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InvisibleBaba Yaga

Registered: 09/13/20
Posts: 698
Long Term Culture Storage On Dehydrated Grain + Revival Tests * 4
    #26943501 - 09/19/20 06:01 AM (1 year, 1 month ago)

Long Term Culture Storage on dehydrated Grain

This is an Experiment
about storing cultures long term (5-10 years). The storage method is dehydrated grain spawn and a first revival attempted after
8 month of dry storage turned out successful which lead me to do this write up. Further tests to revive the same cultures will be carried out in
intervals of a year from now on. This is nothing new, I just want to show a practical/easy way to produce dehydrated grain spawn on a small scale.
It's still early days for my experiment, but the proof of concept is there and cultivation performance as well as long term performance of
the cultures after a few years in storage will have to be determined which is especially of importance for clones and isolates but as long as you are
getting a few fruits to print you are up and running again and maybe reviving 5 year old grains turns out to be easier than getting 5 year
old spores going.

TL;DR: There is a final conclusion on this experiment at the end of the post for those who want to know how this turned out first but feel free
to read through the whole post if you are inclined to try it yourself so you can do things differently and go beyond the point where I ended.



It was brought to my attention by Pastywhyte that drying the grains in a fridge will not work as well or not at all in tropical climates (hot & humid)
which I wasn't aware of. The model and age of your fridge will also have an impact on the efficacy of this method I guess. In these cases
you might want to look into storing cultures in distilled water as this seems to be a good option as well, even at room temperature.
Long explanation cut short: This method worked for me so far and I am not claiming it will be for everyone but feel free to experiment :cookiemonster::elmo:



Well I was curious about alternative methods to store cultures long term. Currently I do use just petri dishes and liquid sample containers which I use as "slants".
This is all good and works pretty well although I would like to reduce my workload eventually by not having to re-slant all my cultures every year or two.

Through some research I found the following two methods which seemed to be the easiest to achieve with what I already have and do:

- storing mycelium in sterilized water or a low nutrient broth

- storing mycelium as dehydrated grain spawn

I mean there were more options mentioned such as storing in sterilized sand and perlite (both soaked in a low nutrient broth), frozen, grown
out on filter paper and then dried etc., but those methods are requiring extra equipment/material and/or do require extra preparation.
At the bottom of this post you can find a list of a few papers on this topic that I found and read during my research, a simple web search
should get you the PDFs.


Aiming for maximum convenience in creating a long term storage medium I finally narrowed it down to the dehydrated grain spawn as this seemed
to be the most robust method to store a culture and most adequate for room temperature storage. There is no moisture/liquid and cooling
involved which I liked cause it's something new as everything else I got here that has mycelium in or on it is either slightly moist, dripping wet or at field
capacity and has to go in the fridge if I want to keep it around for longer.




The Method

The method I use to create dehydrated spawn is quite simple and goes along nicely with usual cultivation work.


STEP 1:

I transfer a good spoon full of spawn (50-80 grains) to a sterile petri dish without agar, the grain can be poured like when doing G2G. Other containers
might work as well as long as they are sterile but I think they should allow for lots of GE in order to dry out faster.


STEP 2:

The petri dish gets wrapped with micropore tape. I gave it a couple of layers and used some tension to avoid the tape from creasing too much and any
creases that formed were flattened out to make a good seal. The photo below is just a dummy dish for illustration purposes.



This protects the plate from getting contaminated during the time in the fridge.


STEP 3:

The dish gets placed in the fridge, best place is one of those covered compartments on the door.



As the cold air in the fridge has naturally a low rH the grains should dry out fairly quick. I do have one of those fridges that has an automatic defrost and I don't know
how much of an influence that has on the drying time. I check on the grain every now and then and remove the plate when the spawn seems to have dried out.


STEP 4:

Once the grains are dry the plates are stored it in an air tight container. I used to put mine in vacuum sealer bags and sealed them without pulling a
full vacuum but I just put them in an airtight container now and replace the micropore tape on the plates with cling wrap.



