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Cleaning up spore prints in 7 steps


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#1 JanSteen

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Posted 09 April 2016 - 09:56 AM

Hey y'all,

 

I didn't find this tek out on the web, but then again, I didn't search very far and wide.

 

Anyhow, this tek is working for me, since I deal a lot with contaminates and I want those out. I use perforator snippets I make from thin cardboard sheets (toilet paper rolls).

Spore syringes in my country suck for some reason, and self-healing holes don't always self heal. This made me think of a fast and easy route to go from spore -> culture.

 

I made a little pictorial for you guys, here you go:

 

JAnsteentek.png

 

 

 

I also found that the dry-verm barrier (which doesn't always work for me) can be replaced with simple, plain A4 paper. Fold it down the sides, sterilize, let cool, and place the agar on top. The mycelium will rip through it in just a few days (especially when using heat from below so condensation keeps the paper moist). The paper barrier also prevents loads of bacterial, yeast and fungal contaminates, since they have a tough time growing on cellulose. I don't know yet how this barrier influences fruiting, but it could just be that the layer is too thick for nice GE or that it dries out too fast. I'll update with pics when the time is right.


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#2 pharmer

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Posted 09 April 2016 - 08:30 PM

when you say A4 paper do you mean photograph printing paper?



#3 wharfrat

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Posted 09 April 2016 - 09:47 PM

gotta work with what you can get, nice write up and pictorial  :biggrin: 


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#4 JanSteen

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Posted 10 April 2016 - 04:25 AM

photograph printing paper usually contains some kind of coating, which would both make the paper too expensive to waste and probably unsuited for fungal growth. If I remember correct those glossy coatings are silicium-based, silicium in its many chemical forms is used to prevent most fungi from growing on plants.

I meant regular, plain A4 paper, the stuff that get's stuck between your printer, and starts wrinkling when you print a large color picture. But I'm using tissue-paper as well in another jar. It seems to be working fine with tissues, but they tend to dry out faster if you remove the bottom heat (the water is just soaked up by the rye again, instead of slightly condensating at the top, keeping the paper moist).

My bet is that newspaper, writing paper, toilet paper and rice-based papers would work just as well.


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#5 pharmer

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Posted 10 April 2016 - 09:29 AM

I think A4 must be a standardized description of size. I have A4 photo paper and just assumed that's what you were talking about. Good to know about the mushroom inhibiting add-ons to photo paper.

 

Good job on this write up. I'm hoping some of the old hands will vet it for accuracy because I really need to get into a habit of cleaning up prints before blindly trusting them to work without being self contaminated. If it's this easy, and will end run the need for agar, this looks like a real winner.



#6 Seeker2be

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Posted 10 April 2016 - 11:43 AM

Very excellent tek , write up and pictorials. .  I saw something similar in The essential guide to cultivating mushrooms by Stephen Russell



#7 Heirloom

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Posted 11 April 2016 - 03:00 PM

JanSteen, very nice pictorial. This will help out a lot of people.

                                    thanks, peace and safety



#8 JanSteen

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Posted 14 April 2016 - 03:12 PM

Thanks for the kind words guys!

 

Most likely, this is something everybody could figure out after months of searching forums and reading books. I did do the forums part, but not the books. Too expensive for this poor student. I combined techniques I found here, and on other fungi-related websites into one. Most likely somebody has done that before me, at least I hope so.

 

I know there are some.. differences between forums, their users and all that. I am not going to get involved by using names or locations.

Basically I used these modified statements from the people I see as the 'vets of fungi'. 

- Cardboard is hard to contaminate

- Wood in agar culture is a great place for a culture to survive tough times in the fridge, even for cubes (so that means they do degrade/metabolize cellulose and/or pectin)

- Going from print -> syringe -> agar -> agar -> growing medium always leaves chance for contaminates; you'll have to expose your media/culture to air at least twice.

- Cardboard agar tek for woodlovers.

- Adding wood flakes to rye jars makes unusual growth happen.

 

Then, from my own knowledge as biotech student (which shouldn't add any value to my claims!):

- Yeasts and bacteria - from human source, the biggest contaminator around! - grow poorly on most cellulose based material, since they lack the enzymes to break it down. If they do grow on there, they would most likely reside on the inner structure than on the outside, since they have bigger survival rates surrounded by moisture and structures. When a colony gets big enough though.. Biofilms etc. etc.

- Cardboard contains nearly no sodium-salts or other nutrients that are favorable for bacteria. Just like with the husks of rye; the bacterial communities only secrete there, while they feed and thrive on the nutritious insides of the rye. Some contaminated jars didn't show contamination until I broke the cakes apart and found the rye to be eaten from the inside out; no fungal metabolites or signs of contaminations in some jars that were fully eaten by contaminants.

