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OfflineMadoxx
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Registered: 05/19/13
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Full laboratory access ideas
    #25130519 - 04/11/18 09:52 AM (6 years, 9 months ago)

Hello community

I have recently started working in a Plant Physiology lab with other associated fields. Luckily i have found out that i have a lot of equipment at my disposal.

I have been growing mushrooms in a home setting for a few years, autoclaving on a pressure cooker and growing mainly on different kinds of grain as spawn and coir, straw, hpoo and verm as bulk in different combinations.
Now i have all of these exciting possibilities at my disposal but no guidance, so i turn to this community in the hopes that we can do some kind of a brainstorming of ideas so that i can explore some more advanced procedures.

I am specially curious on things like
- Germinating two single spores on an agar plate in the hopes of crossing two interesting strains (which will probably be something strange like PESA and Syzygy)

-Liquid culture, to have some great inoculation times

So i want to open up the discussion of all the other possibilities i have with the following equipment; an autoclave, a horizontal laminar flow cabinet, different kinds of glassware like Erlenmeyer tubes, test tubes, petri dishes..., quite a bunch of media to put in these petri plates.

Also, there are quite a few kinds of agar for mycology, I'm wondering which one is the best for cultivating psilocybes, between oatmeal, soy, standard nutrient agar.
Another question which comes to mind is, when autoclaving at home we use a cycle time of 1h30 at 15psi, whereas the cycles on a lab autoclave, even if they are also at 121ºC seem to recommend much shorter times, do we use longer time to ensure that this temperature is achieved on the inside of the substrate jars? or another reason?

Thanks a bunch to whoever can answer a few of my doubts!!!!

Let the talking BEGIN!!!

Note: I wasn't sure on which forum to place this but i guess this one will get to more people who work in labs.


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:awecid:

Edited by Madoxx (04/11/18 10:22 AM)

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InvisiblebodhisattaMDiscordReddit
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Re: Full laboratory access ideas (moved) [Re: Madoxx]
    #25131169 - 04/11/18 03:34 PM (6 years, 9 months ago)

This thread was moved from Advanced Mycology.

Reason:
Single spore germination will require a micromanipulator microscope.
Or you could do dilution and streaking to get single spores in isolation on a dish then verify with microscopy. Using a hemocytometer to verify dilution as well.

Lab autoclaves are not really super useful. Small capacity and are not really made with sterilizing grain or food in mind.

You would want to use it on a setting that doesn't dump the steam at the end of the cycle.

They use shorter times because sterilizing liquids and utensils is very easy. Sterilizing food and grain with space between it is hard. Grain also traps air and entrapped air is the number one cause of failed sterilization in labs. This is why we vent so long. And also have long cooking times.

Sterilization of lab glass, scalpels, and liquids takes only 15m at 15psi. A quart of grain takes at least 90m at 15psi.




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Invisibleelasticaltiger
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Re: Full laboratory access ideas (moved) [Re: bodhisatta]
    #25131200 - 04/11/18 03:55 PM (6 years, 9 months ago)

Quote:

bodhisatta said:
<img src='https://www.shroomery.org/forums/images/moved.gif'> This thread was moved from Advanced Mycology.

Reason:
Single spore germination will require a micromanipulator microscope.




:broke:  :justno:

Use the sprayed plate method like they did in they did in the old days.

http://www.fungaldiversity.org/fdp/sfdp/FD_3_29-38.pdf&ved=2ahUKEwiolaW7iaDZAhVD3GMKHZijAdAQFjABegQIDRAB&usg=AOvVaw28DzcEvDp9GIO9miM0upq3

You can use a microscope to verify but if you do like 10 plates surely a couple of them will give you single spore monkaryotic mycelium.


--------------------
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Afraid to Start Growing From Your Own Prints? Drop it Like a Tiger!
No Pouring. No Syringes. No Cutting. No flaming. No Contamination. No Bullshit.

"The best thing to do while your waiting is to start more stuff. I usually got so much happening that I have tossed projects simply because I didn't have time for them. -Pastywhite QFT

Pastywhite's Easy Agar Tek (PastyPlates)

Tiger Drop Video Demos By munchauzen

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EZEKIEL 23:20

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InvisiblebodhisattaMDiscordReddit
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Re: Full laboratory access ideas (moved) [Re: elasticaltiger]
    #25131451 - 04/11/18 05:53 PM (6 years, 9 months ago)

404 page doesn't exist.

https://www.singerinstruments.com/yeast-tetrad-dissection/

yea these things are about 5K to start. but they're basically made for single spore isolation of yeast cells. but yeast's spores are way smaller than cube spores so you'll have a much easier time.