Here are two plates of grains I prepared







Revival Test Part 1

I've prepared a few plates like this, 10 species in total. For my first test I used Oyster and Semilanceata cultures because I had plates
of these species in cold storage which I roughly made at the same time as the grains, so good to have a some sort of comparison.

The process is very simple just take a grain or two and put it onto an agar dish, just regular standard agar recipe, nothing special.

First Revival Test after 7/8 month in dry storage:

It took a week to show first signs of growth on the grains and I let it grow out for about a week before taking the photos so this is ~2 weeks after the transfer.
left: semilanceata after 7 month dry storage, right:pleurotus after 8 month dry storage




The control cultures from cold storage were doing a little bit better after ~2 weeks from transferring onto new plates.




Side by side after ~2 weeks




Side by side after ~3 weeks




Side by side after ~4 weeks




This definitely gives you something to work with, the new growth looks very clean. The dehydrated grain cultures are also catching up but I
managed to get some bacteria onto my cold stored Ps. semilanceata plate while taking a transfer. Will try these two cultures again in a years
time together with other cultures that I'm going to prepare in the meantime and I will update this thread with more results. If I manage
to revive 2 year old cultures I will start and fruit some of them to see how they are going to perform.





Revival Test Part 2

This is the second round reviving some of the dehydrated grain span I made. This time I am not dropping the kernels to agar but straight into
some LC broth.



The broth is 1% nutrient strength using LME and a drop of corn sirup. In this run I am reviving the following species:

Panaeolus cambodgeniensis, Psilocybe semilanceata, Pliscybe subaeruginosa,
Psilocybe cubensis (amazonian), Lentinula edodes 75 and Pleurotus pulmonarius

and here is the list of the samples and results. This list will be updated as results are coming in.

Grains dropped to LC on June 12 2021

Formula: 0



RESULTS:

The cultures PanCam "B" and PanCam "c" showed growth after 2 days they both were made 4 month ago.

This is PanCam "C" after 2 days and after 10 days, it came out clean when tested on agar and was put to grain showing good growth.




Spawn kernels of Pan cam I & Pan cam II have shown growth after 10 days in the broth.

This is Pan cam II after 10 days and I have put the LC to sterilized horse manure + straw substrate once it was full grown without testing on agar first.




After 4 weeks non of the other kernels showed any growth. I thought dropping the grain straight to LC would revive the cultures faster but maybe this
is not the way to go. I also didn't like that I was not able to verify the cleanliness of the culture without testing the LC on agar. This is why I
will keep dropping the grains to agar from now on as this will show me straight away whether the culture is clean or not unlike LC which leads me to
the next installment of this experiment.


Revival Test Part 3

As said above I am dropping grains to agar again. This time around I am testing the following species:

Panaeolus Cambodgeniensis (x3), Cyclocybe aegerita, Psilocybe papuana, Gymnopilus purpuratus, Psilocybe subaeruginosa,
Psilocybe semilanceata, Psilocybe cubensis, Lentinula edodes 75 and Pleurotus pulmonarius

and here is the list of the samples and results. This list will be updated as results are coming in.

Grains dropped to agar on July 5 2021

Formula: 1

It is not easy to determine when exactly new grows starts, on some kernels it seems that the first growth happens underneath the kernels hull/skin and
it takes a while to get through to the surface. Once the mycelium has broken through though the growth is quite vigorous.

The temperatures are currently a bit low here and I am not home much so all I can manage to do is to keep them at 58-68oF (14-18oC)
which makes for a relative slow growth rate.


