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JMcDoogle
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Preserving cultures using sterile Distilled water in cryovials 1
#19001307 - 10/19/13 08:10 PM (10 years, 4 months ago) |
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Quote:
http://www.fpl.fs.fed.us/documnts/pdf1994/burds94a.pdf
This is the reference article -
Preserving Cultures Using Sterile Distiled Water in Cryovials
Abstract:
Prior to 1985, cultures at teh center for forest mycology research were maintained on a 1.5% malt extract agar test-tube slants. This system not only made it neccessary to transfer the entire collection every year but also permitted genetic change because continual growth occured. In 1985, the method of storing fungal cultures in sterile distilled water in CRYOVIALS was introduced. This study reports on the use of this method for long-term fungal storage. For varying periods of up to seven ( 7 ) years, 151 miscellaneous specials of wood-decaying Basidimycotina were stored in sterile distilled water. Water storage has numerous advantages: culture viability or growth rate is not significantly influenced; isolates can be stored longer; genetic stability is greater; the method is quick, easy, and inexpensive, and requires less space.
Cyrovials ( Internally Threaded & Self Standing
These can withstand autoclaving so if I were to buy them and try something along the lines of distilled water long term storage, I could always uses them as slighty more expensive alternative to the glass vacutainer blood collection tubes.
Heres what Im guessing - seven years, of unrefigerated storage is a possiblity, but even two years unrefigerated would be nice aswell.
Full Sterile - with option of threaded inside, or external - which would you prefer - internal or external threads?
Take a fully colonized grain, or spore print - isolate, isolate, gather your monoculture - inside of SAB/GB/FLOW/FKN LABRATORY
Take 4-6-8 ML of Sterile Distilled Water and put onto your agar monoculture
Scrape it a bit, mix up the myc. Suck it back up and put into the Cyrovial -
Than go about your business and save for 5 years and when needed re-enter onto agar - break down any contams if any - and sustain your strain? Or take fully colonized agar chunk, cut into 2x2 pieces and drop three or four into the cyrovial filled with sterile distilled water, seal and store. They feature a rubber type gasket internally and can be twisted completley air-tight aswell, so no need to wrap them.
Im new to all of this, so this all might be a huge waste of brainpower. Please leave any and all feedback, discreation unneccesary, so... what you think?
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Amanita virosa
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Re: Preserving cultures using sterile Distilled water in cryovials [Re: JMcDoogle]
#19026922 - 10/24/13 05:25 PM (10 years, 3 months ago) |
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That is an excellent article. Thanks for posting. Easy and cheap. I will try this as my culture collection has grown to the point that I don't want reslant once a year. Interesting that it seems to preserve the strain genetically as well. That to me is almost more relevent than the longer storage time. And he'll you could prolly just do it in micro centrifuge tubes. Smaller volume seems ideal for long term storage.
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matsc
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Re: Preserving cultures using sterile Distilled water in cryovials [Re: Amanita virosa]
#19028946 - 10/24/13 11:25 PM (10 years, 3 months ago) |
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This is more or less the procedure we use for long term storage in my lab. 2mL cryo tubes, agar plugs, sterile water. We have a whole bloody room full of the things.
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Terry M
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Re: Preserving cultures using sterile Distilled water in cryovials [Re: matsc]
#19042550 - 10/27/13 08:23 PM (10 years, 3 months ago) |
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My experience with any kind of sterile distilled water storage showed that it doesn't work well with all species. Fortunately, it works reliably with some temperature sensitive species. Volvariella volvacea is an incredible bitch to store. It's the most finicky of all tropicals with regard to cold. I've tried slants at various temperatures, but even the highest I used, not all that much under room temperature, killed it. Fortunately, it responds very well to room temperature sterile distilled water.
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Amanita virosa
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Re: Preserving cultures using sterile Distilled water in cryovials [Re: Terry M]
#19052808 - 10/29/13 04:37 PM (10 years, 3 months ago) |
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Quote:
Terry M said: My experience with any kind of sterile distilled water storage showed that it doesn't work well with all species. Fortunately, it works reliably with some temperature sensitive species. Volvariella volvacea is an incredible bitch to store. It's the most finicky of all tropicals with regard to cold. I've tried slants at various temperatures, but even the highest I used, not all that much under room temperature, killed it. Fortunately, it responds very well to room temperature sterile distilled water.
Good to know terry. What others have you tried and how did they preserve? I was thinking about abm and pinks.
