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InvisibleAlkeides
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Registered: 04/11/20
Posts: 61
Lazarus Mushroom 1
    #26611655 - 04/18/20 10:10 PM (3 years, 9 months ago)

Hello friends, this is my first post on here. Before we begin I want to say all written posts/content, verbal/implied interactions, or media of any format uploaded to this website by this account from this point forward, regardless of legality, is entirely fictitious, including the use of the first person.

I'm brand new to mushroom cultivation and am excited to begin a new hobby!

I ordered a spore syringe off a store on this website's vendors list and unfortunately I did not check the location the spores were shipping from. Three weeks later (prime has really spoiled me) I received a padded envelope patched with green department of homeland security tape. My spores were removed at the US customs office and destroyed and they simply sent me the original envelope filled with a no-action-needed notice of non-permitted spore import.

I ordered another syringe from a shroomery sponsor located inside the US but in the mean time I've been excited to begin, so tomorrow I'm attempting some advanced mycology.

I have the remains of some very pleasant street mushrooms in a ziploc baggy. These mushrooms must be at least 18 months old. I also have a friend who has some fresher street mushrooms I'm sure he'll donate a small amount of.



I'm going to attempt to rehydrate tissue and either restart mycelial growth or germinate old spores.

I do not doubt contamination will be an issue so I've prepped 16 agar plates using a modified version of pastywhyte's no pour agar method. I used brass plumber's inserts with polyfill for GE instead of just micropore as described in Mycologist217's LC tek. Specifically this recipe is 20g/L BRF and agar.



These "plates" will be sterilized tomorrow along with distilled water. The crushed fruit tissue (specifically gills) will be added to distilled water in a SAB then distributed onto plates. There will be plenty of backups for the eventual necessary transfers IF I can get growth/germination in the first place.

I've found threads of people having success using similar methods, including one featuring RR vouching for dried tissue's viability.

I intend to take these from agar to PF style BRF and verm 1/2 pints. If anyone has any tips for increasing viability of desiccated tissue/spores please let me know!

This is all just time killing while I wait for my actual syringe to come in. I'll be posting any progress here, wish me luck!


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OfflineStinkBorg
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Registered: 04/11/20
Posts: 13
Last seen: 1 year, 11 months
Re: Lazarus Mushroom 1 [Re: Alkeides]
    #26611830 - 04/19/20 12:22 AM (3 years, 9 months ago)

Hi Lerman, its a long road of doubt and second guessing yourself starting from a dried tissue sample but it is possible. I actually took dried gill fragments to Munchs easy as 1 2 3 PDA agar for germinating and did a sort of streak across the plate as to dilute the spores, as well as the contamination which i believe was the secret to success in this experiment as at least half of my plates 6 out of 12 had germination and growth viable of taking transfers from. I always thought powderizing tissue and leaving it in there would a worse contam vector than just using tweezers and streaking gill tissue like a spore swab and not leaving the remains in the plate.

My very first plates just started pinning about four days ago and the relief from knowing now that its not just white mold was intense because ive because cleaning up from all the bacteria for about 3 months now. My fault though, i just suck at agar.

Thought id chime in with my first post too since ive kinda been up to the same thing as your asking for a while now.
Good luck!
P.S. screw the syringes, theyre probably filled with bacteria! Ive literally has better success so far with agar and gill fragments
P.P.S. I also wouldn't fuck with adding water to the powdered fragments if thats what you decide to do.


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InvisibleAlkeides
Child

Registered: 04/11/20
Posts: 61
Re: Lazarus Mushroom 1 [Re: StinkBorg]
    #26612151 - 04/19/20 06:40 AM (3 years, 9 months ago)

The streak plate of gill makes sense, maybe the worst/majority of contams would be deposited in the first part of the streak. I'll do a little (non-scientific) experiment and do a few cultures of gill streaks and a few of the "bag dust" slurry w sterile water.

Looks like I should probably make some more plate containers to keep on hand.


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OfflineCootermonkey
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Registered: 01/30/18
Posts: 272
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Last seen: 1 year, 4 months
Re: Lazarus Mushroom 1 [Re: StinkBorg]
    #26612181 - 04/19/20 07:04 AM (3 years, 9 months ago)

Hell If youre gonna get goin with agar you might as well get a case of quart jars and do some grains/wbs/grass seed(whichever you can find easier haha)
The pf tek is really just for startin with spore syringe to get everyone addicted to the hobby by showing how "easy" it *can be done.
But since youre jumpin in agar you can do it the better way and get more(alot more) bang for your buck!