RESULTS:

PanCam "B"

after 5, 9 and 15 days on agar




PanCam "C"

after 5, 9 and 15 days on agar




GymPur

after 5, 9, 15 and 22 days on agar




Papuana

after 15 and 22 days on agar




Poplar

bacterial growth showed after 2 days. Mycelium has started to grow as well after 8 days but it's still to thin to get a good shot of it. The visibility
of the plates are poor but you can see some of the hyphae relecting light on the second image. The mycelium well outgrew the bacteria after 22 days making
it possible to easily transfer.

images after 8, 15 and 22 days




Conclusion for Test #3 so far:

This seems to work better than dropping kernels straight to LC, "PanCam I" showed growth after 10 days in LC broth during the previous
test but still doesn't show anything here yet. This does indicate that it would be necessary to drop more than just a couple of kernels
onto the plates to get a more average result as some kernels might not be viable or take longer to reactivate. In the next round I will
focus on the cultures that haven't produced any growth so far and will play the numbers game by dropping ~10 kernels onto each plate.
In the meantime I will let the currently unproductive plates run for another 4 weeks.



Revival Test Part 4

This will be the last test trying to revive dehydrated grain spawn. This time I will try to revive the grain that hasn't shown any grows in
the previous two tests. What I'm doing different this time is putting a lot more grains on each dish to get a better chance of having viable
kernels on the plates. Each plate has between 30 and 50 kernels. I still got some more dried cultures that I haven't tested yet but will leave
that for sometime later.

Cultures tested this time are: Subaeruginosa from LME LC, Subaeruginosa from BRF LC, Semilanceata, Cubensis, Shiitake 75 and Phoenix Oyster.

Grains dropped to agar on August 11 2021: 

Formula: 2



RESULTS

Sub BRF

3 out of ~50 kernels of the Subaeruginosa spawn made with BRF LC are showing new growth after 7 days on agar. Second image of fully colonized plate after 18 days.




Cube

Finally after 18 days one of the cube kernels is showing growth. This spawn was dehydrated 1 year and 7 month ago. Two more kernels showed growth after
20 days and the plate fully colonized eventually, second picture is a 1st transfer from the grain plate which shows clean growth and finally the grain plate
pinned after a couple more weeks.



All remaining plate were finally tossed after 35 days.


Final Conclusion

Well this wasn't the success I was hoping for but it wasn't a total failure either. I am sure that with a more refined drying method this
would work much better but I was aiming for the easiest way that was available to me at the time. If I would do this again I would dry the
spawn under controlled conditions in a climate chamber with a saturated salt that buffers the humidity level at around 10% so all grains dry
under the same conditions and will end up with the same moisture content. If I could I would also run tests with a freeze drying setup but
this will probably never happen. I'm focusing more on other long term storage methods now and currently I am putting together a collection of
my cultures as agar wedges in distilled water and LCs with a nutrient content of 0.05% which will be tested for viability in a years time.

This was a fun project, lets see how the other methods turn out.

:sun:


--------------------------------------------------------------------------------

Titles of research papers on long term storage of mycelial cultures:
  • An example for comparison of storage methods of macro fungus cultures Schizophyllum commune

  • Evaluation of different storage methods for rapidand cost-effective preservation of Botryosphaeria species

  • Preservation of live cultures of basidiomycetes

  • Preserving cultures of wood-decaying Basidiomycotina using sterile distilled water

  • Saving-fungal-Cultures-Sterile-Water-By-Harris

  • Preservation of fungi in water (Castellani) 20 years

  • Preservation of fungal cultures of medical importance in distilled water




Edited by Baba Yaga (10/05/21 08:25 PM)


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OfflineZakkery
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Re: Long Term Culture Storage On Dehydrated Grain + Revival Tests Part One [Re: Baba Yaga]
    #26943570 - 09/19/20 07:39 AM (1 year, 1 month ago)

Dope. Didn't know this was a thing. I've got plates just sat in my fridge but this is a nice long term option.

Do you think the grain would last a while in the fridge too? I don't have a vacuum sealer just yet.