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Terry M
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Re: Preserving cultures using sterile Distilled water in cryovials [Re: Amanita virosa]
#19056687 - 10/30/13 09:25 AM (10 years, 3 months ago) |
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I don't have any specific notes on abm and pinks, but I wish I had. I was in the process of converting from slants, when I discovered that SDW was unreliable. I got frustrated and failed to make any useful records. But info on storage of these tropicals would be very good to know, as they otherwise require a special temperature. As many have found out the hard way, normal refrigerator temperatures of 40 degrees F will definitely kill them. A mini-fridge controlled to 52 degrees F works perfectly.
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Edited by Terry M (12/24/13 11:42 AM)
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Terry M
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Re: Preserving cultures using sterile Distilled water in cryovials [Re: Amanita virosa]
#19149223 - 11/17/13 11:07 AM (10 years, 3 months ago) |
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Quote:
Amanita virosa said:
Quote:
Terry M said: My experience with any kind of sterile distilled water storage showed that it doesn't work well with all species. Fortunately, it works reliably with some temperature sensitive species. Volvariella volvacea is an incredible bitch to store. It's the most finicky of all tropicals with regard to cold. I've tried slants at various temperatures, but even the highest I used, not all that much under room temperature, killed it. Fortunately, it responds very well to room temperature sterile distilled water.
Good to know terry. What others have you tried and how did they preserve? I was thinking about abm and pinks.
Interesting news:
I was just clearing out some messy space in the basement and found a whole lot of STD vials containing assorted cultures. These are from 2011! That was when I had great hopes for SDW storage, and was buying vials and storing every culture in sight. I found some pinks, but don't think I have any ABM though I'm continuing to look. Did find some paddy straw, though.
I'm going to try and grow out a few of these 2 year old SDW cultures, but I have to be very selective as I don't want to divert a lot of time to this. If you have some other specific species you are interested in SDW storage of, let me know ASAP. I may just have it in my vial collection. Would then try to grow on a plate.
This won't be a rigorous experiment by any means, because I probably only have a couple of samples for each strain. But it may show which species it is possible to store in STD for a long time.
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JMcDoogle
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Re: Preserving cultures using sterile Distilled water in cryovials [Re: Terry M]
#19151576 - 11/17/13 08:08 PM (10 years, 3 months ago) |
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Keep us apprised.
Glad this thread came of some use!
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Tryptoman
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Re: Preserving cultures using sterile Distilled water in cryovials [Re: JMcDoogle]
#19163335 - 11/20/13 01:58 AM (10 years, 3 months ago) |
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Personally I like to transfer about 20cc of LC to some tubes and spin them down in a rough centrifuge. Then just drain off the supernatant and replace with dH2O (3x wash/spin cycle). This removes removes the nutrient broth while allowing you to combine and concentrate your LC samples. Then just store as mentioned above.
Note. If you don't have access to a centrifuge, try positioning your tubes within your laundry during the spin cycle (never tried it but it could work).
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forrest



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Re: Preserving cultures using sterile Distilled water in cryovials [Re: Tryptoman]
#19163412 - 11/20/13 02:50 AM (10 years, 3 months ago) |
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at second-hand-shops you often find these hand driven salad-spin (for drying washed green-salad) bins, wich you could adapt for microcentrifugaltubes
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Terry M
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Re: Preserving cultures using sterile Distilled water in cryovials [Re: forrest]
#19181737 - 11/24/13 08:33 AM (10 years, 2 months ago) |
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Good news!
My Pink Oyster and Paddy Straw have started to grow nicely after 2 days on plates. The SDW vials were made 2 years and 1 month ago. This is particularly important for the Paddy Straw, for which I have never found a cool slant survival temperature. And I've gone as high as 58 degrees F! CORRECTION FROM 68 DEGREES
The mycelium look like the real thing, but if there are any contaminants, I'll see them in the next few days. At some point, I will begin my "27 Year Plan." That's storing slants with a covering of mineral oil at room temperature. This will be my emergency, archival, when all else fails culture backup. Oil covered slants are messy, so it's not for regular use. This technique was described in Perrin, Peter W, 1979 "Long term storage of wood-inhabiting fungi under mineral oil," Mycologia, 71:867-869. Perrin revived all 30 cultures after 27 years of storage. I'm planning on using PP 12x50mm culture tubes and 12 mm dual position closure caps. Can get 1,000 for only $100.
Edited by Terry M (11/24/13 09:57 AM)
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Amanita virosa
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Re: Preserving cultures using sterile Distilled water in cryovials [Re: Terry M]
#19181780 - 11/24/13 08:52 AM (10 years, 2 months ago) |
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Quote:
Terry M said: Good news!
My Pink Oyster and Paddy Straw have started to grow nicely after 2 days on plates. The SDW vials were made 2 years and 1 month ago. This is particularly important for the Paddy Straw, for which I have never found a cool slant survival temperature. And I've gone as high as 68 degrees F!
