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Skysearch
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Registered: 09/21/19
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Seeking Advice on Growing with Agar
#26344925 - 11/25/19 03:19 PM (4 years, 2 months ago) |
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Hello everyone,
I have just started getting into cultivation and done a fair amount of research and decided agar to grain to tub is my best bet. I made 25 plates of agar and inoculated 8 of them directly with a syringe of APE in a SAB, parafilmed the dishes, and put them in a sterilized plastic container. I have waited one week and I have checked my dishes and 4 out of 8 are growing vast bacterial colonies, 3 of them show no growth, and 1 of them appears to have somewhat successfully colonized. I was pretty much swimming in 91% rubbing alcohol the entire time inoculating and I'm sure I used enough inoculant to get things going from what I have gathered. My unused plates also show no growth so I know the contamination must have happened somewhere along the line while inoculating. Can someone please let me know what I can do to improve my success rate? Any advice is appreciated
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SpunkyMonkey88
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Re: Seeking Advice on Growing with Agar [Re: Skysearch]
#26344957 - 11/25/19 03:40 PM (4 years, 2 months ago) |
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Google "what alcohol concentration is most effective at killing germs" but to sum it up for you 91% alcohol is highly ineffective...
Your technique needs to be adjusted.
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SpunkyMonkey88
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Registered: 10/08/19
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Reread how to handle agar in a SAB and use 71% alcohol...thata all I got man. Did u flame your instruments?
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Skysearch
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I did flame my instruments and upon further reading I think I need to take another trip to the store and make some adjustments to my sterile tek. Also have read that syringe to agar may be a cause for contams, would it be better if I started with PF tek and started agar from tissue, or does syringe usually work out fine and my sterile tek just blows?
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curious.psychonaut
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Re: Seeking Advice on Growing with Agar [Re: Skysearch]
#26344982 - 11/25/19 03:59 PM (4 years, 2 months ago) |
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> swimming in 91% rubbing alcohol
- Better to dilute down some more. Higher alcohol concentrations denature proteins too fast, resulting in protective membranes around cells that can last until evaporation, which happens quickly at 91%. 70% penetrates cell walls better and is considered best for decontamination.
- Also keep in mind that many things survive alcohol exposure anyway. It can help, but is in no way a sterilizer. "Swimming" in it can produce drops/droplets which can carry contams.
> I have waited one week It's best to transfer from spore-noc'd dishes as soon as you see growth that you can reasonably cut & extract. (Later transfers should generally be allowed to grow, so you can assess the culture better and make sure there are no contams. Except when there *are* contams and you want to save the culture, then always transfer ASAP.)
> I'm sure I used enough inoculant to get things going The recommended amount from a syringe is one drop in the center of the dish. Less liquid is better. (Best is dry transfer from spore print of a tiny amount of spores.)
> Can someone please let me know what I can do to improve my success rate?
If it's a bad syringe there's little you can do except noc a few more times with less liquid and then try to clean up the best-looking cultures. The best procedure should include
- wiping the syringe with alcohol (as it will come above the receiving petri)
- flaming the needle and squirting a bit to cool it (not in the receiving petri)
- not letting hands come over receiving dish (even if gloved & sanitized), just the syringe
-------------------- My LAGM2020 grow log
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SpunkyMonkey88
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Registered: 10/08/19
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I wouldn't even use the syringe directly over the agar, adding a drop of water to agar usually moves around alot until it germinates. I'd put that drop on a sterile swab then do a stream across or an "S" partern with it, that would also eliminate having to put a dirty syringe over the plate... that's just me tho..
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Skysearch
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This is all very useful, thanks for the replies
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ChardRich
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Registered: 04/16/19
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Re: Seeking Advice on Growing with Agar [Re: Skysearch]
#26345019 - 11/25/19 04:18 PM (4 years, 2 months ago) |
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As suggested in your other post about this, try spore prints and an inoculation loop. Your syringe sounds like it was dirty af.
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