What: How I do long term culture storage and propagation in LI. Basically blenderless li + Long Term Sterile Water Storage.
Why: I love LI and have great success with it. I combine the ideas of Blenderless LI and Long Term Sterile Water Storage and can just pour a super speedy jar of reliable culture any time I like. The LI can last for years at room temperature and using a Master jar and an Inoculation jar, I easily maintain and use a culture indefinitely with no additional hassles.
To me it seems easier and more idiot proof than LC. It could be argued that it is not as good for expansion as LC. I would argue that it is just as expandable, it is just that the expansion takes place on the agar plate where you can keep a better eye on it instead of in the liquid culture where mold has a better chance at hiding.
How: Store a clean agar culture in sterilized water in media bottles. The mycelium will continue to grow on the agar suspended in the water until it runs out of nutrition or air. Then it will go into stasis until it gets revived with more air and food.
This is how I perpetuate all of my favorite cultures:
Steps
- Sterilize distilled water in three quarter full media bottles.
- Get a very clean plate of a culture I want to preserve and propagate.
- Dice up the colonized part really fine (~1cm cubes) when it reaches about 75% coverage.
- Drop the finely diced myceliated agar into 2 of the prepared media bottles. One is Master the other is the Inoculation bottle.
- Shake the bottles once a day or month or not at all. Doesn't seem to matter much.
- When ready to use, shake vigorously and pour 1 to 10 ml from the Inoculation bottle into prepared grain any time after the first week.
- When the Inoculation bottle gets low, put a drop or two from the Master bottle on a couple of new plates and start over.
Infinite-ish culture. Cheap and easy, except for the media bottles.
Detailed instructions
Sterilize distilled water in three quarter full media bottles.
- I use 100ml to 500ml media bottles, doesn't really matter.
- Fill them 3/4 full with distilled water.
- With lids slightly loose, PC for 15 minutes at 15psi after venting.
- Let them cool in the unopened PC.
- Tighten lids and store until needed. If you tighten the lids while they are still hot, they will pull a vacuum as they cool and draw in air when opened. Probably okay in front of a hood, but sketchy in a SAB.
- They will stay sterile for a really long time.
Get a very clean plate of a culture to preserve and propagate.
- Clean a culture that you love on agar.
- My process for selecting is spores-> brf puck 1-> agar 1-> grain 1-> bulk 1-> clone to brf puck 2-> agar 2-> grain 2-> bulk 2-> if culture is good either use a pretty agar 2 to LI OR clone to brf puck 3-> agar 3 to LI
Each of the agar steps above include as many transfers as needed to get clean symmetric growth and keep culture active.
Dice up the colonized part pretty fine (~1cm cubes) when it reaches about 75% coverage.
  
Drop the finely diced myceliated agar into 2 of the prepared media bottles.
- One is Master the other is the Inoculation bottle.<pix>
Shake the bottles once a day or month or not at all. Doesn't seem to matter much.
- You will start to see growth on the agar and breaking off of the agar when shaken.
No Shake Light shake Vigorous daily shaking
- All three colonized fast, though the light shake and vigorous shake were faster on grain at first but the unshaken caught up soon enough.
 
Day 0 and day 9 of each.
When ready to use, shake vigorously and pour 1 to 10 ml from the Inoculation bottle into prepared grain any time after the first week.
When the Inoculation bottle gets low, put a drop or two from the Master bottle on a couple of new plates and start over.
- At first, I used a flame sterilized loop to fish out a bit of myc, but prefer a more ideal tool. These work great: (pimple popper tools)>. I flame sterilize them. Tilt the LI bottle in the flow or a SAB and grab a small bit of mycelium to transfer to a plate.
- Then let it grow out to ensure it is clean.
- At 75% repeat the steps above to make a new inoculation LI.
- If it is not clean, repeat the transfer to agar a few times determine if the problem is with the master or the sterile procedures at transfer. If the master is contaminated, do the usual agar clean up and create a new Master and Inoculation bottle as above.