The mycelium look like the real thing, but if there are any contaminants, I'll see them in the next few days. At some point, I will begin my "27 Year Plan." That's storing slants with a covering of mineral oil at room temperature. This will be my emergency, archival, when all else fails culture backup. Oil covered slants are messy, so it's not for regular use. This technique was described in Perrin, Peter W, 1979 "Long term storage of wood-inhabiting fungi under mineral oil," Mycologia, 71:867-869. Perrin revived all 30 cultures after 27 years of storage. I'm planning on using PP 12x50mm culture tubes and 12 mm dual position closure caps. Can get 1,000 for only $100.
Awesome!! That's great news for all of us.
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Pestile

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Re: Preserving cultures using sterile Distilled water in cryovials [Re: Terry M]
#19183160 - 11/24/13 03:27 PM (10 years, 2 months ago) |
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Very interesting!
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shopdropper
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Re: Preserving cultures using sterile Distilled water in cryovials [Re: Pestile]
#19183497 - 11/24/13 04:44 PM (10 years, 2 months ago) |
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very interesting. why wood inhabiting fungi? i wonder how well this would work for Claviceps. can you post that source paper please?
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Terry M
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Re: Preserving cultures using sterile Distilled water in cryovials [Re: shopdropper]
#19183632 - 11/24/13 05:19 PM (10 years, 2 months ago) |
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Quote:
shopdropper said: very interesting. why wood inhabiting fungi? i wonder how well this would work for Claviceps. can you post that source paper please?
Sorry, can't post the entire paper, as it's owned by the journal. But here's a public link to the first page, and it's only a 3-page paper. http://www.jstor.org/discover/10.2307/3759204?uid=3739888&uid=2&uid=4&uid=3739256&sid=21102986781717
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MrGumball
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Re: Preserving cultures using sterile Distilled water in cryovials [Re: shopdropper]
#19183796 - 11/24/13 05:57 PM (10 years, 2 months ago) |
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Quote:
shopdropper said:can you post that source paper please?
Volume 1-100 of Mycologia is available for free access at Cyberliber: An Electronic Library for Mycology.
Edited by MrGumball (11/24/13 05:59 PM)
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shopdropper
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Re: Preserving cultures using sterile Distilled water in cryovials [Re: MrGumball]
#19184926 - 11/24/13 11:06 PM (10 years, 2 months ago) |
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thanks.
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wildernessjunkie
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Re: Preserving cultures using sterile Distilled water in cryovials [Re: shopdropper]
#19187128 - 11/25/13 01:53 PM (10 years, 2 months ago) |
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Im curious to see how these SDW cultures turn out. This would be a good solution for me if it does.
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lipa

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Re: Preserving cultures using sterile Distilled water in cryovials [Re: Terry M]
#19200367 - 11/28/13 11:33 AM (10 years, 2 months ago) |
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Quote:
Terry M said: Good news!
My Pink Oyster and Paddy Straw have started to grow nicely after 2 days on plates. The SDW vials were made 2 years and 1 month ago. This is particularly important for the Paddy Straw, for which I have never found a cool slant survival temperature. And I've gone as high as 58 degrees F! CORRECTION FROM 68 DEGREES
The mycelium look like the real thing, but if there are any contaminants, I'll see them in the next few days. At some point, I will begin my "27 Year Plan." That's storing slants with a covering of mineral oil at room temperature. This will be my emergency, archival, when all else fails culture backup. Oil covered slants are messy, so it's not for regular use. This technique was described in Perrin, Peter W, 1979 "Long term storage of wood-inhabiting fungi under mineral oil," Mycologia, 71:867-869. Perrin revived all 30 cultures after 27 years of storage. I'm planning on using PP 12x50mm culture tubes and 12 mm dual position closure caps. Can get 1,000 for only $100.
Works very well with Phellinus and all pleurotus. I have a good 4 years with them in sterile distilled water and I pull them every year and run them. Started 4 years ago with 10 vials each. I have not had good luck with shiitake.
Lipa
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wbastz
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Re: Preserving cultures using sterile Distilled water in cryovials [Re: Terry M]
#19208250 - 11/30/13 11:31 AM (10 years, 2 months ago) |
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Hi Terry,
I recently recovered a culture of Volvariella volvacea that was stored at room temperature in a screw cap test tube since 23/12/2001 (almost 12 years).
The mycelium was growing nicely on agar , but unfortunately I lost it in the next day due a failure of the thermostat of the incubator, when the temperature reached 59 ° C.
The mycelium was kept in distilled water , without any piece of agar, just a big cluster of mycelium. The tubes I use has the following dimensions: 100 x 15mm diameter with 4 ml of distiled water in every tube.
The same material stored on agar and on the same date of inoculation died.
Cheers,
Wilson
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