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[*] posted on 24-4-2011 at 16:33
Agar substitutes


I have always wanted to grow fungus and bacterial cultures, but I have no means of obtaining agar. I looked everywhere and I can't find it.

But I found gelatine which I read about that I can be used as agar substitute. I would like to grow oyster mushrooms tissue culture (mycelium, spawn) on gelatine. How should I prepare it?

Thanks for answers.
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[*] posted on 25-4-2011 at 08:53


Did you try Asian food stores? It's sold in these translucent crumpled sheets that look like dried plastic. You can also try vegetarian jello with no flavors/colors.

As to how to prepare gelatin as an agar substitute, I'm not too sure since I've never tried, but maybe try autoclaving the gelatin soln and pouring it while hot? Or maybe you can even pre-pour the plates/flasks and then autoclave?

Btw, have you consided liquid culture, in honey or corn syrup? It's very easy and mycellium grows very fast. You can clone or use spores. It's also great because you can just draw up some myc from the LC to expand the culture without ever having to open it like you do an agar plate. I had a perfect LC recipe written down somewhere, tried and true.

I also realized that oyster mushrooms grown on cardboard and newspaper taste like nothing. Is there a more nutritious substrate that will make them taste better? lol I do like those decorative pink oysters :D

[Edited on 4/25/2011 by Saerynide]




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[*] posted on 26-4-2011 at 04:21


I bought my agar from the chinese grocery store and added some beef broth to it, it works. You can get agar at carolina biolgical as well as many other science supply web sites it's not hard to obtain.
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[*] posted on 27-4-2011 at 01:55


I haven't found any asian or science stores near me, so that's not available. :(

Saerynide, could you post your LC recipe? Also, you just grown oyster mushrooms from little tissue and cardboard? This seems interesting, also maybe you could soak the cardboard in some nutrients like phosphates, some nitrate.. ;)
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[*] posted on 27-4-2011 at 10:34


I found it, after much digging in my lab book and revisiting some fun times :P

Makes one 1/2 pint LC:

3.4 g clover honey and 100 ml boiled Britta-filtered tap water (filtered, then boiled) was added to a 1/2 pint canning jar.

A tiny hole (~2 mm across) was punched into the lid. Cloth bandaid tape (the kind that comes in a roll made of plasticky cloth with adhesive on one side) was placed over the hole to provide a makeshift gas-permeable sterile filter and the band was screwed tight onto the jar (but not super tight or the jar'll break when heated).

Al foil was then used to cover the jar top - mold a square of foil onto the jar so that at least past the band is covered with foil. This serves as an additional gas-permeable contam barrier (will keep contams from falling onto the bandaid tape/hole, but air and CO2 can pass around the foil)

Jar was steamed over a high rolling boil for 35-40 min (measured after boiling commenced). The bottom of the jar was just touching the water in the pot. When finished boiling, do not uncover the pot or expose the contents to outside air until it is completely cold (I wait over night)

To innoculate, loosen foil cap and peel off bandaid tape to the edge of the hole, but dont expose the hole (this makes it easier in the next step when you need to really quickly peel off the tape to inject). Wipe with IPA, and replace foil cap lightly.

When ready, swiftly peel off tape, inject 0.5 - 1ml spore soln into hole (IPA and flame needle), stick back down tape, and wipe with IPA and replace foil cap.

If cloning a mushroom, wipe down the stem of your mushroom with IPA. Then sterilize (and resterilize) a razor blade, your hands and a syringe needle. Keep the razor and needle wrapped in an IPA soaked cotton ball. Then take a deep breath (so as to not breathe on the sample) Quickly slice down the base of your mushroom stem to split it lengthwise, then carve out a tiny thin rectangle of tissue from the center of the stem and pick it up by impaling it with the needle. If done very quickly and properly, this sample is sterile.

Still holding your breath, quickly lift off the foil cap (do not turn it upside down), lift off the tape just enough to expose the hole, drop the tissue sample through, but DO NOT touch the sides of the hole (for this reason, make sure you a cut piece of tissue that doesnt require "stuffing" through the hole). Stick back down tape, wipe down with IPA and replace foil cap. Breathe.

