Ausi Panaeolus cyanescens Liquid Culture/Spawn bag Log

[bluehelix]

Well, decided to try my hand on Austrailian Panaeolus Cyanescens (PC) using the liquid culture (LC) and spawn bag techniques that worked so well for me with cubensis. So far PC appears to grow as fast as cubes and does great with liquid culture techniques.

The LC was a 4% (by weight) sugar solution of 50% dextrose and 50% light malt. It was brewed on a coffee machine and pressure cooked for 25 minutes in a quart jar. The quart jar was covered with a metal lid with two holes. One hole the size of a penny in the center and one small bb-sized hole off to the edge. Both holes were covered with breathable cloth medical tape and the lid was covered with a filter disc and metal band.

The LC was stirred constantly at varying RPMS from 60 to 300. It was done in 7 days. Attached are three pictures showing the LC mature at days 3, 5, and 7.

[bluehelix]

Well, decided to try my hand on Austrailian Panaeolus Cyanescens (PC) using the liquid culture (LC) and spawn bag techniques that worked so well for me with cubensis. So far PC appears to grow as fast as cubes and does great with liquid culture techniques.

The LC was a 4% (by weight) sugar solution of 50% dextrose and 50% light malt. It was brewed on a coffee machine and pressure cooked for 25 minutes in a quart jar. The quart jar was covered with a metal lid with two holes. One hole the size of a penny in the center and one small bb-sized hole off to the edge. Both holes were covered with breathable cloth medical tape and the lid was covered with a filter disc and metal band.

The LC was stirred constantly at varying RPMS from 60 to 300. It was done in 7 days. Attached are three pictures showing the LC mature at days 3, 5, and 7.

The next step was making the spawn bags. My spawn bags in my case do not hold spawn but rather the final fruit-out substrate. They were loaded with a mix of the following:

6.5 parts compost (via dry weight)
2 parts WBS (via dry weight)
2 parts rye grass seed (via dry weight)
3% Stevia leaf (percentage via volume)
0.2% black mustard (percentage via volume)

Stevia tea leaf is a sweet leaf that adds glycosides which reportedly increase potency. Black mustard is a very rich source of oil and is much like adding a shot of vegetable oil to the mix to round out the nutritional matrix of the substrate.

Moisture was balanced by feel with all elements but the WBS. The WBS was added last and mixed in dry, so it could slightly dry out the overall mix a little when cooking. A drier mix is important to accept the LC injections without getting things too wet. All bags were pressure cooked for 4 hours, then sealed with an impulse sealer without letting air in, and hung up with rubber-footed wood clamps to suck the air into the filter patch and allow them to cool. After totally cooled the bags were removed from the clamps and injected through a tape-reinforcement on the bag with 140ml of liquid culture using a huge syringe. Immediately upon removal of the needle, the hole was sealed with hot glue and the entire bag well mixed by kneeding it.

The pictures attached show the explosive growth after only 48 hours. I expect the bags to be completely done in 7 to 10 days (total). I'll post more pictures as they complete.

Thanks, Waylitjim. You all are making me blush.

Yeah, the stir plate can be your friend too. For about $25 to $50 on Ebay, you can get one. The nicer ones, closer to $50, tend to look new and should be direct drive and silent. Also, don't forget the rubber tubing over the magnet middle to silence the glass-to-magnet clinking sound.

With a stir plate on constantly I've seen liquid cultures really take off unlike anything. 5 to 7 days from spore to so dense you can barely see the magnet isn't uncommon with cubes. These ausi pan cyans create mycelium a little less dense than cubes in liquid culture, but they are fast little buggers too.

My second secret weapon are vacutainers. These are little test tubes that are sterile inside and feature a self-sealing port on top. They come with a vacuum inside that will suck your liquid culture literally out of your syringe for safe storage. Later you can poke the lid, push in a couple ml of air, and draw the solution back out! All of this can be done without any clean room.

When in the vacutainer, you can easily store liquid cultures for months, even years, in the refrigerator. You can store spore solutions in them too. For a few cents a piece, you can't go wrong! On Ebay you can find 1,000 red-top (that's what you want) for about $25 plus shipping. Or you can always find them $10 for 100 here:

http://www.culturekits.com/index.cfm...3F5E7BF943.cfm

PS - Pictures courtesy of Agar on The Shroomery.

