CHATPRIVACYDONATELOGINREGISTER
DMT-Nexus
FAQWIKIHEALTH & SAFETYARTATTITUDEACTIVE TOPICS
Working with wild spores - Panaeolus cyanescence Options
 
intosamadhi
#1 Posted : 3/16/2021 3:24:22 AM

DMT-Nexus member


Posts: 117
Joined: 05-May-2008
Last visit: 13-Apr-2021
Location: 2nd star to the right
Does anyone have any handy advice for working with spore prints made from wild mushrooms (ie non-sterile)? I am struggling to get the spores that I have collected to sporulate. I've gone through dozens of agar plates and all of them end up horribly contaminated with little to no signs of mycelium.

I've tried plain water agar, as I saw a video about using this as a way to get a small amount of mycelium to form without having bacteria overtake it too quickly, but that didn't work at all.

I've tried dropping spores on soaked and sterilized cardboard but nothing here either.

I'm using PDA for my plates. I sterilize everything with a pressure cooker and work in a still airbox trying to keep things as sterile as possible (which amounts to dousing everything with 70% ethyl alcohol). I've ordered a UV-C lamp which I will try on my next round of plates.

I managed to isolate what looked like healthy mycelium from one plate and have been growing that out and even made what looks like a healthy LC from it, but because I've only managed to do this once, I am a bit worried that it may not even be pan myc, and so would prefer not to rest all my efforts on one success.

I've attached pictures of the specimens I made the prints from, some of the plates, and some of what I hope is healthy pan MYC. Any comments or tips welcome!
intosamadhi attached the following image(s):
pan-myc3.jpg (322kb) downloaded 82 time(s).
pan-myc2.jpg (1,044kb) downloaded 82 time(s).
pan-myc1.jpg (1,191kb) downloaded 80 time(s).
agar1.jpg (122kb) downloaded 78 time(s).
agar2.jpg (138kb) downloaded 78 time(s).
agar3.jpg (261kb) downloaded 80 time(s).
IMG_20210207_084853.jpg (1,154kb) downloaded 76 time(s).
 

Trippy glass for trippy people.
 
downwardsfromzero
#2 Posted : 3/16/2021 2:25:22 PM

Peeing into the abyss

Chemical expertSenior Member

Posts: 5601
Joined: 30-Aug-2008
Last visit: 09-May-2021
Location: square root of minus one
The healthy myc you have does very much look like Pan myc.

As it appears you have access to fresh mushrooms, consider cloning tissue from inside the carpophores.

You might want to look into using antibiotics in your agar in order to increase success rates with using spores.

There are methods involving adding hydrogen peroxide to the agar for decreasing contamination rates when transferring mycelium. H2O2 will kill spores so it cannot easily be used at the start of an agar run with spores.

What kind of technical set up do you have? Laminar flow hood? SAB/glovebox? What PC or autoclave?
Ora, lege, lege, lege, relege et labora

“There is a way of manipulating matter and energy so as to produce what modern scientists call 'a field of force'. The field acts on the observer and puts him in a privileged position vis-à-vis the universe. From this position he has access to the realities which are ordinarily hidden from us by time and space, matter and energy. This is what we call the Great Work."
― Jacques Bergier, quoting Fulcanelli
 
intosamadhi
#3 Posted : 3/16/2021 2:52:14 PM

DMT-Nexus member


Posts: 117
Joined: 05-May-2008
Last visit: 13-Apr-2021
Location: 2nd star to the right
downwardsfromzero wrote:
The healthy myc you have does very much look like Pan myc.


Well that's a relief! Very happy I've seen enough bacterial and mold growth now to know the difference, but because I've only gotten myc from one plate inoculated with spores and dozens of failures I keep doubting.

One thing I've noticed with all the plates that I've transferred the healthy mycelium onto is that the agar eventually starts turning dark after a couple of weeks. Is this just the agar drying out ?

downwardsfromzero wrote:
As it appears you have access to fresh mushrooms, consider cloning tissue from inside the carpophores.