Incubate at 78-89F - these are temps I measured and it fluctuated between this range throughout the day.

According to my notes, in 2 days, translucent whitish fuzz was seen sprouting from the tissue sample, making it look like a fuzzy gelatinous ball.

As the myc grows, the LC becomes lighter in color. When the LC is almost colorless, the nutrients have been depleted.

It is also possible to stab a mushroom (first wiped with IPA) with a sterile syringe, draw up some cells into the syringe, and then inject into the LC. However, in my experience, this has not worked because I can never pull anything into the syringe except contams from the surface of the mushroom...


Some notes:

- The LCs should be CRYSTAL clear light sunshine yellow. If its even the slightest teensiest turbid, toss it. When I first started, I would think "well maybe it'll be ok, it's not *too* bad lookin". Turned out nope.. it's ALWAYS bad :(

- Do NOT shake (at all) or swirl (too hard) the LCs. Steaming is not 100% sterile (but its been a very good substitute for an autoclave so far :D) and if liquid touches the hole, it could contam.

- Spores take longer. I much prefer cloning, but cloning is also easier to contam.

- You can also make one of those silicone self-sealing injection ports if you plan on injecting spore soln, but IMO, I tried it once and it contamed, but the bandaid tape holes have a high success rate for me, even though I got the occasional contam from accidently shaking the contents.

- Sucking up the myc from the LC when its ready can be really hard, especially if the myc is very strong. I've heard some people add a piece of broken glass to the LC before steaming so it can be used to cut up the myc when the LC is shaken. I would assume they use a shake resistant injection port :P

As to why 3.4 grams, it just seems to be the magic number that works well every time. Maybe there is a better amount out there, but I just stumbled upon this number while trying several concentrations in the begining and this one worked the best for me, so I just stuck to it. Too high a conc and it nothing grows. Too low a conc and nothing grows :(

And finally, my failure rate the first time was high. Second time was significantly better and by third time, it was no problem :D


As for Oysters on cardboard/newspaper, you might find this interesting:

http://www.ncbe.reading.ac.uk/ncbe/materials/microbiology/PD...

This isnt the way I did it. I just used newspaper and magazines that went through a shredder (tiny squares), added some boiled water to make a bag full of soggy paper, sterilized by steaming/boiling again, and innoculated with a few spoons of grain spawn. They looked pretty, but tasted very bland :P (At least they didnt taste like magazine inks)



[Edited on 4/27/2011 by Saerynide]




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[*] posted on 28-4-2011 at 12:20


Thanks a lot for that information, I have wanted to do this for a few years and didn't know how to do it at home. Now I am going to try that maybe even tomorrow and I will report the results :D

So, after LC becomes almost colorless I should cut the mycelium from LC with maybe sterilized knife and then put it into the grain to make spawn? What do you use to make grain spawn? I am thinking about using brown rice as substrate, though maybe sawdust would work too. Maybe even putting LC with mycelium into toilet paper would work too :D
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[*] posted on 29-4-2011 at 07:28


I think with grains, an autoclave or pressure cooker is almost mandatory and steaming just doesnt cut it. - I'm guessing its because grains are just that dirty and also dont transfer heat well because they cant be too soggy (I didnt make the grain spawn I used, I got it from my prof).

I've never tried opening up the LC to outside air, but I think your idea of innoculating the toilet paper directly with the LC sounds interesting. Brown rice and sawdust definately work as substrates. I've never used sawdust, but brown rice flour cakes are great because they are easy to sterilize:

To sterilize brf jars:

Stack jars into supermassive pot on a steaming rack so they dont touch the bottom and put ~1.5 inches of boiled water in the bottom.