Heres bods everything you need in one spot, can read up n decide which direction to take while doin agar https://www.shroomery.org/forums/showflat.php/Number/24144021


--------------------
MyLAGM2020Growlog


Edited by Cootermonkey (04/19/20 07:04 AM)


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OfflineStinkBorg
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Registered: 04/11/20
Posts: 13
Last seen: 1 year, 11 months
Re: Lazarus Mushroom 1 [Re: Alkeides]
    #26612222 - 04/19/20 07:24 AM (3 years, 9 months ago)

Id love to see the results from said experiment. Good info on reviving old spores/mycelium is super interesting to me and a little sparse on the forum.

And yeah pretty much what id see on the first part of the streak would be a satellite colony of some mold and by the end of the streak was usually germination.

Im rooting for ya! Make those street shrooms rise from the dead :wink: lol.


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InvisibleAlkeides
Child

Registered: 04/11/20
Posts: 61
Re: Lazarus Mushroom 1 [Re: Cootermonkey]
    #26612277 - 04/19/20 07:55 AM (3 years, 9 months ago)

Hey cooter I totally agree mono tubs are obviously the way to go, however I already bought all the PF stuff so I'll at least be doing one round of PF. I also am totally new to sterile technique and mycology in general, and my home is a drafty mold box, so I figure PF may be more forgiving considering if I lose a few jars I still have others that may be viable.

Honestly I've let this hobby expand from just doing it the "cheap easy" to whatever fills my time during the covid lockdown.


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InvisibleAlkeides
Child

Registered: 04/11/20
Posts: 61
Re: Lazarus Mushroom 1 [Re: Alkeides]
    #26613830 - 04/19/20 08:33 PM (3 years, 9 months ago)

Alright fellas, an update.



Agar dishes were PCed for 45 minutes at 15 PSI then allowed to cool for most of a day. 8 plates were brought into the sanitized SAB. Plates were prepared as follows:

1,2 - West coast gill
3,4 - West coast slurry
5,6 - East coast gill
7,8 - East coast slurry

Gill plates were rubbed with gill material, slurry plates were streaked with sterilized (not distilled) water slurry of bag dust and discarded gill material. Plates were placed in a cardboard box on a shelf to incubate at room temperature in darkness.




Previous threads attempting this technique note mycelial regrowth can take as long as 3 weeks, I assume I'll at least have contaminate growth before then but if the plates remain clean I'll keep them in incubation for a month or two.

This was my first time attempting inoculation, using sterile technique, or using a SAB. Some notes:

-PCing significantly reduced the tension of the lid over the no pour plates. One plate even couldn't be unwrapped without removing the lid. Pastywhyte uses round PP containers, maybe that was the difference. Future preparations will use different containers.



-There was A LOT of condensation inside these plates, unsure what can be done about that other than switching to pouring sterilized agar. May make the switch at some point.



-I think Bod was the one who posted about using a wet towel and raised work surface in the SAB. I used a nonstick cookie rack I found for cheap, it worked well but smaller objects (like the shotglass pictured) were too unstable.




-I neglected to wipe down the SAB before beggining work after spraying down the inside, made viewing work very difficult. I can only assume other people give a quick wipe to the face they intend to view through.

-I showered between loading/spraying the SAB and beginning work then dressed with a longsleeve shirt, nitrile gloves, hat, and fabric facemask to reduce my risk as a contam vector.

-I need a larger, clearer SAB with better lighting provisions.



-The learning curve for sterile technique is extremely steep, the last few plates I prepared were handled much better than the first few.


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InvisibleAlkeides
Child

Registered: 04/11/20
Posts: 61
Re: Lazarus Mushroom 1 [Re: Alkeides]
    #26619491 - 04/22/20 08:04 AM (3 years, 9 months ago)

Update: no growth fungal or otherwise visible on any of the dishes. I also prepped a control agar dish in open air and did a streak of my finger (albeit recently washed) on it to compare contamination to, it also shows no growth. May be due to ambient temperatures being low, house ranges from 50F at night to 65F during the day. I moved the dishes closer to a heat source to incubate.


