on bleached rag/towel. Sterilize on 90% alcohol flame lamp until the tip glows red for a while (also heating the start of the handle and everything leading up to it). Remove the lid, hold the lid, cut the agar into rice size wedge. Take the piece out on the scalpel, close lid. Open lid on receiving, put myc facing up on the center of the dish. Close lid. Doing all this with as little movement as possible, slowly. Like a tortoise.
For the No-Pours I recall the wedge got stuck on the scalpel edge, couldn't simply wipe it to the agar below so I loosened it on the lip of the PP5 receiving container. That might not be so clean, but it wouldn't come off the tip 
I always wipe then flame sterilize the blade, never the other way around. Doing this between each transfer. Ill also spray more bleach on my hands and inside the SAB in-between transfers. I always wear a mask and gloves though sometimes I reuse the nitrile gloves and re-sanitize them with bleach.
Sometimes I would put the lid adjacent the dish so I could hold the transfer plate in one hand to make it more stable, maybe I dropped somethings onto the plates.
Basically I shouldn't A) move too fast B) have unsterile items above any open plate or inner lid. Including my hands.
Edited by huey.bluey (09/15/23 12:08 PM)
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Sun-flare
🍄Padawan



Registered: 11/05/18
Posts: 136
Last seen: 7 months, 5 days
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Re: What are these contaminants and is any of this salvagable [Re: huey.bluey]
#28470069 - 09/15/23 01:40 PM (1 year, 3 months ago) |
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Quote:
huey.bluey said: Hello I have 6 distinct plates that show random spherical abnormalities away from what Ive transferred. These are all T1's from spore plates.
I notice these white rings appear around the edges and away from the transferred material. Can someone identify what these contaminants are called? Also could I try transferring away from most of these contaminants or should I just compost the agar within them and try again from T0?
Thank you for the help
1) 2) 3) 4) 5) 6)
Plate 4,5,6 have some good growth IMO. You can make transfers and clean them. It is good to preprare your plates at least few days before using them. To see if you have properly sterilized them. I usually aim for 7+ days, which may be overkill… Also I would also leave the bleach and stick to the iso.
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