Initially, this was my plan, but these little guys are so small I didn't see how I would be able to get any clean tissue from them. You reckon if I slice one of the caps with a sterile razor blade there'll be enough clean tissue in there to work with?

downwardsfromzero wrote:
You might want to look into using antibiotics in your agar in order to increase success rates with using spores.


Can you recommend anything that is easily available?

downwardsfromzero wrote:
There are methods involving adding hydrogen peroxide to the agar for decreasing contamination rates when transferring mycelium. H2O2 will kill spores so it cannot easily be used at the start of an agar run.


I looked into this, but since my transfer plates seem to remain healthy, it didn't seem so relevant for getting more spores to grow.

downwardsfromzero wrote:

What kind of technical set up do you have? Laminar flow hood? SAB/glovebox? What PC or autoclave?


Bear minimum at the moment. This is the first time I am growing and so have been building things up gradually. Just an SAB and PC. I've played around with the idea of building one of these cheapo flow hoods (ie strapping a HEPA filter to a box fan), but so far the SAB seems to be good enough. I'm prepared to keep investing in this as I start to see some results. Starting with Pan's is probably not the best move, but they are what's available around here.
 
downwardsfromzero
#4 Posted : 3/16/2021 3:37:26 PM

Peeing into the abyss

Chemical expertSenior Member

Posts: 5601
Joined: 30-Aug-2008
Last visit: 09-May-2021
Location: square root of minus one
It looks like you're doing pretty well with what you've managed to get through the process so far, tbh. I find it very cool that you have access to Pan. cyan.!

Useful article: https://www.researchgate...-fungi-on-growth-medium
You'll see gentamycin and chloramphenicol recommended for bacteria, and cycloheximide for fungi. Unfortunately, cycloheximide is now recognised as being extremely toxic Confused


You might just have been lucky but there's a chance you've selected for more vigorous and contam resistant genetics with the myc that you have.


One more thing - you should ditch those plates with obvious contams much more quickly. Dump them in a bleach bath for safety's sake, particularly the one with the big black blob.

Would you say the contamination appears at the inoculation sites or is there a chance that some of it is airborne?
Ora, lege, lege, lege, relege et labora

“There is a way of manipulating matter and energy so as to produce what modern scientists call 'a field of force'. The field acts on the observer and puts him in a privileged position vis-à-vis the universe. From this position he has access to the realities which are ordinarily hidden from us by time and space, matter and energy. This is what we call the Great Work."
― Jacques Bergier, quoting Fulcanelli
 
Kumarajiva
#5 Posted : 3/16/2021 3:42:26 PM

DMT-Nexus member


Posts: 96
Joined: 05-Apr-2019
Last visit: 10-May-2021
Stash the agar with bacterial contam somewhere safe for couple of month and check up on them later. there is a good chance the bacteria will die out and mycelium will survive and will consume whatever is left of that contam and you will have nice fat blanket of myc to copy to another agar dish

Additionally, i see clean agar there (as much as the pictures allow the first, second and third pic).
cut little chunks of this agar and move it onto new plates. And repeat this after new plates have been colonized as many times as needed until the culture is clean.
(Use separate blades for each agar dish to avoid cross-contam)
Gate Gate Pāragate Pārasaṃgate Bodhi Svāhā
 
shroombee
#6 Posted : 3/16/2021 9:16:43 PM

DMT-Nexus member


Posts: 156
Joined: 16-May-2014
Last visit: 11-May-2021
Location: CA
intosamadhi wrote:
You reckon if I slice one of the caps with a sterile razor blade there'll be enough clean tissue in there to work with?

Even using a sterile razor blade, when you cut you will push contaminants from the surface into the interior tissue. Ideally, you can peel the stem open from the bottom (like string cheese) and scrape a tiny bit of tissue to transfer to agar. The sample can be smaller than a grain of rice.