Cover up smoke detectors (they will go off - I take it that copious amts of water vapor means there is a serious fire), open the kitchen windows, and remove items you might not want to get damp. Then turn heat full on to get a (almost violent) rolling boil and generate steam for 1hr 30-45 min. There should be so much steam that the pot lid barely stays on the pot and instead kind of floats on a cushion of steam :D This is much more intense than sterilizing for LCs because the brf jars do not transfer heat as well as the LCs. However, one caveat. Dont turn up the heat too high such that the BRf burns. I don't think its possible on an electric stove, but a gas stove can cause the sides of the pot to get seriously hot. I always did this on an electric, and I think gas calls for very careful experimentation...

A LOT of steam will be generated over the course (enough to fog up and condense water on all the windows in my kitchen and living room and make the whole place feel damp), so really make sure you remove all items that can be damaged by excessive humidity first.

At 1 hr in, you will most likely have to top up the water level. I do this by cracking open the lid and pouring in pre-heated-to-boiling water so as to not drop the temp and interrupt steam generation. I usually judge when to add water when the boiling starts to sound less violent and more "normal" :P

Again, dont open the pot until 12 hrs later because it must be allowed to cool very slowly.

Im sure a pressure cooker makes mycology orders of magnitude easier. Funny because my housemate had a pressure cooker, but I was way too scared of it to use it :P

[Edit]: I forgot to mention, but I dont think you can open a BRF jar to non-sterile air because it is very prone to contams. You must inject for BRF jars or use a hood. However, once they are snow white and fully colonized, they are contam resistant.

For the magazine shreddings, I just quickly and carefully opened the bag (while keeping the opening turned downwards) to scoop in the colonized grain and it was ok. I think sawdust and toilet paper should be ok too since they dont have much nutrients unlike BRF.


[Edited on 4/29/2011 by Saerynide]




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[*] posted on 2-5-2011 at 08:44


Thanks for the info. I tried to do something last Friday night, but I think I wasn't so clean like you described.

If I remember correctly this is what I did:

I took two lowball glasses and one small transparent plastic box. Then I cleaned my hands and used cotton wool soaked with brandy to clean the glasses and box. Before that I washed them with soap and let the brandy evaporate. Meanwhile, I took syrup medicine spoon (5g) and took almost full spoon of honey. I added it to like 1/3 - 1/2L water (I am not currently sure how much was it, I think a little bit under 0.5L) and mixed it with honey. After that I boiled it, but some honey dissolved maybe burned (I tried to drink some of that water, it hat sweet but at the same time strange taste,like burnt). The solution was light brown. I cooled it to room temperature and added it to those jars. Disinfected with brandy, aluminium foil was used as cover for lowball glasses and the original plastic cover was used for the box. I took some tissue and added it to the solution. Then I put them to clean dark place. Today I checked them, I think in one glass mushroom tissue looks very little bigger, but i didn't see any big growth. Sometimes I notice the tissue at the bottom and sometimes is floating at the top. There is maybe some yeast contamination in one glass because I see few very small bubbles forming, but that is not too much. I just opened the cover of aluminium foil a little bit by accident and I think I noticed strong mushroom smell instantly. DId I actually do something right or is that a failure?
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[*] posted on 2-5-2011 at 09:58


I'm sorry if my previous posts were confusing. I assumed it was obvious that the medium went into the jars before sterilization. Let me clarify:

You should mix the honey with the filtered+boiled water into the canning jars, and then sterilize the jars filled with the honey water.

Though it is entirely possible to do it the way you did (sterilize medium and containers separately and then fill), it is significantly harder because you risk contamination by exposing both the insides of the jar and the entire liquid surface to outside air when you pour. If you have a laminar flow hood, then doing it this way will be no problem at all.

You might ask why it is ok to expose the tissue sample and the LC inside to air when cloning but not ok when pouring. True that while cloning, you are exposing the sample and LC to air. However the chance of contamination is much smaller in the case of cloning because the sample and the hole have a very tiny surface area. On the other hand, when you pour, you must remove the lid, and the pouring liquid will create a lot of turbulence and will mix with the air, pulling in contams.

The honey also burnt in your case because the medium was boiled on the stove directly. If the jars are prefilled and steamed without allowing them to directly contact the pot bottom, the steam and water will keep the jars at 100C and the medium wont burn :)

I don't know if a newly started LC should smell like mushrooms, since opening the LC before its fully colonized will most likely destroy it, and thus I've never done so.

