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ShroomBoy
Stranger
Registered: 02/17/09
Posts: 13
Last seen: 7 years, 20 days
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Spore washing experiment
#9842863 - 02/22/09 08:16 AM (15 years, 28 days ago) |
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I am asking because I received a contaminated syringe from Ralphster , look at this post;
I would like to find a way to wash the rest of my syringe to cultivate that brazilian cubensis strain . What would you recommand?
I am wondering if it is possible to unload a syringe in a sterile glass, working in a highly sterile environment, and then add a drop of chlorine to kill all germs inside the water containing the spores, then give it an hour or two in that sterile environment to let the chlorine evaporate, then refill the syringe, and inoculate with the decontamined syringe. Is it possible?
I have also read about a diluted bleach experiment on this post and a filtration experiment on this post. The filtration seems difficult because you need some specialized equipment, but the bleach technique seems interesting. What about chlorine? Any experienced grower can help me on this one?
Thank you for your precious help, youre the only ones I know on the web who has knowledge to help me on the subject.
Thank you
ShroomBoy
Edited by ShroomBoy (02/22/09 09:15 AM)
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obi
Du Bois
Registered: 07/19/06
Posts: 582
Loc: Europa
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Re: Spore washing experiment [Re: ShroomBoy]
#9842872 - 02/22/09 08:25 AM (15 years, 28 days ago) |
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I have no experience with 'spore washing' but why don't you follow RRs advice and transfer away from the contaminants?
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neopet nub
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Re: Spore washing experiment [Re: obi]
#9842920 - 02/22/09 08:51 AM (15 years, 28 days ago) |
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Links are broken FYI
-------------------- Ego death from weed!
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califarmer619
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Registered: 10/17/08
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Re: Spore washing experiment [Re: neopet nub]
#9842941 - 02/22/09 09:02 AM (15 years, 28 days ago) |
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innoculte some agar and isolate good sectors to create a clean culture. I know nothing about washing the spores. Im sure that if somone could revive a single spore then this could be done
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ShroomBoy
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Registered: 02/17/09
Posts: 13
Last seen: 7 years, 20 days
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I know his advice sounds really good, but the mycelium development actually has stop growing on the agar and what has developed does not appear to be very rhizomorphic, so I will need to inoculate once more from the contaminated syringe. That does not make sense. So I need a cleaning method for the syringe. I know this is complex, but we are in the advanced mycology section, I believe these kind of problems deserve to be investigated seriously. As we develop our expertise, future growers will benefit this expertise and so our comunity.
I will eventually do the bleach method and report my experiment. I simply wanted to share if any had knowledge about cleaning spores.
Thank you
ShroomBoy
Edited by ShroomBoy (02/22/09 09:16 AM)
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JaComet
Old Hand
Registered: 11/12/02
Posts: 347
Loc: Out Yonder
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Re: Spore washing experiment [Re: ShroomBoy]
#9845480 - 02/22/09 05:53 PM (15 years, 28 days ago) |
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I recall a technique for primary cleaning of wild prints using syringe filters of two different grades. The first allowed spores and smaller particles to pass through. The second trapped the spores and allowed bacteria and endospores to pass. The filter was then backwashed to recover fairly clean spore solution for plating out. All with your best sterile conditions, of course.
Forget the micron sizes used.
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Workman
1999 Spore War Veteran
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Re: Spore washing experiment [Re: ShroomBoy]
#9847259 - 02/22/09 10:13 PM (15 years, 27 days ago) |
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Cleaning the spores from a contaminated syringe can be futile. Bacteria in the syringe can multiply to huge numbers and will kill dormant spores. Spore washing is a known technique but always involves dry or freshly collected spores, not those stewing in water for who knows how long.
Antibiotic agar is your best chance, assuming any spores still live. Good luck!
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JaComet
Old Hand
Registered: 11/12/02
Posts: 347
Loc: Out Yonder
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Re: Spore washing experiment [Re: Workman]
#9848610 - 02/23/09 05:28 AM (15 years, 27 days ago) |
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Quote:
Workman said: Bacteria in the syringe can multiply to huge numbers and will kill dormant spores.
Good point. Well taken.
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Terillius
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Re: Spore washing experiment [Re: ShroomBoy]
#9850012 - 02/23/09 12:45 PM (15 years, 27 days ago) |
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I always add bleach when I make syringes from prints. A couple squirts of 1:10 bleach water from a spray bottle in the bottom of a shot glass is enough to kill bacteria without killing all the spores.
Dilution of the remaining liquid might help with bacteria also. What was a murky nutritious muck would become a desolate wasteland if you doubled the volume of water.
You might also try starting them on cardboard. Chances are slim that your bacterial infection will grow on some good old corrugated.
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ShroomBoy
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Re: Spore washing experiment [Re: Terillius]
#9850696 - 02/23/09 03:02 PM (15 years, 27 days ago) |
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Thank you for these precise informations JaComet, Workman and Terrilius, this knowledge enlarge our spectrum of understanding the mushroom universe. I know few have investigated into syringe cleaning, but I find this challenging and allows us to develop our collective knowledge in that area.
I contacted Ralph again, he told me that his syringes are made fresh, just prior to shipping and not stored. Once I received the syringe, I stored it in a refrigerator. So I don't fear bacteria in the syringe have multiplied to huge numbers and killed dormant spores.
Since syringes filters are difficult to find and expensive, I am thinking about spraying diluted bleach water 1:10 in a clean glass, then unloading the contaminated syringe in that sterile glass, working in a highly sterile environment, then give it an hour or two in that sterile environment to let the chlorine evaporate (chlorine evaporates at -34.4 °C), then refill the syringe, and inoculate with the decontamined syringe. Is it possible? What do you experts think of that?