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InvisibleBaba Yaga

Registered: 09/13/20
Posts: 698
Re: Long Term Culture Storage On Dehydrated Grain + Revival Tests Part One [Re: Zakkery]
    #26943580 - 09/19/20 07:52 AM (1 year, 1 month ago)

nah you wont need a vacuum sealer necessarily I think, as I said I don't pull a real vacuum in the bags its just to store it air tight like you
would store dried fruits. wrapping the dishes a few time with cling wrap and putting them in a drawer would be fine as well I suppose, but I just
started this long term experiment and will be curious to see what happens next year. My plan is to get away from cold storage so i will keep
everything at room temp.

And by the way i do this parallel to my usual plate/slant in the fridge storage. If I can revive 3 year old grains then I will call it good
enough to switch methods. 


Edited by Baba Yaga (09/19/20 08:09 AM)


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OfflineForresterM
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Re: Long Term Culture Storage On Dehydrated Grain + Revival Tests Part One [Re: Baba Yaga]
    #26943613 - 09/19/20 08:25 AM (1 year, 1 month ago)

I've always thought dried mycelium would be good for storage, but never tested it.  Nice write up!

I think this would be an excellent method for tropical species like pink oysters that have trouble in the fridge as well.


--------------------
Have some medicinal mushrooms and want to get the most out of them?  Try this double extraction method.


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InvisibleBaba Yaga

Registered: 09/13/20
Posts: 698
Re: Long Term Culture Storage On Dehydrated Grain + Revival Tests Part One [Re: Forrester]
    #26944878 - 09/19/20 10:26 PM (1 year, 30 days ago)

Quote:

Forrester said:
I think this would be an excellent method for tropical species like pink oysters that have trouble in the fridge as well.




Yes I would try and use it on Pans as well, some do have a hard time bouncing back after a year in the fridge.


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InvisiblePastywhyteMFacebook
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Re: Long Term Culture Storage On Dehydrated Grain + Revival Tests (moved) [Re: Baba Yaga]
    #26952824 - 09/24/20 03:58 PM (1 year, 25 days ago)

This thread was moved from Mushroom Cultivation.

Reason:
Moved upon request of OP. I suggest it might be an idea to look into vacuum drying for the grains. Many people find that their fridges are damp, not dry. This may also be a more axenic friendly and a lot faster way to get the grains dry.


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InvisibleBaba Yaga

Registered: 09/13/20
Posts: 698
Re: Long Term Culture Storage On Dehydrated Grain + Revival Tests (moved) [Re: Pastywhyte]
    #26953086 - 09/24/20 06:51 PM (1 year, 25 days ago)

Thanks Pasty for your input, have edited the OP.


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InvisiblefahtsterM
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Re: Long Term Culture Storage On Dehydrated Grain + Revival Tests (moved) [Re: Baba Yaga]
    #26953557 - 09/25/20 12:10 AM (1 year, 25 days ago)

I use dried grains for storage as well.. I use special tyvek lids for drying the grain out.  I like the vacuum sealing.. gonna give that a go this next season.

Here’s the link to the lid set up https://www.shroomery.org/forums/showflat.php/Cat/0/Number/6886717/an/0/page/0

More procedure on extracting the glc later in the post.. might give ya some ideas

Faht


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PICTURE GALLERY

EVERYTHING FAHT, CLICK ME!




PHENO HUNTERS


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InvisibleBaba Yaga

Registered: 09/13/20
Posts: 698
Re: Long Term Culture Storage On Dehydrated Grain + Revival Tests (moved) [Re: fahtster]
    #26953724 - 09/25/20 02:33 AM (1 year, 25 days ago)

Thanks Faht, great stuff! Definitely some inspiration there, will also look into Agar's thread but can't access it yet (no permission) have to wait a couple of days.

I thought about small jars with just bands and filter discs, but I don't have any discs atm, also no tyvek. I will look into
getting some tyvek suits for asbestos removal, they are pretty heavy duty or maybe get some cellulose filter sheets for sterile filtering
from a brewers shop or so, I might be able to get that stuff local, wanted to try some with my grain jars anyway.