Lazlo, I like the Shroomery, but since I posted my Ecuadorian grow log last time there, I thought I'd share this grow with this board for a change. Both boards are great for a little bit different reasons.

Update: today the bags are coming along really well! I would estimate 35% colonization at this time (day 3), and speeding up. They should make it under 10 days as expected. This is my first Pan Cyan grow, so my fingers are crossed!

Update: Day 4 and the bags were about 45% to 65% done. The differences were because I injected with 140, 120, 100, and 80ml since I was running low of liquid culture. Today, I decided to mix the bags up to even the growth out between them so they finish about the same day. Considering the growth I've seen so far, I figure the bags should be ready to lay in the tray in 4 or 5 more days.

Pardon my ignorance, but what do you mean when you say the LC was "brewed on a coffee machine?"

I think Hippe3 was the first I knew to popularize the idea of using a coffee machine to mix the sugars in the solution, heat up the solution readying it for pressure cooking, and filter it all in one. Here's how I do it:

Put three or four coffee filters, each stacked inside the other like they come, on a gram scale and fill them with measured dry powdered sugars of light malt and dextrose (see picture; each sugar 2% by weight, making a 4% sugar solution). Put the filters, full of the powdered sugars, in the coffee machine and pour the water for the liquid culture in top of the machine. In my machine, I need two extra cups of water than I expect in the liquid culture because of the water lost in the vapor. What brews up is a delicious cup of the finest Columbian coffee... just kidding. Really the liquid culture support solution brews up reasonably filtered of the non-soluble stuff in malt powder--don’t have a clue what that stuff is--that would otherwise cloud the mix.

I take that, pour it in a quart jar, put my Teflon magnet in, cover with a metal lid drilled and taped as shown in the picture, cover with a filter disc (shown in picture but can be replaced with Tyvek material), screw it all down with a metal band, put foil over it all, and pressure cook for 25 minutes. After I remove it from the pressure cooker, I remove the foil and let it cool. Once the solution cools completely--don't try to rush it--I briefly take off the band, slide the filter disc over a little to expose the inject hole, and inject either liquid culture or spore solution. The medical tape is a reasonable filter for very quick things like an injection to prevent mold spores from falling in, so you don't need a flow hood or glove box but work fast. Although not necessary, I also spray with Oust if I have it sometimes (depends on my mood). Once the injection is done, slide back the filter disc and screw it all down with the metal band as before.

PS - The breath hole in the metal lid could be about twice as big, but I didn't have a way to drill a larger one. I don't seem to have any trouble with my liquid cultures maturing very fast even with the small hole, though.

Having a stir plate at home would be so SWEET.

Good stir plates on Ebay have been a little hard to find lately, but yesterday there was a perfect stir plate for mycology that I found on Ebay that sold for $31:

http://cgi.ebay.com/Thermolyne-Cimar...QQcmdZViewItem


That's about a $300 stir plate there in like-new condition that sold for about $30! And that happens all the time. You just have to search carefully.

How do you break up those pretty little mycelia balls before you suck them up in the syringe?

That depends on the type of mycelium balls (aggregates). The mycelium balls you see there are soft gelatinous aggregates, so they were easily sucked up in my 16-guage needle. I was pretty scared they were going to clog up the needle too since I had not seen that form of aggregate before, but this was my first time with a spore inoculation of pan cyans too. The cubensis forms I’ve seen were more like a heavy blizzard of snow that formed even more quickly than the pan cyans. I had a cubensis liquid culture finish in about 4 days (see the picture of a 3.5 day culture that was being constantly stirred at about 600 RPM). And I’ve had other edibles in LC too that formed other types of aggregates.

The faster developing mycelium seems to form a snow like slurry. As the culture is stirred more vigorously or for longer, aggregates will form. If you stir too long, the aggregates can harden which can be a big problem since they can clog the needle.

I think the exact morphology of the mycelium is a complex function of the stir rate, original amount of inoculants, whether it was a spore solution or liquid culture inoculation, type of mycelium, temperature of incubation, length of incubation, the types of sugars used, and other things. In other words, you should just keep an eye on it. If it looks like it's forming hard aggregates, you'd best use it right away or change something. If it works out, which it usually does, do the same thing next time.

