|
recklesshouseplant
Houseplant


Registered: 11/14/20
Posts: 10
Loc: In a pot of soil
|
An attempt at liquid cultures from spores without a pressure cooker
#27039097 - 11/14/20 04:24 PM (3 years, 2 months ago) |
|
|
Hello readers, thanks for stopping by my first post here on this lovely forum.
Today I made an attempt at creating liquid cultures, from spore prints, without the use of a pressure cooker. I am acutely aware that this is very likely to be a fruitless endeavor, and will likely result in contamination.
I had some free time on my hands, and plenty of spores so I decided to attempt it anyway. I'm creating this post to share my experience with you. As of Nov 14th 2020, the results are not in yet but I will begin by describing my method and as time goes on I will edit this to include the results of my experiment. EDIT 16th Nov 2020: There appears to be some activity. Scroll down to my reply to this post to see photos.
So let's begin, shall we?

I began by poking holes in the center of my jar lids using a hammer and a nail. You'll notice there are 10 of them. I figure, why not try making a bunch of them given that chances of success are low. Additionally, I'll be able to quantify my failure rate in a more granular fashion. If one succeeds, we will know this process has an approximate 10% success rate. If none succeed, well shit. We will still learn that my process has a 0% success rate.

I then put on a baseball cap tightly, and put on a surgical mask and some gloves. Not pictured, I sanitized a casserole and its lid using an 85% ethanol 15% glycerol disinfecting solution. Then, using the same sanitizing solution and new paper towels, the jars and their lids were sanitized.
I tried to very carefully place the jars and jar lids in the casserole after sanitizing each of them, making sure not to touch anything with any other part of my body than my gloved hands.
I sanitized the lid once more and placed the lid on top of the casserole and left the room for 10 minutes.

A picture of the jars, their lids, and the casserole in question. The hope is of course that these are sterile at this point. (I can hear those of you with experience giggling!)

After the casserole had sat with it, and its contents covered in sanitizing solution for 10 minutes the lid was cracked as little as possible to fill it with water from my tap. The temperature of the water it was filled with was cold. The lid was carefully replaced and the entirety of it was placed on high heat to boil. Those of you who are opening these pictures in full-resolution may notice that the casserole lid looks dusty at this point. That's because it is, I didn't bother to sanitize the top of it.

Approximately 90 minutes to 2 hours later, some preparation. First, I put on fresh nitrile gloves, a surgical mask and a tight baseball cap to contain my hair.
I then douched a paper towel in sanitizing solution and proceeded to sanitize my gloved hands, the phone I was about to take pictures with, the surface I was about to do work on.
I placed down a fresh towel, on top of which I placed an oven grate, some tongs, a teaspoon and some pre-cut sheets of aluminium foil, and I placed my honey container nearby. I wiped all of these down in a sanitizing solution except for the sheets of aluminium foil.

I then removed the lid on the casserole. I used the tongs to remove a lid, placing it on the grate. Then, I removed a jar making sure that it was filled with water but with a few centimeters of air left at the top. Promptly I filled a heaping teaspoon with honey and added it to the jar. Working as fast yet calmly as I could I placed the lid on top of the jar and screwed it tight. I wiped down one side of an aliuminium foil square in sanitizing solution and wrapped it tightly over the jar as pictured. I repeated this process for every jar.

I removed a lot of water from the pot so that the water would only reach approximately halfway up the jars and placed the jars back into the water. I left them there to boil for an additional hour, after which I removed the casserole from the heat and placed it elsewhere to cool. It probably took around 6 hours for it to reach room temperature.