And you really should invest in canning jars :D Al foil lids on lowball glasses (even with plastic wrap) are rather unlikely to suceed without a real autoclave and proper cell culture equipment (because I've tried lol). I've seen flasks with Al foil lids that have been properly autoclaved in our research lab and they still developed mold puffs, so foil lids without some kind of sterile filter (even if its just bandaid tape) is not a very good on its own.

You can find canning jars at the dollar store or $8.95 at big supermarkets will get you a flat with 12 jars. You can keep reusing the jars and metal parts as long as you wash them out very well with soap between each culture. If you are paranoid or had a serious contam, you can soak them in bleach water or boil them before reuse.

You're on to a good start, so dont give up even if your culture turns turbid/powdery in the next few days. If they contam, pour them out, resterilize everything, and try again. Do it once or twice and you will get it right :D

[Edit]: Dont throw out your LCs yet. Wait until they either grow, or contaminate. That way, you will at least get to see what a contamination looks like first hand in the worst case, and what a growing culture looks like in the best case :D

[Edited on 5/2/2011 by Saerynide]




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[*] posted on 2-5-2011 at 10:43


Quote: Originally posted by Random  
I have always wanted to grow fungus and bacterial cultures, but I have no means of obtaining agar. I looked everywhere and I can't find it.


Hi. Re. the original question for agar substitutes, I unearthed the following suggestions. These are from reliable sources, but not things that I've tried myself:

3% cellulose slurry (any sort of clean/washed cellulose fibre)
sand
psyllium,
fiberglass batting
vermiculite
possibly acrylamide gel
washed peat moss,
probably washed coffee grounds

starch and gelatin tend to be broken down by enzymes secreted by most things making that less of an option

You would need to nutritionally supplement most/all of these. I expect that a potato/dextrose agar recipe could be pillaged for the nutritional component.




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[*] posted on 3-5-2011 at 13:56
A lot less expensive the Agar-agar


Quote: Originally posted by Random  
I have always wanted to grow fungus and bacterial cultures, but I have no means of obtaining agar. I looked everywhere and I can't find it.

But I found gelatine which I read about that I can be used as agar substitute. I would like to grow oyster mushrooms tissue culture (mycelium, spawn) on gelatine. How should I prepare it?

Thanks for answers.

The Economist
Apr 20th 2011
| From the print edition | Science and Technology

Bioremediation
Bottom feeders
A novel way of dealing with an unpleasant problem


For your delight and delectation

DESPITE their name, disposable nappies are notoriously difficult to
dispose of. Studies of landfills suggest they may take centuries to
rot away. But Alethia Vázquez-Morillas of the Autonomous
Metropolitan University in Mexico City thinks she has found a
method of speeding the process up.

As she and her colleagues describe in Waste Management,
cultivating the right type of mushroom on soiled nappies can break
down 90% of the material they are made of within two months.
Within four, they are degraded completely. What is more, she
says, despite their unsavoury diet the fungi in question, Pleurotus
ostreatus (better known as oyster mushrooms), are safe to eat.

To prove the point she has, indeed, eaten them.

The culinary use of oyster mushrooms was one reason why she
picked them for the experiment. The species is frequently used in
stir-fries and is often added to soups. The other reason was that
Pleurotus ostreatus is widely used in what is known as
mycoremediation—the deployment of fungi to clean up waste. It is,
for example, already grown on agricultural materials such as heat
and barley straw, and industrial waste like coffee grounds and the
leftovers from making tequila. Dr Vázquez-Morillas and her
colleagues were trying to extend the oyster mushroom’s own
culinary range.

The reason nappies are difficult to break down has nothing to do
with their use. Even a clean nappy would hang around for a long
time in a dump. The main ingredient of a nappy is cellulose, an
annoyingly persistent material. Pleurotus, however, grows on dead
or dying trees in the wild and is thus well provided with enzymes
that break cellulose down. And, since Mexicans alone throw away 5
billion nappies every year, there is plenty of material from this
source for them to get their mycelia into.