I do have some loose guidelines I’ve applied. For example, when I see the magnet load down with mycelium, I turn up the RPMS until it shakes it loose and then keep the RPM elevated. On the other hand, if I see hard aggregates forming, I turn down the stir rate since it seems that high stir rates tend to promote this sort of thing (no you can't break them up by turning up the stir rate real high at least not with the typical octagonal magnets; I've tried that). And if the culture is over a week old and I see aggregates, it's best to just use it then since it won't become any more dense with time, only more a pain in the ass as the aggregates harden.

And if I may be so bold, what kind of camera took those sweet closeups?

I have a 5 megapixel Olympus C-5050 Zoom. I love it a hell of a lot better than my old Nikon 5000 because it has a very bright lens in comparison. The Nikon was okay and had a great macro lens, but it had this very typical digital camera problem where the camera really requires a professional flash attachment for indoor shooting or else you won't get decently fast exposure times (it'd take like 1/8th a second which is way too slow for anything that moves). Incidently, I am not a photography buff or photographer nor do I pretend to be. I know the most basic concepts of photography but mostly I just point and shoot unless I am in a really odd lighting situation that requires me to manually adjust the focus or aperature.

PS – Attached you see what a Ecuadorian cubensis liquid culture looked like only 3.5 days after inoculation (it was actually used the next day). As you can see, cubensis liquid cultures can develop, if constantly stirred, screamingly fast and look like snow. I suspect the snow-like mycelium is rhizomorphic whereas the pan cyans, even on agar, don’t form rhizomorphic mycelium that I know of.

BuckarooBanzai, I use the brewer's grade POWDER, not syrup. It's the Cooper brand, which is well known throughout the world. I am guessing the stuff that the brewer's use is probably of a higher quality than lab stuff as it’s likely fresher.


UPDATE
It's day 7 since I injected the Ausi Pan Cyan bags with liquid culture, and today, all the bags are well colonized (see picture below). After the casing cooled, I laid the trays.


The casing soil was sterilized. The casing is the basic 50/50 Tek balanced to a pH of 7.2 (as measured by a calibrated pH meter accurate to +/-.01). Balancing was via aragonite crushed coral, a chip form of calcium carbonate, and powdered food grade calcium carbonate. 10-15% (per volume) coco coir was added as well to improve the overall texture and add some nutrients which the mycelium can grow into.


I then laid the trays. Upon opening the bag, I found the mycelium smelled slightly strange, not bad just different than cubensis. On top of the heavy mushroom smell, it had a slight peppermint smell or something which was odd. Tray depth was laid to approximately 2". The casing soil was applied to a depth of 1/4" (just enough to cover the substrate). Each tray was covered with plastic wrap with was banded down and taped down the center with masking tape. Using a small knife, the masking tape was cut and the cut was spread about 1/8" to 1/4" wide. I taped over the gap with cloth medical tape.


All trays have been elevated off the shelf to allow air to circulate under the tray in order to prevent overheating, common after casing if plastic wrap is used. Overheating can encourage bacterial blooms in sterilized casings and result in a loss of mycelium vigor, so it's important to prevent it. I am monitoring the temperature of the containers with a temperature controller set to 84F. If the trays exceed this temperature, the controller turns on a small desk fan to slowly cool the trays.

Due to limitations in my setup, the trays will not be incubated in darkness.

UPDATE


The tray core temperatures were soaring high (85F) and the thin layer of casing was immediately penetrated by all the trays. Half could wait another 24 hours for a fuller penetration so they'll go in tomorrow. But half went to the fruiting chamber today. I was amazed at how closely this mycelium looks to contamination when first coming through. It looks like a gray mold when it penetrates compost (not grain), but I am pretty positive it's just the way the mycelium looks because I saw it look that way in the bags too.

After placed in the fruiting chamber, I went through and micromanaged the casing surface as I usually do. That includes moving uncolonized casing to areas that are poping through and adding casing if necessary. The idea is to even out the casing application using the mycelium growth underneath as a guide.

It seems that the momentum of this grow is fairly high which is usually a good sign. At this point, I think little if any casing incubation is needed given the casing is so thin and the momentum of this mycelium is high with liquid culture. Of course other grows could be different, so I don't think casing incubation should necessarily be eliminated for pan cyans.

Thank, waylitjim. I don't think I deserve anything, but I am hoping for it! Mushrooms are way to wonderful for me to deserve them.