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InvisibleAlkeides
Child

Registered: 04/11/20
Posts: 61
Re: Lazarus Mushroom 1 [Re: Alkeides]
    #26623030 - 04/23/20 05:52 PM (3 years, 9 months ago)

Made a small TiT style incubator and left the cultures to incubate since yesterday. Observations:
Plate 1 - no growth visible
Plate 2 - 2 light spots and 2 dark spots, all sized ~1mm
Plate 3 - 1 light spot <1mm
Plate 4 - 1 light spot too small to confirm whether sediment or biological
Plate 5 - 2 light spots, 1 ~2mm, the other under 1mm
Plate 6 - numerous light spots <1mm
Plate 7 - 1 light spot <1mm
Plate 8 - 2 light spots <1mm
Control - no growth visible (somehow)

Is there a way to post on my thread without bumping it?


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OfflineStinkBorg
Stranger
Registered: 04/11/20
Posts: 13
Last seen: 1 year, 11 months
Re: Lazarus Mushroom 1 [Re: Alkeides]
    #26623066 - 04/23/20 06:08 PM (3 years, 9 months ago)

Very nice updates, im excited to see this play out.

If i could juat share a few tips, not that it may mean much, just whats helped me the most on my similar journey.

Winco (if you live near one) sells these small twist top PP tupperware containers that dont need any modifications. Just screw the lid on like only a 1/4 of the way. And if you leave them in the pressure cooker overnight, 95% of them will have little to no condensation issues. Im almost positive theres a good write up about them already. Its what i personally use just so I didn't need to switch up my technique.

letting the room settle for 10-15 minutes before working in the SAB helped my contam rate quite a bit.

My house has about the same temps and swing patterns and i haven't found any need for any incubation. Cubes seem to be actually pretty tough.

Just wanted to share some limited personal experience with ya. Im watching this thread for sure tho. :thumbup:


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InvisibleAlkeides
Child

Registered: 04/11/20
Posts: 61
Re: Lazarus Mushroom 1 [Re: StinkBorg]
    #26623106 - 04/23/20 06:23 PM (3 years, 9 months ago)

thanks for the tips stink.

I actually found a killer deal for some culture dishes on amazon, theyre only 35mm but 300 sterile for $40 when i bought. And theyre brand name eppendorfs, not some nameless chinese stuff, snag them if you can before the price changes too much, there the "Eppendorf 30700112".

I also did prep and load up my box then went and did some chores and showered so the room had a little less than an hour to settle.


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InvisibleAlkeides
Child

Registered: 04/11/20
Posts: 61
Re: Lazarus Mushroom 1 [Re: Alkeides]
    #26630558 - 04/26/20 06:44 PM (3 years, 9 months ago)

Update

Continued growth observed on all plates, control growth has exploded. Looks to be dactylium, which unfortunately means it could be dactylium on all the other plates but hey I started this project assuming I'd need to clean some cultures. Plates 2 and 5 exhibit dark spots, unknown contam. It's hard to take photos through these shitty plates but I tried:

Plate 4



Control Plate



New plates were prepared out of 1/2 pints today, hopefully I can make some transfers once these grow out enough to be able to differentiate between contams and any potential cubes. A legitimate spore syringe should be arriving tuesday night, i'm going to immediately start PF from multispore but I have two fruiting chambers so I can devote one to isolates from this project and other experiments.


Edited by Alkeides (04/26/20 09:24 PM)


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InvisibleAlkeides
Child

Registered: 04/11/20
Posts: 61
Re: Lazarus Mushroom 1 [Re: Alkeides]
    #26640036 - 04/30/20 10:02 PM (3 years, 9 months ago)

Update

Plates have been incubating 11 days and all display significant visible growth and contamination.

Today plates were all observed for potential cube mycelium and transfers made to new poured culture dishes. Sterile technique was observed as practical, I use a different smaller SAB for cleaning contaminated cultures than I do inoculation and clean culture work. Scalpel(s) were flame sterilized between each transfer even from the same dish and gloves and pastyplates were sanitized with 70% iso before being brought into SAB. New plates use a 20g/L BRF recipe, same as the pastyplate's they're transferred from.

I used my newfangled SAB documentation rig to take photos before and after transfers were cut (except I forgot a before picture for plate 7). I also included a photo of the control pastyplate I wiped a cleaned finger on. I saw some bacterial but mostly fungal contams. If anyone can ID any of them I'd love to learn.

Transfers will be allowed to fully colonize in incubation unless obviously contamd and I'll post here the results.

Photos:

Plate 1



Plate 2



Plate 3



Plate 4



Plate 5



Plate 6 (2 transfers)



Plate 7 (forgot a before photo)



Plate 8 (2 transfers)



Control Plate



Would love any input from someone more experienced than I. If you think I'm wasting my time... I wouldn't disagree.