Notes:
I have bought 100 ml media bottles on the 'zon. But the prices can fluctuate wildly, so any size is fine if the price is right.
Q: Is using distilled water an absolute must? Is demineralized/deionized water not good enough?
A: tldr: Ideally, no. For short term use, probably. For long term storage, maybe.
Demineralized and deionized water both leave behind trace biological and particulate matter that distillation removes. Since we are going to sterilize the water any way, you might think that this is good enough. However, although the sterilization process will kill any nasties, it will not remove them or their byproducts. So you will not end up with true stasis until any live mycelium consumes or ignores any remnant materials or harmful byproducts.
Senescence - Yes it is a real thing. In one study of 150 species of fungus, 26 species, (less than 20%) were found to senesce in 10 to 50 generations of agar subcultures. The rest had not after 50 generations and the end of the study. I do not know the rate of senescence failure of active mushrooms. The studies that I could find did not include them. I understand that a sufficient number g2g transfers will eventually result in poor performing cultures. However, in this method one media master bottle can produce thousands of agar plates in the first generation of subcultures over several years. That still leaves many generations and years to go on masters before needing to go back to spores and find a new culture. So you have a few minutes to work on that.
Some details on where this started.
LC from LI trial - Didn't really become an LC but it led to this idea when combined with the cryo vial storage in sterile dh2o threads.
Also, 'Cryovial Tek': Long-Term Storage of Cultures with Cryogenic Vials from Mycoplex.
See also Verum Subsequentis's thread on Master/Slave propagation, Munchhauzen's Blenderless LI & the end of Pastywhyte's EzLC for the section on Super EzLC.
What I have in LI storage (complete):
- Cubensis Starry Night APE -labar
- Cubensis SWPE - A potent non-blobbing lineage
- Hericium Erinaceus Lion's Mane - My first gourmet lineage -sponsor
- Ochraceocentrata - ElvishKlingonSith
- Panaeolus Cambo Komodo -PsiloPsychin
- Panaeolus Cyan Bunnell FL -PsiloPsychin
- Subtropicalis Bell Cap -RockinRobot
- Subtrobicalis "slow" -Hindsight-YoshiTrainer
- Subtropicalis "fast" -Melgo
- Tampanensis Pollock -Ambrose
- Tampanensis Pollock (regerm for potency) -Ambrose
What I am currently working to get into storage:
- Cubensis Burringbar AUS wild -3AM
- Cubensis Florida -Bra
- Cubensis Golden Teacher -Womble
- Cubensis Jack Frost -labar
- Cubensis Phantom-f5 ghost x PE -3AM
- Cubensis Slippery Crossing AUS wild -3AM
- Cubensis Stokers AUS wild -3AM
- Cubensis x7x+ -Nektar61
- Mexicana Chicon Nindo -PsiloPsychin
- Natalensis -YoshiArmano
- Panaeolus Bisporus Coleville WA-sponsor
- Panaeolus Bisporus Washington -Bra
- Panaeolus Bisporus - Mackload
- Panaeolus Cyan British Virgin Islands (BVI(ttbvi)) -sponsor
- Panaeolus Cyan Brooks FL -PsiloPsychin
- Panaeolus Cyan Burringbar AUS wild -3AM
- Panaeolus Cyan Kona Hi Rainbow Eucalyptus -PsiloPsychin
Waaay too many. I have to use a multidimensional spreadsheet to keep track of the progress of each and next steps. I will NEVER do this many at once again. Well..., unless it becomes my full time job. A man can dream, right?
Update: It got out of hand and I had to put some to cryo vial storage before reaching an ideal culture. In theory, I can pull them out again and continue to work them to my satisfaction before going to LTLIS (Long Term Liquid Inoculant Storage).
Edited by ReverendMyc (06/24/25 12:18 PM)
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