- Cardboard inoculated with agar wedges of cubes do great, are easy to work with - just tear a piece off, instead of cutting, leaving out one more contaminate source; the knife - and growth is vigorous when the cardboard is kept moist. 

- Cardboard is easy to rehydrate; just add boiled toilet paper or boiled cardboard to the dish. And it's easy to see when it dries out; the edges turn a lighter color. This makes managing them easier. With agar, you'll just have to trust your gut, knowledge or experience to know when it's drying out (Sometimes agar doesn't shrink horizontally but only vertically, especially when using cling wrap).

- Cardboard is stackable and/or foldable which increases the effective surface of your petri dish with 200-300%; you can have more fungus in the same dish. Which translates into less space used, less materials needed and more jars to be inoculated with one dish.

- Cardboard can be of a lot of colors, adding better visibility to growth, status and furry/fuzzy things.

- Cardboard is CHEAP and a waste material, it can be found everywhere.

- Glue on cardboard is made with modified starch, which bacteria and yeasts CAN break down. Try to avoid glue. Also because it's treated with anti-fungal agents sometimes.

 

 

Some findings I thought I'd share:

Proven effective on:

- Trichorderma (which means this could still be a contaminate!), G. Applanatum, Reishi, B+, amazonian, Z-strain (B+ derivate?), Mexicana A, wine cap (strophoria?), pink oyster.

 

Oddities:

Cubes go very, very rhizomorphic on cardboard. Something that takes at least 3 transfers on agar for me.

Applanatum doesn't, but I guess that's it's thing: staying fuzzy.

Oyster goes the long route, leaving rhizomorphic growth out completely, and ending up stringy. I don't know how that's different on agar, since I haven't been able to get a uncontaminated oyster culture. It can also pin really fast from cardboard, so leave those in the dark or some shrooms might pop out of the petri dish.

 

 

Weird things observed when reversing this technique (placing cardboard on old - some contaminated - agar plates for the sake of experimenting):

  •  In Mexicana, the yeast on agar plates grows along with the fungus, this made me think that they were symbiotic. After some brain crushing, I figured out that Mexicana probably excretes more cellulase than it can take up sugars, thus leaving sugars for yeasts to feed on.  Now I think the symbiotic relation is bullshit, why else would Mexicana secrete deadly metabolities when yeast infections occur? This information could prove useful in the need for biofuels; let mexicana break down wood, paper, plant materials, and let yeast make the oil/ethanol from the leftover sugars. Anyhow, mexicana seems to be able to break down either pectin, or cellulose but not both, since I can't dissolve plant tissue with metabolites or fungal secretions from mexicana. It does however weaken the cells structure. I don't own materials to further test this.
  •  
  •  Seemingly dead agar plates (left untouched for.. ehr.. 6 months? at room temp. dried out, contaminated, pinned, total armageddon) seem to revive in 2-3 days after adding wet cardboard. Whether it's trich, contaminants or actually cubes is something I still need to find out. Not all species/strains of fungi do this of course, I'll report the findings when there is more to tell. I'm just doing this on the side when I have nothing better to do.
  •  
  • Sugars/nutrients in dried out LME agar are absorbed by the cardboard, making it a wonderful place for contaminates to grow, I haven't found a single one though.

Edited by JanSteen, 14 April 2016 - 03:17 PM.

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#9 JanSteen

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Posted 18 April 2016 - 02:18 PM

Reversed technique (wet cardboard on old agar plates) did something weird; old (presumed/assumed) dead B+ cultures are pinning. Mind you, this plate was a contaminated salvage culture from june 2015. I was salvaging this culture because it overtook trichoderma with relative ease; something I haven't ever read about.. But that figures, I mean, we don't want contaminated cultures simply because we want results. We do want the most contaminant-resistant culture out there, but nobody seems to want to spend time looking for it. Neither did I.. So it pinned on agar and I just kept the plate around for months, closer to a year, I forgot about it completely. After adding cardboard, new pins are showing up out of the blue. On the agar, and not on the cardboard. The fungus doesn't even seem to grow on the cardboard at the moment, but it benefits the added moisture. This could very well be a figment of my imagination, I want to say that just for clarification; after almost a year, I can't be sure if those white(¿Shouldn't they be brown if they died?) pins have been there all along, or if they just appeared in the past days. I will monitor however this will continue and report back here.

 

Z-stain from june 7th, 2015, seems to be growing onto the cardboard. It still could be trich, this plate was contaminated too, but here, the same happened as with the B+. However, this looks suspiciously much like trich. Fuzzy and suspicious.

 

Strophoria is dead, as dead as dead can be. 

 

Reversing doesn't work on all strains, so it seems. Well, what did I expect? These cultures are close to a year old, contaminated and unattended. I expected nothing and I got some surprises.

Don't try this at home, because it may not work for you. Or maybe it will. I don't have sufficient results and evidence to completely back my claims since I haven't been paying much attention to salvaging plates/cultures.