Edited by Alkeides (04/30/20 10:21 PM)


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InvisibleSmartattack
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Registered: 12/21/18
Posts: 3,776
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Re: Lazarus Mushroom 1 [Re: Alkeides]
    #26640058 - 04/30/20 10:14 PM (3 years, 9 months ago)

Yikes man, id certainly work on being able to get a better start than that in the first place rather than work from a mess like that!


--------------------
* Smarts videos :teacher:
* :thumbup: Planet of the APES:thumbup:
 
I'm a fungal white supremacist.


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InvisibleAlkeides
Child

Registered: 04/11/20
Posts: 61
Re: Lazarus Mushroom 1 [Re: Smartattack]
    #26652573 - 05/06/20 09:26 AM (3 years, 9 months ago)

Update

All transfers showing no significant growth except 8A which has colonized beyond the little slice of agar it was transferred with. Certainly fungal but the center has a dark core to it underneath mycelium, could be a contaminate culture, could be a contam cased with mycelium, could just be a trick with how the light hits that agar slice. Too early to tell. Pictured:



PS Obviously I now know to pour my agar when its cooler hahaha


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OfflineStinkBorg
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Registered: 04/11/20
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Last seen: 1 year, 11 months
Re: Lazarus Mushroom 1 [Re: Alkeides]
    #26655936 - 05/07/20 08:00 PM (3 years, 9 months ago)

Wow so much content this is super cool!
These look a lot like the experiments I once ran. I wanted to reply sooner ive been thinking about this thread non stop. Just have been getting too sidetracked.

Congrats on getting to using actual pitri dishes. That's a nice upgrade i need to get around to doing lol.

I wish i could see with a little more clarity because some of these pictures look really promising at least to me. I kinda think you definitely have some usuable mycelium in there. Looks like you may have transfered some already! I only encountered a small amount of strong rizomorphic growth once on my first plate and from then on it was all tomentose  This led me to think it was also cobweb/dact or something similar. But i kept looking for sorted/organized white growth and took transfers. And eventually when i took it to grains it grew much more like what you'd see in the pictures online.

If you do have cube myc, over time its pretty likely that it could take over the whole plate given enough time and if you keep them around long enough, i had a few bacteria filled and mold filled plates that looked seemingly clean or taken over now a few months down the road. It was kinda cool to watch. Id kinda doubt its anything you should consider using no matter how much it turns around .

I think you've got some promising results at least through the pictures. It doesn't look grey enough to me to be worried but im surely no expert on this.

Id love to see you to continue to work on this i really think you've got a fighting chance. maybe do a grow log on your spore syringe too when you get it :smile:


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InvisibleAlkeides
Child

Registered: 04/11/20
Posts: 61
Re: Lazarus Mushroom 1 [Re: Alkeides]
    #26729702 - 06/08/20 06:57 PM (3 years, 8 months ago)

Update

Transfer 8A and 8B have fully colonized their tiny (35mm) homes.



Certainly looks like shroom myc. I have some adult sized petris coming in and when they arrive I'll transfer and try to clean up these (probably) dirty cultures. In the mean time each one is getting a sample dropped in a vtek style quart. If I can get them to fruit right there I'll call this whole thing a success.


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OfflineA.k.aM
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Re: Lazarus Mushroom 1 [Re: Alkeides]
    #26729714 - 06/08/20 07:01 PM (3 years, 8 months ago)

There you go man now you have your own clone of dealer shrooms.


--------------------
LAGM2020


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InvisibleAlkeides
Child

Registered: 04/11/20
Posts: 61
Re: Lazarus Mushroom 1 [Re: Alkeides]
    #26850903 - 07/28/20 10:50 PM (3 years, 6 months ago)

mycelium has been expanded, first vteks are starting to fruit:



Underwhelming but I have a good looking monotub coming in too.

I'm calling this a success. I have since harvested a few tubs grown from shroomery vendor spores, so while not time efficient this just goes to show you don't necessarily need to buy spores (or have any prior cultivation experience) to produce cultures from street shroomies.


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InvisibleAlkeides
Child

Registered: 04/11/20
Posts: 61
Re: Lazarus Mushroom 1 [Re: Alkeides]
    #26866593 - 08/06/20 02:19 PM (3 years, 6 months ago)

Tubs coming in, best flush I ever had. Genetics may have been worth the work on these guys.


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