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InvisibleAndyHinton


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Re: Full laboratory access ideas (moved) [Re: bodhisatta]
    #25131559 - 04/11/18 06:47 PM (6 years, 9 months ago)

Agreed with bodhi, probably nobody at that lab will let you sterilize grains or germinate cubes spores.

You can easily make LC at home. All my stuff is adapted to a home environment so I can cleanly transfer macro amounts of liquids, make reusable ported plates, etc. See if you like my spore-to-shroom tek. You have good account history.

On horizontal hoods, it's sacrilegious to say it here, but I really prefer vertical ones. You get the most working volume relative to the filter size, which is important because HEPA filters only last 2-3 years. The clean air also showers the media.

On media, malt extract is the default in mycology. You can use potato dextrose, lysogeny broth, etc. It doesn't matter as long as it has sugar and minerals. Unless you need special media for some purpose like glycerol for cryopreservation, charcoal for old spores, Murashige and Skoog for mycorrhizae, etc.


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Edited by AndyHinton (04/11/18 08:07 PM)

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OfflineMadcapper144
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Re: Full laboratory access ideas (moved) [Re: AndyHinton]
    #25131618 - 04/11/18 07:07 PM (6 years, 9 months ago)

I ran across a crazy post like a month ago using rattle snake Venom and some crazy procedures that were way over my head to "splice" two kinds of mushrooms lol.


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Buy the ticket, take the ride!! :fearandloathing:

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OfflineMadoxx
Aspiring Mycologist


Registered: 05/19/13
Posts: 111
Last seen: 3 years, 8 months
Re: Full laboratory access ideas (moved) [Re: elasticaltiger]
    #25131685 - 04/11/18 07:29 PM (6 years, 9 months ago)

Awesome feedback  :amanita2:

Quote:

bodhisatta said:
<img src='https://www.shroomery.org/forums/images/moved.gif'> This thread was moved from Advanced Mycology.

Reason:
Single spore germination will require a micromanipulator microscope.
Or you could do dilution and streaking to get single spores in isolation on a dish then verify with microscopy. Using a hemocytometer to verify dilution as well.

Lab autoclaves are not really super useful. Small capacity and are not really made with sterilizing grain or food in mind.






yeah there is no way in hell I'm getting away with finding/using comfortable such a great microscope around the lab... someday. BUT, that neubauer chamber method seems the most solid method i can think of, verifying the plate straight with a microscope feels like i'm going to be looking for a needle in a haystack.

You just burst my bubble with the autoclaves to be honest man, i was hoping to store my PC for some time. I thing there is an oldschool one with a manual draining and venting knob, maybe that could work? Also why does it have to keep the steam in after the cycle? If you use 15psi for 90min shouldn't that be enough?


Quote:

elasticaltiger said:


Use the sprayed plate method like they did in they did in the old days.






Sounds interesting to look into, shame about the broken link, is this what you are referring to?
http://www.fungaldiversity.org/fdp/sfdp/FD_3_29-38.pdf On page 36, it is a crazy intelligent method.

(Also i like how the paper open with 'These methods are specifically
designed for mycological laboratories which are not necessarily well funded' ----> Try this at home kids)

I agree Andy vertical feels more natural when working but i guess both feel miles better than any SAB around  :rotfl:  :naughty: I was amazed to find oatmeal agar, malt it is for the ages then, later on i might give others a try when i have some tissue to inoculate with.
G.r.e.a.t. TEK....:cool:


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:awecid:

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Invisibleelasticaltiger
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Re: Full laboratory access ideas (moved) [Re: Madoxx]
    #25131688 - 04/11/18 07:32 PM (6 years, 9 months ago)

This is it basically how the spread plate method works in pictures. It's basically the same as dillution except all the dilution occurs on the agar.



This is how spore works got their monokaryotic cultures to create Albino Penis Envy


--------------------
First time growing cakes? DON'T make a Shotgun Fruiting Chamber

The Shmuvbox. - The Old TC's Like it :shrug:

Afraid to Start Growing From Your Own Prints? Drop it Like a Tiger!
No Pouring. No Syringes. No Cutting. No flaming. No Contamination. No Bullshit.

"The best thing to do while your waiting is to start more stuff. I usually got so much happening that I have tossed projects simply because I didn't have time for them. -Pastywhite QFT

Pastywhite's Easy Agar Tek (PastyPlates)

Tiger Drop Video Demos By munchauzen

Van Gogh would’ve sold more than one painting if he’d put tigers in them.―Bill Watterson

EZEKIEL 23:20

Edited by elasticaltiger (04/11/18 07:33 PM)

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OfflineMadoxx
Aspiring Mycologist


Registered: 05/19/13
Posts: 111
Last seen: 3 years, 8 months
Re: Full laboratory access ideas (moved) [Re: elasticaltiger]
    #25131720 - 04/11/18 07:47 PM (6 years, 9 months ago)

Right, i just got a flashback from microbio lab, seems like a painfree method, i guess its pretty good to get single spores on the last stoke. After doing it you would let then germinate and transfer to another plate at a safe distance like so?