My fridge is hard to beat though, it is so dry that it will dry out the
outer leaves on a white cabbage if left in there for a week and it will make a hand full of small fruits cracker dry in 3-4 days, that gave me the
initial idea to use it for drying my grain spawn. It's pretty empty most of the time though apart from a box of eggs and a block of cheese
and my slants and dishes lol.


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InvisibleBaba Yaga

Registered: 09/13/20
Posts: 698
Re: Long Term Culture Storage On Dehydrated Grain + Revival Tests (moved) [Re: fahtster]
    #26953750 - 09/25/20 03:06 AM (1 year, 25 days ago)

How long does it take for your myc-water to bounce back into action when
taken from dried grains?


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InvisiblefahtsterM
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Re: Long Term Culture Storage On Dehydrated Grain + Revival Tests (moved) [Re: Baba Yaga]
    #26954046 - 09/25/20 10:18 AM (1 year, 24 days ago)

I’d say 2-3 more days than usual for the bounce back... myc is super resilient.

Oh yeah, the fridge is great for drying stuff out.  Lol, I actually have a little write-up about using the fridge to get cracker dry fruits in the summer when the humidity is high (it was before I had a dehydrator) ...now I just gotta remember where I put it.. here she is:

Quote:

I've posted this before but there are some new updates on it!! and they are good. lol ok, so when i've stated this before i said you should just stick the baggie in the fridge in the crisper drawer with the bag open and they would get about 10% drier in the summer when the RH  in the air is too much that your mushies don't dry completely no matter how long you have them under a fan. well... I was looking at this old crusty piece of pizza that i didn't cover in the fridge and it's rock hard after like three days... i can snap the thing in half. So i got the idea to use a breathable container to put them in the fridge with. i.e., a tyvek  mailer. yet another use for tyvek  from the P.O.!!  just put your partially dried mushies in a tyvek  mailer....



now just seal it up and toss it in your crisper! they will be cracker dry in probably less than a few days. some other benefits: it's sealed and you can't see whats in it... if someone saw it they probably wouldn't go ahead and break the seal on it. AND once you are done using it for drying, you can use the envelope to make self-healing jars lids or something. lol

fahtster




From this Thread

Great minds and all that :wink:. I will say that this last year I decided to store my grain jars in the fridge over the summer while I took a break to see if I could just start them up again in the fall (I use to store them at room temperature in a drawer.. I wanted to slow any growth down kinda like a slant)... it was a bad idea with my set up.. moisture definitely still got on the jars and made it’s way through my tyvek filters.. there was also glue residue on the filters from t tape I used to cover and make the lids.. it was moldy and made its way onto the grain.  That’s why I like your vacuum seal idea so much.. I’m going to use it to thwart the problem I had this summer.  Also gonna figure out a glueless route for the lids.

Faht


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EVERYTHING FAHT, CLICK ME!




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Edited by fahtster (09/25/20 10:41 AM)


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InvisiblefahtsterM
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Re: Long Term Culture Storage On Dehydrated Grain + Revival Tests (moved) [Re: fahtster]
    #27249296 - 03/12/21 05:40 AM (7 months, 5 days ago)

Just revived a 3 month old dried out clone on grain.. GLC’d the grains just like they were


Faht


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PICTURE GALLERY

EVERYTHING FAHT, CLICK ME!




PHENO HUNTERS


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InvisibleBaba Yaga

Registered: 09/13/20
Posts: 698
Re: Long Term Culture Storage On Dehydrated Grain + Revival Tests (moved) [Re: fahtster]
    #27249305 - 03/12/21 05:49 AM (7 months, 5 days ago)

Noice! 
:highfive:


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OfflineLukeTheDuke187
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Re: Long Term Culture Storage On Dehydrated Grain + Revival Tests (moved) [Re: Baba Yaga]
    #27270324 - 03/26/21 03:27 PM (6 months, 21 days ago)

Have you tried to dehydrate the grains before storage with a dehydrator maybe on a low temp setting?


