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Help, White contamination on Culture

Posted: Wed Dec 02, 2020 6:14 pm
by JustinSmith
I'm a complete noob in tissue culture, this is my first try and sadly it failed. My cultures started to turn brown after just 3 days and white mold like contamination. I followed the guide by PhytoTech closely... I used 10% bleach and tween to sterilize my explants for 10 min . Also added ppm to the media. From what I read over the web the browning may be caused by to long time in the sterilization solution. But the white mold I don't seem to understand how it survived the 10 min soak time. They seem contradicting ... Because the long soak time should have killed all the contaminants. Also there is no visible contamination around the rest of the media seems to suggest the transfer was sterile. It's just that the explants were still contaminated. I will try 5 to 6 min soak next to prevent the browning of the explants or maybe some activated charcoal.. but I don't know how to deal with the mold ... I have been googling for days now... hoping for some direction and advice. Hope this forum could give me some insight thank you

Re: Help, White contamination on Culture

Posted: Thu Dec 03, 2020 5:13 pm
by hungry carnivores
VFT's never work using explant; can only realistically use in-vitro divs or seeds. Seeds are not a bad way to go, order some from Matt/Leah at FTS. Then, a 5 min soak in 5% bleach, move it to methanol/ethanol (Pure), then into an SDS solution ( I have used dishsoap). Then, rinse 4-5 times using autoclaved/microwaved sterile water. Plus, seeds grow pretty fast.

For me, the best and only explants I have taken are Heli pitchers. Rip them off, sterilize, and stab into media.

Did you use PPM? If so, I'd be down to trade plants with you for some supplies like agar, MS, and ppm.

Re: Help, White contamination on Culture

Posted: Thu Dec 03, 2020 11:21 pm
by jose
Matt got all his VFTs into TC using explants. HC: You’re using a bit of an odd sterilization procedure. Have you had luck with strikes on the heli pitchers?

Sterilization procedures vary drastically depending on size of explant and cuttings. Sounds like the OP burned the explants with bleach without killing the bacteria. This could be possibly due to rough cuts

Re: Help, White contamination on Culture

Posted: Fri Dec 04, 2020 3:22 am
by hungry carnivores
Yeah. I mean in a home lab lol. Wistuba can even get nepenthes leafs to differentiate. My sterilization process has worked twice for helis. They contaminate up the media due to symbiotic fungi, but methanol normally takes care of that.