The idea that the result might be sold and eaten may be
controversial but it is not absurd. The nappies the researchers
used were contaminated only with urine, not faeces. A healthy
person’s urine is sterile and Dr Vázquez-Morillas also treated the
nappies with steam, to make sure. Such treatment would kill the
nasty bugs in faeces, too, though, so mushrooms grown on treated
nappies should, in theory, be safe to eat.

In practice, overcoming the yuck factor might be an insuperable
barrier to marketing nappy-grown fungi, and the cost of the steam
treatment could make the exercise futile. Mycoremediation of this
sort does not, however, depend for its success on selling the
results. Merely getting rid of what would otherwise hang around
indefinitely is worthwhile. And of the fungi themselves, Dr
Vázquez-Morillas observes, “they are cleaner than most of the
vegetables you can find in the market, at least in Mexico.”


djh
------


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[*] posted on 3-5-2011 at 18:01


So first he'll need a source for nappies. That implies a 'wife' in the picture somewhere. Are they over the counter? Sometimes maybe.

Mushrooms as packaging is another interesting fungus tale. Although if you think about it there could be down sides to their plan of using colonized material as packing stuff. Very green though. In fact, if you're too lazy to dispose of the spent mycelial mass a group of bongo playing hippies come over and consume the stuff for you.





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[*] posted on 3-5-2011 at 19:16


Quote: Originally posted by food  
So first he'll need a source for nappies. That implies a 'wife' in the picture somewhere. Are they over the counter? Sometimes maybe.

You could go down the street to the Old People Home and
collect their Depends. You can get free samples, albeit
unused.

http://www.depend.com/
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[*] posted on 3-5-2011 at 21:01


Quote: Originally posted by The WiZard is In  

You could go down the street to the Old People Home and
collect their Depends


... hadn't thought about that. Which was probably a good thing.

filthy rags will play a part in my next mycological adventure, as a matter of fact. I'm going to grow out some oyster mushroom on shredded newpaper. All the news that's fit to plant.




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[*] posted on 4-5-2011 at 07:06


Quote: Originally posted by The WiZard is In  
Quote: Originally posted by food  
So first he'll need a source for nappies. That implies a 'wife' in the picture somewhere. Are they over the counter? Sometimes maybe.

You could go down the street to the Old People Home and
collect their Depends. You can get free samples, albeit
unused.


Awwww that's disgusting!! Hhahaha. I guess I'm not enough of a scientist to over come my prejudice against food grown from human waste! :P




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[*] posted on 7-5-2011 at 01:04


Well, I would rather take clena toilet paper than nappies :P

I am starting to see some small white things floating in the water along the main part that was used to clone. That part didn't increase, but this white stuff could be mycelium which is growing there.

I read on some pages that when fluffy pieces of mycelium appear I should store it in the fridge or put it on the substrate. So, should I wait now or put it on the substrate?

[Edited on 7-5-2011 by Random]
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[*] posted on 11-5-2011 at 07:14


Man, been too busy lately to visit the forum! Yep, when its done (ie, liquid is mostly coloress, lots of fluffy balls of mycelium) you should either use it or stick it in the fridge :D



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[*] posted on 11-5-2011 at 14:11


It looks I have been succesful then :D Liquid is yellow, but more pale. There are many 1-3 mm pieces of mycelium floating in the solution and in the smaller glass there are few smaller and two big pieces of mycelium floating (though, one has a black dot - contamination?). Is this ready for putting it on substrate? I am thinking about cooking some barley or maybe brown rice in water and then decanting it to just leave it moist. Should I ust pour liquid culture over that and seal the jar?

[Edited on 11-5-2011 by Random]
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[*] posted on 13-5-2011 at 10:32


A black dot eh? Hmmm I dont recall seeing black dots exactly in my LCs but I do remember sometimes, it looked like I had dark colored regions because really dense mycellium. If the black dot is submerged, I don't think it is mold because it would be unlikely for a mold to fruit under liquid. If it were floating, then it could be a puff of mold. Molds usually take over very fast compared to mushroom mycelium though.