The pic is from some bacterial colonies but just to get the idea of placement.


--------------------
:awecid:

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InvisiblebodhisattaMDiscordReddit
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Re: Full laboratory access ideas (moved) [Re: Madoxx]
    #25131976 - 04/11/18 09:50 PM (6 years, 9 months ago)

Problem is spores like to clump together. Ive diluted and done streaks but always saw groups of spores not single ones. Hence suggesting the manipulator and verification.



Of course the growth looks thin and weak right away and that's when you need to transfer. Low and behold its dikaryotic already.

Or find one spore

But there's already mycelium around.

If anyone says they got monokaryotic growth from just doing streak plating I want pictures to back it up because you would take pictures if you did something that cool anyway.

Vendors have a ridiculous history of embellished stories and plain old made up ones. So i take it with a grain of salt. They don't necessarily give out their methods and secrets

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OfflineMadoxx
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Registered: 05/19/13
Posts: 111
Last seen: 3 years, 8 months
Re: Full laboratory access ideas (moved) [Re: bodhisatta]
    #25135808 - 04/13/18 01:27 PM (6 years, 9 months ago)

Well I'll look in every corner of the lab for a manipulator but i doubt the will have one, they touch on a lot of things like plant pathology, entomology, mycorrhiza where I'm at etc. so not so much micro equipment around. Might have to try that Korean technique and cross my fingers.

Also I'm just going to bump this thread slightly so ideas keep on coming in, with some picture of the autoclaves at the lab see what you guys think.

Candidate #1

The one i am probably going to end up using.
Old school baby with the awesome feature of having two knobs where you can regulate drainage and steam venting. Would this solve the venting problem in autoclaves?




Control panel

Timer detail




Steam vent


Candidate #2

Electronic AC mainly used for liquid waste but can also take solids, feels a bit to risky to start tampering with the interface on something so complex.




Candidate #3

Just for fun, check out this beast where you autoclave up to and including a 10yo kid. There is no way i am using this but it would certainly be practical.



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OfflineFishLevelMidnight
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Re: Full laboratory access ideas (moved) [Re: bodhisatta]
    #25135973 - 04/13/18 02:26 PM (6 years, 9 months ago)

Quote:

bodhisatta said:
Problem is spores like to clump together. Ive diluted and done streaks but always saw groups of spores not single ones. Hence suggesting the manipulator and verification.



Of course the growth looks thin and weak right away and that's when you need to transfer. Low and behold its dikaryotic already.

Or find one spore

But there's already mycelium around.

If anyone says they got monokaryotic growth from just doing streak plating I want pictures to back it up because you would take pictures if you did something that cool anyway.

Vendors have a ridiculous history of embellished stories and plain old made up ones. So i take it with a grain of salt. They don't necessarily give out their methods and secrets




I think it would be worth adding 0.1% detergent in the spore solution before streaking or serial dilutions, I bet you could overcome the spore clumping issue by having a final concentration of 0.1% triton X-100 or tween-20...

Hoping to have time to try this out in the summer and see if I can do some more controlled matings.

Have you thought of this bod or tried something similar?


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InvisiblebodhisattaMDiscordReddit
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Re: Full laboratory access ideas (moved) [Re: FishLevelMidnight]
    #25136006 - 04/13/18 02:39 PM (6 years, 9 months ago)

No, but i agree with the detergent or surface tension modifiers.
I tried using a vortex genie to vibrate the spores loose but that didn't really work.

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OfflineFishLevelMidnight
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Re: Full laboratory access ideas (moved) [Re: bodhisatta]
    #25136022 - 04/13/18 02:44 PM (6 years, 9 months ago)

yeah I wonder if a sonicator could be used briefly in the detergent or if that would just break them open...


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OfflineMadoxx
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Re: Full laboratory access ideas (moved) [Re: FishLevelMidnight]
    #25151025 - 04/19/18 03:37 PM (6 years, 8 months ago)

Update: So on Tuesday luck was on my side big time and i got JUST enough time to create these awesome spore syringes in a horizontal air flow cabin. Transferred three strains as you can see in the picture; Syzygy, PESA and Thai.
I'm willing to trade with y'all or give away as these spores only make sense to me if they colonize the world. So  :pm:  :pm: :sunny::sunny::sunny:



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:awecid:

Edited by Madoxx (04/19/18 04:33 PM)

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