So, some readers might be wondering about my fruiting chamber. It's the same grow chamber I've used for years now for all sorts of mushrooms. Below you can see the basics of the chamber. I use a Vick's cool mist attached to a garden hose as my 100% RH source. The high humidity cool mist is mixed with unfiltered room air in a mixing PVC tee. I can control the speed of the fan that brings in the room air, and so I basically control the exact humidity of the chamber. For cubensis (and Pan Cyans this time) I shoot for a chamber humidity around 92%-98%, never allowing it to reach 100%, which means the input is mixed down to maybe 85%-90% RH and the rest of the humidity is generated by the casings. I have observed that green mold is almost entirely caused by overly high humidity in combination with weak mycelium growth, so I am always really careful to monitor and adjust the humidity. Humidity is measured, by the way, with an accurate digital hydrometer called the Caliber III. They are a bit pricey at a little over $20, but they always pass the salt test dead on and are about as good as it gets for under a couple hundred dollars.

The actual chamber is huge rubbermaid (50 gallon I think) with the lid modified to provide a clear vinyl window (they sell that stuff at Walmart). The window was cut to size, hot glued on the lid frame, and the sealed with duct tape. I also have a single side window for mushroom-level observation (the forest-of-mushrooms view). Also, the lid has been made a little more air-tight with low density foam weather stripping tape on the underside.

[shobimono]

That lc looks really nice!!
Can't wait to see how this turns out, please keep us updated.

This is good stuff... keep it comin Blue...

[bluehelix]

The next step was making the spawn bags. My spawn bags in my case do not hold spawn but rather the final fruit-out substrate. They were loaded with a mix of the following:

6.5 parts compost (via dry weight)
2 parts WBS (via dry weight)
2 parts rye grass seed (via dry weight)
3% Stevia leaf (percentage via volume)
0.2% black mustard (percentage via volume)

Stevia tea leaf is a sweet leaf that adds glycosides which reportedly increase potency. Black mustard is a very rich source of oil and is much like adding a shot of vegetable oil to the mix to round out the nutritional matrix of the substrate.

Moisture was balanced by feel with all elements but the WBS. The WBS was added last and mixed in dry, so it could slightly dry out the overall mix a little when cooking. A drier mix is important to accept the LC injections without getting things too wet. All bags were pressure cooked for 4 hours, then sealed with an impulse sealer without letting air in, and hung up with rubber-footed wood clamps to suck the air into the filter patch and allow them to cool. After totally cooled the bags were removed from the clamps and injected through a tape-reinforcement on the bag with 140ml of liquid culture using a huge syringe. Immediately upon removal of the needle, the hole was sealed with hot glue and the entire bag well mixed by kneeding it.

The pictures attached show the explosive growth after only 48 hours. I expect the bags to be completely done in 7 to 10 days (total). I'll post more pictures as they complete.

[waylitjim]

Lookin' good Blue, it's nice having you around

[BennyBlanco]

why dont my lc's ever look like that

[waylitjim]

Benny, keep in mind that Blue Helix is a 100 yr old myco-ninja.
I asked him about his artform, and he revealed to me his secret weapon,
The Stir Plate.

Originally Posted by bluehelix

Waylitjim, the stir plate is Fisher Scientific Isotemp 7"x7" plate model.
It's like the attached picture but larger. It is a non-heating ceramic-top
plate that magnetically stirs from 60 to 1200 RPM. It's also totally silent.
You can get that kind of thing on Ebay for about $40.

I am also using reflective bubble wrap on the bottom and wrapping the jar
with it too. I control temperature via a controller that is connected to a
heat bulb. It turns on and off keeping the temp around 84F. This part is
totally not necessary and I did it just because I had the stuff around.

[bluehelix]

Thanks, Waylitjim. You all are making me blush.

Yeah, the stir plate can be your friend too. For about $25 to $50 on Ebay, you can get one. The nicer ones, closer to $50, tend to look new and should be direct drive and silent. Also, don't forget the rubber tubing over the magnet middle to silence the glass-to-magnet clinking sound.

With a stir plate on constantly I've seen liquid cultures really take off unlike anything. 5 to 7 days from spore to so dense you can barely see the magnet isn't uncommon with cubes. These ausi pan cyans create mycelium a little less dense than cubes in liquid culture, but they are fast little buggers too.

My second secret weapon are vacutainers. These are little test tubes that are sterile inside and feature a self-sealing port on top. They come with a vacuum inside that will suck your liquid culture literally out of your syringe for safe storage. Later you can poke the lid, push in a couple ml of air, and draw the solution back out! All of this can be done without any clean room.

