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Justsomedude
Stranger
Registered: 11/23/11
Posts: 29
Last seen: 9 years, 9 months
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100% failure. Where am I going wrong?
#18767558 - 08/28/13 08:24 AM (10 years, 5 months ago) |
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It's been a very long time since I posted but I've run into a major problem. Right now 100% of my work is contaminated with green. It's been getting steadily worse over the last few months. I need some help determining where the contam is coming from.
Here is my procedure
Dunk jars and lids in 10% bleach solution for an hour Rinse and clean wbs in bucket until water runs clear remove sunflower seeds Fill add boiling water to bucket of clean seed and seal the lid Let steep for an hour Strain seed from boiling water onto large screens and let sit for 1 hour or until water stops draining Scoop seed into pillow cases and massage to remove remaining water Scoop thin layer of vermiculite into bottom of gallon jars Scoop seed into jars until 3/4 full Place metal lid with 2 pencil sized holes punched onto jar Place synthetic filter disks over top of metal lid Place and tighten metal mason jar ring Cover top of jar with tin foil cap Pressure cook at 15 psi for 90 minutes Remove jars from pressure cooker and shake Allow jars to cool Unscrew mason jar ring slide filter disk back to reveal edge of punched hole Insert needle of multispore syringe and inject 1 or 2 cc of solution in a swirl Replace ring and tighten
How I made spore solution Pressure cook water at 15 psi for 30 min allow to cool Pressure cook a butter knife (spore scraping tool) in foil Full syringes with water wrap in foil and pressure cook Place cooked water spore print and other tools inside glove box Clean inside of glove box with 99 isopropanol Open tools and spore print Open cooked water Scrape sporeprint into clean water with tool Seal spore solution and shake. Attach brand new needles to syringes and draw solution Seal small hole in spore solution jar with micropore tape and refrigerate Any time I need to draw more syringes they get a brand new needle
Contam is happening in the jars. Does my procedure suck or is this just bad solution??
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Kizzle
Misanthrope


Registered: 08/30/11
Posts: 9,855
Last seen: 14 hours, 43 minutes
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Re: 100% failure. Where am I going wrong? [Re: Justsomedude]
#18767654 - 08/28/13 09:04 AM (10 years, 5 months ago) |
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The spores probably.
You should flame sterilize whatever you're transferring the spores with and it wouldn't hurt to flame the needles as well, even if they're new. There's a lot of opportunity for contamination between PCing a tool and using it.
Also I would only make one syringe at a time and limit the amount of spores for each syringe. More spores means more chances for contaminated spores and making multiple syringes from the same solution means if one gets contaminated they all do.
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Justsomedude
Stranger
Registered: 11/23/11
Posts: 29
Last seen: 9 years, 9 months
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Re: 100% failure. Where am I going wrong? [Re: Kizzle]
#18767703 - 08/28/13 09:21 AM (10 years, 5 months ago) |
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I was somewhat thinking the sporeprint maybe wasn't clean which in turn made bad solution. Previous solutions have been fine but the tail end of the last one seemed to contam things so I made a new batch. Since the new batch I've had complete failure. Guess I should redo that part.
Flame sterilization has to be until metal parts are literally red hot?
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Kizzle
Misanthrope


Registered: 08/30/11
Posts: 9,855
Last seen: 14 hours, 43 minutes
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Re: 100% failure. Where am I going wrong? [Re: Justsomedude]
#18767764 - 08/28/13 09:45 AM (10 years, 5 months ago) |
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You should> Then you'll know for sure they've reached a high enough temperature to guarantee they're sterile. That'd probably hard to with a butter knife. A paper clip would probably work better for transferring spores.
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Justsomedude
Stranger
Registered: 11/23/11
Posts: 29
Last seen: 9 years, 9 months
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Re: 100% failure. Where am I going wrong? [Re: Kizzle]
#18767890 - 08/28/13 10:18 AM (10 years, 5 months ago) |
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Will add flame to my procedure. I guess I'll have to switch to bleach to clean my glove box rather then the alcohol otherwise when I flame the needle and scraping tool after wiping the box down ill have a ball of fire in my living room.
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