ShroomBoy
Edited by ShroomBoy (02/23/09 04:18 PM)
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lipa
Registered: 07/24/07
Posts: 2,684
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Re: Spore washing experiment [Re: ShroomBoy]
#9851133 - 02/23/09 04:19 PM (15 years, 27 days ago) |
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Like workman said anti-agar will be you're best bet. If it follows the mycelium after germination just take a wedge up and turn it over on another anti-agar plate sandwiching the mycelium between the two and push it down into the agar firmly so the hyphea has to grow through the anit-agar. It will clean up well afterwards. That is if there is no other molds you have to deal with. Sometimes works by making the mycelium travel up through cardboard too although the former works better most of the time. You can also use the piping hot agar tech that RR uses after they germinate.
Lipa
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Terillius
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Re: Spore washing experiment [Re: ShroomBoy]
#9851289 - 02/23/09 04:43 PM (15 years, 27 days ago) |
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Quote:
ShroomBoy said: Since syringes filters are difficult to find and expensive, I am thinking about spraying diluted bleach water 1:10 in a clean glass, then unloading the contaminated syringe in that sterile glass, working in a highly sterile environment, then give it an hour or two in that sterile environment to let the chlorine evaporate (chlorine evaporates at -34.4 °C), then refill the syringe, and inoculate with the decontamined syringe. Is it possible? What do you experts think of that?
Why do you need to let the bleach evaporate? Once the bleach is gone you have lost its protection. If the bleach isn't going to kill the spores in a few hours of soaking then it can't hurt to just immediately inoculate.
I think it has a good chance of working, but you may want to keep going with agar so you don't waste any grain (if that isn't your plan already).
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No_Life_G33k
Now with 10%less noobness
Registered: 03/08/05
Posts: 356
Last seen: 13 years, 11 months
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Re: Spore washing experiment [Re: ShroomBoy]
#9884938 - 02/28/09 02:53 PM (15 years, 22 days ago) |
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Quote:
ShroomBoy said: I am asking because I received a contaminated syringe from Ralphster , look at this post;
I would like to find a way to wash the rest of my syringe to cultivate that brazilian cubensis strain . What would you recommand?
I am wondering if it is possible to unload a syringe in a sterile glass, working in a highly sterile environment, and then add a drop of chlorine to kill all germs inside the water containing the spores, then give it an hour or two in that sterile environment to let the chlorine evaporate, then refill the syringe, and inoculate with the decontamined syringe. Is it possible?
I have also read about a diluted bleach experiment on this post and a filtration experiment on this post. The filtration seems difficult because you need some specialized equipment, but the bleach technique seems interesting. What about chlorine? Any experienced grower can help me on this one?
Thank you for your precious help, youre the only ones I know on the web who has knowledge to help me on the subject.
Thank you
ShroomBoy
would it be bad form to nominate this for the 'I fucked it all up" thread????
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kaliman
Researcher
Registered: 02/12/08
Posts: 47
Last seen: 3 months, 27 days
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Re: Spore washing experiment [Re: ShroomBoy]
#9888214 - 03/01/09 02:09 AM (15 years, 21 days ago) |
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I have done that chlorine thing last year at it worked somehow. Kind of 50% success. But its a risk and maybe not a necessary one. I may sound silly but the best way to go is to ask Ralphster for a replacement and start again? Or buy a new syringe as they are not really that expensive, the time, money effort invested in cleaning your culture will be greater than that. Otherwise the best option can be to clean the culture and then you got the teks of gentamycin agar and sandwich agar, that should be in the archives. I only would go into cleaning if its a rare or unavailable strain.
Good luck!
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ShroomBoy
Stranger
Registered: 02/17/09
Posts: 13
Last seen: 7 years, 20 days
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Re: Spore washing experiment [Re: kaliman]
#9909982 - 03/04/09 12:01 PM (15 years, 18 days ago) |
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Thank you for all these advices, I guess this strain is not rare enough that I will make all these efforts to save it. I still have some other strains left, I will use them instead. But I believe this discussion was really helpfull to develop on the topic of decontaminating a contaminated syringe.
I had another idea to add to this topic on decontamination, I guess if you expose your syringe to an intense UV light for a good moment, it might work. UV is known to kill all germs and contaminants, it could possibly kill all contaminants inside the syringe. UV would pass through the plastic of the syringe and kills contaminants. If anyone could discuss on this idea, this might be interesting.
I really enjoy the scientific aspect of mycology.
Thank you all,
Edited by ShroomBoy (03/04/09 01:43 PM)
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fastfred
Old Hand
Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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Re: Spore washing experiment [Re: ShroomBoy]
#9910561 - 03/04/09 01:46 PM (15 years, 18 days ago) |
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UV will not penetrate plastic. It doesn't even penetrate most types of glass all that well.
-FF
-------------------- It drinks the alcohol and abstains from the weed or else it gets the hose again. -Chemy The difference between the substances doesn't matter. This is a war on consciousness, on our right to the very essence of what we are. With no control over that, we have no need to speak of freedom or a free society. -fireseed "If we are going to have a war on marijuana, the least we can do is pull the sick and the dying off the battlefield." -Neal Levine (MPP) I find the whole "my drug should be legal but yours should be illegal" mindset disgusting and hypocritical. It's what George Bush and company do when they drink a cocktail and debate the best way to imprison marijuana users. -Diploid
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