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OfflineLogicalElk
Stranger

Registered: 05/27/20
Posts: 5
Last seen: 3 years, 4 months
[Need some Help] Back on track Agar Salvaging Spores logbook
    #26700235 - 05/27/20 07:37 PM (3 years, 7 months ago)

Hi guys.

After a long break in mycology I am trying to come back to the hobbie.

Sadly back then I didn't properly store my spores syringes. I made them +1,5 years ago and stored them in a dark place at room temperature so I am afraid the viability is not the best. What follows in this post is basically me trying to salvage that lovely mushies from the pass of time and contamination, I am quite desperate to be honest because I won't be able to source another syringe or print (I live in a shithole country + Covid19).

Before explaining all my experiments so far I apologize: I haven't waited enough for the agar to reach 47C, and almost all my petris got a lot of condensation, not allowing me me to share with you proper pictures.
From 27/05/20 I am only going to work with properly poured agar plates.


In the next few days I'll be editing all this post in order to make it more clear and I'll try to add the pictures of the plates mentioned.

Objective: Salvaging and isolating live mycelium by any means from the spore syringes!

Experiment 1:

02/05/20

I tried noccing the MS solution directly to a sterile popcorn jar. It always worked with properly stored (refrigerated) 6 month old syringes.

09/05/20
This time, out of 11 jars I got 6 without neither growth nor contam, and 5 with both grow and bacteria :frown:, tossed all the jars but 2 with mycellium and bacteria to do further isolation.

Conclussion: The syringes developed bacteria and maybe another nasty microorganisms, but I still have some viable spores in my Syringes.

Experiment 2:

11/05/20
Sooo I've decided to get into agar to get a proper and clean mycelium: I made some BRF agar plates  (400ml water, 9g agar-agar, 6g BRF) and inoculated them with 1 drop each in the middle of the other MS syringe (equally old).

14/05/20
3 days after that I've noticed a really faint, lighting patterned growth in 11/11 plates with no signs of contamination (YAY!) 6 days after that the mycelium was still super faint but expanded all over the plate and started to form knots. This is how almost all plates looked: https://imgur.com/a/Fk9d45f
sadly the next day that knots turned to be green mold, I must admit that I screwed up not properly parafilming or taping the petris right after inoculating, my suspicion is that the mold was not on the syringe but on the air (look at the pattern of moldy spots). Tossed without opening all the petris.

Conclusion: My syringes are viable! Next time I'd better parafilm right away after noccing the petris, gotta repeat this experiment.

Experiment 3:

18/05/20
I picked a healthy grain from experiment 1 and transferred it to the middle of the dish with 50mg/L of gentamicin BRF agar in order to clean the mycelium from those nasty bacteria. I made 2 plates like this.  (Named this dishes G1 and G2)

Also in another 2 plates of the same agar instead of transferring and letting the grain in the dish I scrapped in zig zag fashion a healthy looking mycelium grain. (R1 and G2)

24/05/20
In the G series the mycelium looked nice and expanded in a cleanly ring fashion from the middle grain.
In the R series the mycelium did not grow as well and got overrun by what looks like bacteria/yeast (gentamicin resistant bacteria?). Seems like the mycelium doesnt like to be scraped or at least it takes more time to recover than a transfer.
So this time I took a wedge from G1 and placed to another gentamycin BRF
agar petri dish, repeated 3 times and called this G`1A, G`1B, G`1C.

28/05/20

Today I can see the mycelium growing slooooowly on G`1A, G`1B, G`1C BUT also all the plates look contaminated with bacteria/yeast meaning that maybe G1 looked clean but actually got contaminated with some sneaky contam.

Future experiments:
1) Repeating experiment 1 noccing the agar with a drop and spreading it with a inoculating ring in a zig zag pattern.
2) Finding a way of isolating G1 mycellium from the yeast/bacteria that are resistant to gentamicyn. Maybe taking a tiny aerial mycellium bit and transferring to another plate?
3) Going back to the roots and PFteking one of my old syringes in order to get at least 1 precious fruit and get some semi-clean spores to work with.



Thank you for your answers/comments/advice and sorry for my rusty english, not my first language. If i succeed in this daunting task I'll toast for you all!


Edited by LogicalElk (05/27/20 08:34 PM)


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Invisiblepanne cyanne
albino queen
Female

Registered: 04/15/20
Posts: 145
Re: [Need some Help] Back on track Agar Salvaging Spores logbook [Re: LogicalElk]
    #26702255 - 05/28/20 05:06 PM (3 years, 7 months ago)

you may just simply need new spores.

as for the agar plates condensation or any other container condensation ,
simply place all contianers straight out of the cooker into a plastic tub with desiccant for 24 48 hours.
all condensation will vanish.


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OfflineLogicalElk
Stranger

Registered: 05/27/20
Posts: 5
Last seen: 3 years, 4 months
Re: [Need some Help] Back on track Agar Salvaging Spores logbook [Re: panne cyanne]
    #26702335 - 05/28/20 05:50 PM (3 years, 7 months ago)

That's the problem, sadly I am not able to source any more spores, that is why I am trying to save the ones I've got.

Nice tip! I have some silica gel desiccant mini bags (the ones used to ship stuff). Do you think one will be enough for 10 plates or should I use more?


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Invisiblepanne cyanne
albino queen
Female

Registered: 04/15/20
Posts: 145
Re: [Need some Help] Back on track Agar Salvaging Spores logbook [Re: LogicalElk]
    #26702381 - 05/28/20 06:14 PM (3 years, 7 months ago)

i use like whole cans of damp rid or drierite.
works fast.
not sure how well packets work.
im always shocked more people dont do this.


Edited by panne cyanne (05/28/20 06:14 PM)


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