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#10 roc

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Posted 05 May 2016 - 04:57 PM

Thanks for sharing this!



#11 JanSteen

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Posted 09 May 2016 - 02:25 PM

Update on replacing the verm barrier with five layers of (tissue) paper:

It works okay contamination wise, however growth seems to be much slower than usual. This is caused by the lack of proper GE due to the nearly impermeable seal the fungus+paper forms. At least, that's why I suspect. Mycelium on a mesh makes a finer mesh, therefore inhibiting gas exchange. Sounds logical.

Compared to other jars, the paper-layered one is about 70% slower (it takes 2 weeks or more to colonize a jar using G2G whereas a normal jar takes a day or three, ten at most). 

 

Maybe with cardboard, or something more airy, less layers or other types of paper it'll be faster. 


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#12 JanSteen

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Posted 11 May 2016 - 12:52 PM

I didn't have any spore syringes and/or prints, so this will have to do.

 

Cubes (B+) on cardboard:

gallery_149763_3_695469.jpg


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#13 roc

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Posted 15 October 2016 - 10:53 AM

WOW!



#14 hyphaenation

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Posted 15 October 2016 - 08:15 PM

Excellent! Nice rhyzos...
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#15 CatsAndBats

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Posted 16 October 2016 - 08:11 AM

Damn @janSteen! I'm ALL over this. Well done.

 

high5.jpg


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#16 Hash_Man

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Posted 16 October 2016 - 09:54 AM

excellent janSteen

Informitive well written, not unlike the good TEKs of years gone by,

Not only is it a viable option for cleaning up spores or isolation technque, its good info for our friends with limited resources in other parts of the world, or for that mater, myself when I'm finacially limited.

Edited by Hash_Man, 16 October 2016 - 11:12 AM.

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#17 Heirloom

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Posted 17 October 2016 - 12:11 PM

I would like to see more posts JanSteen, this one is very informative.



#18 JanSteen

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Posted 17 October 2016 - 02:25 PM

Thanks for the comments guys! It means a lot to me. Words > likes if you ask me. As far as these petri's go: I've never opened them, I just let in some air whenever they were out of oxygen (petri's concaved from the top). They have fruited and germinated from those fruits' spores, but the nutrients are all gone now. It's fun to see them survive these kind of heavy treatments. I'll be making some new jars this winter with these cultures, to see if anything contaminates. Just for the fun of it, since I have some leftover rye and empty jars. If you guys would like it, I can post some pictures of what they look like now. Some petri's have died, some still have some moisture and seem to be alive.

#19 Hash_Man

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Posted 17 October 2016 - 05:29 PM

I like to see someone (maybe me) to try this to see how it might work for isolation, you know kinda like the fahtster isolation tek but better.
http://archives.myco...s/5/147159.html
146115.jpg

I like that what JanSteen said that cardboard has a certain resistance of some of our most common cantams and I beleive him, look at Azues, AlienI and P. Cyanescens . . you don't have to get all careful once it gets on wood or cardboard, the difference is you start right with the cardboard . . fuck I bet I could right now go to my kitchen microwave some cardboard in a Ziplock put a drop of multi-spore in the center and wait for some ropes to work with . . the cardboard is relatively low nute, which is what you kinda want when cleaning spores.

Is it just me or what?

@ JanSteen does that sound feasible?

Edit: the advantages I'm seeing is the ease of all this.

.

Edited by Hash_Man, 17 October 2016 - 06:01 PM.


#20 JanSteen

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Posted 18 October 2016 - 03:22 PM

It's not the resistance of cardboard, it's just that most bacteria from human source can't break down cellulose. Resistance would be something like: carboard excreting poisons/toxins or something. You could try something like that isolation tek you showed. But to me isolation means taking rhizos from agar. You could take those rhizo ropes from cardboard, I'm sure, because I did it.. But it would be more difficult since the cardboard tears easily. As for the tek you mentioned: I would just use a sonificator (like the ones used to clean jewelry) instead of shaking. Sonification doesn't damage the lids as much as shaking corn flying on and off the lid. But do sonification only shortly, since it would destroy large parts of fungus. But then again, cardboard soaked in water + fungus that secretes cellulase enzyme, doesn't need much shaking after just a few hours at optimum temperature. The cardboard would just degrade pretty fast, within hours maybe. As long as there are co-factors around for cellulase to do its job. I think that's Mg+ but I'm not sure. It's been a while. Spore prints/solutions on cardboard do work. And cardboard to cardboard is pretty straight forward. So yeah, it sounds feasible. I'm not really into isolation anymore, since I just shoot up everything with multispore and that works just fine. I do however plan on doing some isolation of reishi this winter. That stuff doesn't seem to like my agars all that much, so I think I'll switch to 100% cardboard and see if that works.
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