It was now time to introduce the contents of these jars to spores. I didn't take any additional pictures here because it's a pain in the ass to work in a still air box and an even worse pain to take pictures of your work while doing it.
I wiped down the entire box I was going to use as a still air box in sanitizing solution. I also wiped down the table I was going to work on. I removed the jars and placed them on the table as pictured. I flame sterilized my scalpel, and left it suspended in the air resting on an old stick of RAM to cool (it has handy slots that make it so that the scalpel wont roll). I removed my print from its plastic bag and unfolded all but the final crease so that opening it would be quick later. Then, one by one I loosened up the foil covering a jar, and unscrewed it most of the way. I opened the spore print and scraped a tiny amount of spores onto my scalpel. With my other hand I removed the lid on the jar and whacked my scalpel on the edge of the jar such that the spores dropped into the liquid. I screwed the jar lid back on and once again tightened the aluminium foil around the jar lid. I then pressed my thumb over where the hole in the jar lid was and gave it a good shake.
I repeated this for every jar and here we are. Oh, and I used two separate spore prints. 5x RustyWhyte and 5x Natalensis. I did not flame sterilize my scalpel in between changing spore prints, which is something I perhaps should have done but I'm here to describe what I did, not what I wish I had done. Now all that is left to do is to wait and observe the results.
If you actually bothered to read this far, thank you for your time! Be sure to check back soon, as I will edit this post with observations once they make themselves present. Have a good weekend!
Edited by recklesshouseplant (11/16/20 04:13 AM)
|
recklesshouseplant
Houseplant



Registered: 11/14/20
Posts: 10
Loc: In a pot of soil
|
Re: An attempt at liquid cultures from spores without a pressure cooker [Re: recklesshouseplant]
#27041551 - 11/16/20 03:16 AM (3 years, 2 months ago) |
|
|
It has been 36 hours since inoculation. In some of these jars there is now some activity. I'm not quite sure if it is mycelium or not, though. Would love your input on that. I've definitely grown something
Edit Nov 16th 2020 : As of half an hour ago I squirted this onto some brown rice. There's no better way to find out what this is than trying to introduce it to grain. Well, I suppose I could toss some onto agar as well, of course. Maybe later, I made 10 jars and only used 4 inoculating the brown rice.
First, a photo of a jar that has shown no activity, to get a reference for the initial color of the solution in the current environment I'm taking photos:

Now, 5 photos of jars that have shown some activity. Most have been shaken up a little bit before photographing:




Edited by recklesshouseplant (11/16/20 10:04 AM)
|
recklesshouseplant
Houseplant



Registered: 11/14/20
Posts: 10
Loc: In a pot of soil
|
|
Nothing has happened yet to my brown rice. It's only been 24 hours though, so maybe that's to be expected.
I find the color of my ""liquid culture"" to be intriguing, at this point the solution is very clear but the mycelium, or whatever is in there still retains a brown tint.
Today I received some new glass petri dishes and cooked up a batch of agar on them.
I figured, why not drip a few drops of this on agar, just to see if anything happens?
|
van hatton
Still a noob


Registered: 11/23/14
Posts: 5,617
Loc: Michigan
|
|
-------------------- If I ever give out misinformation please inform me so I can have the correct information. Tmethyl said: Chuck Norris once roundhouse kicked a monotub that wasn't pinning fast enough. The force of the kick rearranged the genetics of the mushrooms, we now call them Penis Envy. Caps McGee said:
Fun part is figuring out what works best for you
|
recklesshouseplant
Houseplant



Registered: 11/14/20
Posts: 10
Loc: In a pot of soil
|
|
It's been 4 days since I put drops of the LC to agar. There is no sign of any growth on the brown rice, and neither on any of the agar plates. Even if it were to show growth later, there is no point to this procedure as a MSS would produce as quick or even faster results.
Feel free to use this as an example of what not to do
|
reflux
Stranger
Registered: 10/01/20
Posts: 4
Last seen: 2 years, 11 months
|
|
nice write up, questionable methodology
|
eatyualive
Eat's You Alive :)



Registered: 08/17/01
Posts: 19,026
Loc: In Your Head
|
Re: So far, not so good [Re: reflux]
#27057569 - 11/25/20 06:14 PM (3 years, 2 months ago) |
|
|
in before
|
mushhead
Potato Devourer



Registered: 08/22/14
Posts: 2,215
Loc: Dimension X-124
|
|
|
A.k.a
Stranger



Registered: 10/27/19
Posts: 16,782
Loc: Gaming the system
Last seen: 4 hours, 2 minutes
|
Re: So far, not so good [Re: mushhead]
#27057640 - 11/25/20 07:01 PM (3 years, 2 months ago) |
|
|
Whatever’s in the jars can’t be mycelium, that’s way too quick especially for spores. I’d guess sediment probably but idk why only some have it if that’s the case.
No growth on the plates is good, it should mean they were sterilized effectively.
So now it’s just a matter of how clean were the spores.
--------------------
LAGM2020     
|
|