As soon as you see mycelium growth on the agar, transfer tiny pieces from the edge of the growth to new agar plates (use a new sterile blade for each transfer). Wait for growth, and continue transferring to new plates until you have a clean culture. Don't dip the clone sample in peroxide or bleach as that will temporarily slow down the growth. The strategy is that healthy mycelium can initially outrun bacteria so you have a chance to get clean growth at the edge of the mycelium to transfer to new agar.

Good luck!
 
intosamadhi
#7 Posted : 3/17/2021 5:43:40 AM

DMT-Nexus member


Posts: 117
Joined: 05-May-2008
Last visit: 13-Apr-2021
Location: 2nd star to the right
downwardsfromzero wrote:
It looks like you're doing pretty well with what you've managed to get through the process so far, tbh. I find it very cool that you have access to Pan. cyan.!
One of the benefits of living in Asia Very happy

downwardsfromzero wrote:
Useful article: https://www.researchgate...-fungi-on-growth-medium
You'll see gentamycin and chloramphenicol recommended for bacteria, and cycloheximide for fungi. Unfortunately, cycloheximide is now recognised as being extremely toxic Confused

All I can find locally for this is eye drops that contain chloramphenicol. I generally prefer doing no-pour-agar plates (ie sterilize the agar in the plates or jars in the PC rather than pouring into them after sterilization) because pouring inside my tiny SAB is a nightmare. Do you think I would need to add these drop after or could i just drop them in when I am prep'ing the agar?


downwardsfromzero wrote:
You might just have been lucky but there's a chance you've selected for more vigorous and contam resistant genetics with the myc that you have.
I hope so, because the plate that I pulled this from actually looked weird and bubbly when I first did the transfer and I thought it was a bust. But after a couple of days, the healthy stuff had spread across the plate, so I did a few more transfers from that and cleaned it up. This isolate which I am playing with has pretty vigorous growth, it colonizes the plates quickly and evenly, whereas the other transfers showed patchy and uneven growth, and some would just stall. Is that usual for pans?

downwardsfromzero wrote:
One more thing - you should ditch those plates with obvious contams much more quickly. Dump them in a bleach bath for safety's sake, particularly the one with the big black blob.
will do, this batch was a bit of a mess because I had just ordered these glass petri's but I couldn't get the damn parafilm to circle the plate and seal them properly, probably because they are tiny 60mm dishes and the parafilm kept slipping off the edge. So i got fed up and just dropped them in a zip lock. I will probably ditch them all.

downwardsfromzero wrote:
Would you say the contamination appears at the inoculation sites or is there a chance that some of it is airborne?
Definitely inoculation site. You can kind of see that in the pic of the jars above where there are rings of whiteish contam encircling the little black specs. Eventually, satellite colonies and all sorts of other nastiness appear if I leave them long enough, but the day after inoculation you can clearly see stuff spreading from each spore chunk.

I was also wondering if overdoing it with the spores also limits my chances of success, I scape loads onto the plates, sometimes just leaving them where they land and sometimes using an inoculation loop to smear them around. One would assume that more spores = greater chance of success, but in this case, because they are so dirty the more spores there are the more contam as well?

I was also wondering, since pan's love straw and manure, rather than starting with agar I could try to inoculate some sterilized manure or straw in small jars and then transfer from that if anything takes to clean it up?
 
intosamadhi
#8 Posted : 3/18/2021 1:48:52 AM

DMT-Nexus member


Posts: 117
Joined: 05-May-2008
Last visit: 13-Apr-2021
Location: 2nd star to the right
intosamadhi wrote:
Do you think I would need to add these drop after or could i just drop them in when I am prep'ing the agar?
Found the answer in that article. Chloramphenicol can withstand autoclaving so I will just add it to the agar before. It says it should be used at 50mg/l concentration. So one of these little eye drop solutions (20mg) will only last for about 400ml of agar.
 
 
Users browsing this forum
Guest

DMT-Nexus theme created by The Traveler
This page was generated in 0.044 seconds.