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InvisibleBaba Yaga

Registered: 09/13/20
Posts: 698
Re: Long Term Culture Storage On Dehydrated Grain + Revival Tests (moved) [Re: LukeTheDuke187]
    #27270345 - 03/26/21 03:49 PM (6 months, 21 days ago)

Well you want to keep it sterile and I can't see how that could be achieved using a dehydrator. You could have the dishes sitting on a shelf
if you're in a reasonable dry climate but I would only put a few grains in each dish.


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InvisibleBaba Yaga

Registered: 09/13/20
Posts: 698
Re: Long Term Culture Storage On Dehydrated Grain + Revival Tests (moved) [Re: Baba Yaga]
    #27347354 - 06/14/21 12:47 AM (4 months, 4 days ago)

OK, here we go again.

Revival Test Part 2

This is the second round reviving some of the dehydrated grain span I made. This time I am not dropping the kernels to agar but straight into
some LC broth.



The broth is 1% nutrient strength using LME and a drop of corn sirup. In this run I am reviving the following species:

Panaeolus cambodgeniensis, Psilocybe semilanceata, Pliscybe subaeruginosa,
Psilocybe cubensis (amazonian), Lentinula edodes 75 and Phoenix oyster

Grains dropped to LC on 06/12/21

There is a table in the OP where I will update the results.


The pans are taking the lead with first signs of growth after only 2 days but the samples are much younger than the rest at 4 month old,
will be good to see if this is going to change once the pan samples are getting older. I hope this is not mold and the cultures are going
to be tested on agar of course.


First growth of the 4 month old pan cambo "C" on wheat kernels after only 2 days.



:sun:


Edited by Baba Yaga (07/13/21 11:05 PM)


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InvisibleBaba Yaga

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Posts: 698
Re: Long Term Culture Storage On Dehydrated Grain + Revival Tests (moved) [Re: Baba Yaga]
    #27354847 - 06/19/21 08:42 AM (3 months, 30 days ago)

UPDATE 06/19/21:

Have put the two Pan LC cultures to agar today to check for cleanliness.

Still no growth on the other grains after a week, might be because the
jars are sitting at or below 60F or 15C. (Not home much lately so no
heating the house either.)

Will keep them a bit warmer, wait another week and report back.

Otherwise I'll try again on soft agar.


Edited by Baba Yaga (06/22/21 02:36 AM)


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InvisibleBaba Yaga

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Re: Long Term Culture Storage On Dehydrated Grain + Revival Tests (moved) [Re: Baba Yaga]
    #27358364 - 06/22/21 03:02 AM (3 months, 27 days ago)

UPDATE 06/22/21:

Spawn kernels of Pan cam I & Pan cam II have shown growth after 10 days in the broth.
There was nothing when I looked this morning but now there is clearly visible growth.


This is Pan cam II:



I really thought that the oyster would have poked out their head by now.

This would probably go a lot faster if kept at normal room temp so at the moment I am
not focusing on the speed cause this test was kept at relatively low temps.
There is growth is all that counts.


This is sample PC "C" after 10 days:



I have put some myc of this on agar but its not enough growth yet to snap a pic. I'm kinda tempted to just put it to a couple of grain jars.
I know, lazy right?!


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Re: Long Term Culture Storage On Dehydrated Grain + Revival Tests (moved) [Re: Baba Yaga]
    #27359622 - 06/23/21 06:43 AM (3 months, 26 days ago)

Thanks a lot for sharing


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InvisibleBaba Yaga

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Re: Long Term Culture Storage On Dehydrated Grain + Revival Tests (moved) [Re: gt40]
    #27386658 - 07/14/21 12:39 AM (3 months, 5 days ago)

Revival Test Part 3 is in progress now and has started on July 5th.

This time I am dropping grains to agar again. Have a look at the OP for the first update on this. I will ad further updates as results are coming in.


Edited by Baba Yaga (07/14/21 04:37 AM)


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