I cant give any practical advice on barley or rice grains, since I've never tried doing it on my own. Be sure to share your results :D

You can also mix up some brown rice flour and vermiculite with a bit of water and lightly pack it into jars, and then sterilize. The vermiculite makes the medium for airy. Make sure you dont add too much water. It shouldnt be soggy, but damp and grainy, kind of like sand for a sand castle.

I suppose since you've already been pouring your culture open to air, you might as well continue pouring. I cant say how sucessful that will be, since I've never tried. For the next grow, I definately recommend canning jars and syringes :P




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[*] posted on 1-9-2011 at 23:00


Knox Gelatin never worked for me very long as an agar substitute, too many things will digest it. I have used knox gelatin to make a primitive photographic emulsion with silver salts, although due to it unrefined nature, it makes for a slow (ISO 5) glass film plate for a box camera.
PolyAcrylamide gel might work, it decomposes over time into ammonia, nitrogen and CO2. I have a bunch of the stuff, it's how I got my potted plants through this heat wave + Drought we've had down here in Texas. I have accidently grown mold on PolyAcrylamide gel when I laced it with a low dose of miracle grow so I could grow some plant cuttings.
I would say grind it fine in a clean coffee grinder (powderize it). You can get the polyacrylamide at Home Depot, Loews, and other garden centers, it goes under the name "WATER CRYSTALS, etc."




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[*] posted on 23-11-2011 at 14:56


You can find a lot of information on mushroom cultivation on the Shroomery. They have some really good guides.
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[*] posted on 9-12-2011 at 19:05


I have done liquid cultures before, and I used malt extract. I would imagine you could prepare an agar plate (if you're using the gelatin) similarly. You can buy malt extract from your local beer/wine brewery hobby shop. And as the person above me said, visit the Shroomery. Lots of very good info to be had.
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[*] posted on 1-4-2012 at 14:08


@Saerynide, I tried your technique for growing fungus with honey and water, with surprising results!

I modified your technique a little, adding a few sterilisation steps. I was expecting mediocre results given that the honey I used was not the best.

According to my lab notebook:
1. Punch a hole in the lid of a mason jar.
2. Rinse both the jar and the lid with warm water, dry, wipe the inside with IPA and close.
3. Set a pot of water to boil.
4. Measure 100g of filtered water directly into the jar.
5. Wipe a plastic spoon with IPA and fetch half a teaspoon of honey (3.4g)
6. Put honey in jar, close and let sit, no stirring.
7. Cover the hole with one of these.
8. Cut a piece of foil and wipe it with IPA.
9. Place the whole thing in a pot of water brought to a roaring boil.
10. Let sit for 50 minutes
11. Let cool for as long as necessary, undisturbed.
12. Inoculate.

I inoculated some Penicillium roqueforti (from Danish blue) and got these results after 3 days at room temperature:
IMG_0524downsize.jpg - 77kB

The picture is not great, I can't take pictures from above because the lid is opaque. I'll probably take a picture from above once the nutrient medium is depleted. As far as I know, I didn't contaminate the culture yet.

If you're wondering how I inoculated this fungus, I took a long needle and flame sterilised it, I then scraped the blue part of a chunk of cheese I cut off. When I unpeeled the bandaid, I was met with an unpleasant surprise, a drop of water closed off the hole. So I had to take a paper towel and dry off the opening, insert the fungus and close it off, all while holding my breath.




"Ja, Kalzium, das ist alles!" -Otto Loewi
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Eddygp
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[*] posted on 3-4-2012 at 12:49


Wow! Those fungi are amazing, Yeti! I wonder if it would grow on a LC with milk...;)
I will see if I make some of my own!!!!:)
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Eddygp
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[*] posted on 9-4-2012 at 02:06


I managed to grow some white mycelium with a LC of my invention. It uses some sugar, a bit of (pH-neutralized) vinegar, very little starch and (to see the fungi better) green food colouring.
Shall I upload some photos?




there may be bugs in gfind

[ˌɛdidʒiˈpiː] IPA pronunciation for my Username
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