When in the vacutainer, you can easily store liquid cultures for months, even years, in the refrigerator. You can store spore solutions in them too. For a few cents a piece, you can't go wrong! On Ebay you can find 1,000 red-top (that's what you want) for about $25 plus shipping. Or you can always find them $10 for 100 here:

http://www.culturekits.com/index.cfm...3F5E7BF943.cfm

PS - Pictures courtesy of Agar on The Shroomery.

[sandman]

Daaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aamn

[Lazlo]

Fed up with the Shroomery Blue? I'm glad you're around here. Lots of nice information comming from you. You still haven't tried the vanilla LC yet huh? lol! Just kidden! Looking good man.....

Nice nice nice! Thanks for the info Blue!!!

I like those vacutainers.

your lookin pretty good man..
damn...i love that magnetic stirrer you
have...lol...i want one..

[bluehelix]

Lazlo, I like the Shroomery, but since I posted my Ecuadorian grow log last time there, I thought I'd share this grow with this board for a change. Both boards are great for a little bit different reasons.

Update: today the bags are coming along really well! I would estimate 35% colonization at this time (day 3), and speeding up. They should make it under 10 days as expected. This is my first Pan Cyan grow, so my fingers are crossed!

[BennyBlanco]

yeah I just picked up some of them vac tubes 100 for 13 99 now I just got to get my stir plate

Extremely informative, blue! Thanks for sharing here at topia!

[bluehelix]

Update: Day 4 and the bags were about 45% to 65% done. The differences were because I injected with 140, 120, 100, and 80ml since I was running low of liquid culture. Today, I decided to mix the bags up to even the growth out between them so they finish about the same day. Considering the growth I've seen so far, I figure the bags should be ready to lay in the tray in 4 or 5 more days.

[Hippie3]

archive material

nice work
thx for sharing

[bluehelix]

Geez, guys, the thread is just starting! Who knows how it will all end up? Let's archieve it once the picking the fruit part comes, okay?

[BuckarooBanzai]

DUDE...VACUTAINERS....the mind reels with possibilities for those lovely little presealed and ported test tubes....oh THANK YOU FOR THAT IDEA!!!!!!!

Pardon my ignorance, but what do you mean when you say the LC was "brewed on a coffee machine?"

That is a pretty collection of puffballs...

[lost_onabbey_rd]

well blue.. the A.M tag is just for sorting things out later... it doesn't go straight to the archives...
but from the way things are going, and you obvious knowledge and well written post.. i'm sure when it's all said and done this thread will end up in the vaults
LOST

[Hippie3]

lol
plz let us worry about the archive end of stuff,
you just keep posting the good stuff.

[bluehelix]

Pardon my ignorance, but what do you mean when you say the LC was "brewed on a coffee machine?"

I think Hippe3 was the first I knew to popularize the idea of using a coffee machine to mix the sugars in the solution, heat up the solution readying it for pressure cooking, and filter it all in one. Here's how I do it:

Put three or four coffee filters, each stacked inside the other like they come, on a gram scale and fill them with measured dry powdered sugars of light malt and dextrose (see picture; each sugar 2% by weight, making a 4% sugar solution). Put the filters, full of the powdered sugars, in the coffee machine and pour the water for the liquid culture in top of the machine. In my machine, I need two extra cups of water than I expect in the liquid culture because of the water lost in the vapor. What brews up is a delicious cup of the finest Columbian coffee... just kidding. Really the liquid culture support solution brews up reasonably filtered of the non-soluble stuff in malt powder--don’t have a clue what that stuff is--that would otherwise cloud the mix.

I take that, pour it in a quart jar, put my Teflon magnet in, cover with a metal lid drilled and taped as shown in the picture, cover with a filter disc (shown in picture but can be replaced with Tyvek material), screw it all down with a metal band, put foil over it all, and pressure cook for 25 minutes. After I remove it from the pressure cooker, I remove the foil and let it cool. Once the solution cools completely--don't try to rush it--I briefly take off the band, slide the filter disc over a little to expose the inject hole, and inject either liquid culture or spore solution. The medical tape is a reasonable filter for very quick things like an injection to prevent mold spores from falling in, so you don't need a flow hood or glove box but work fast. Although not necessary, I also spray with Oust if I have it sometimes (depends on my mood). Once the injection is done, slide back the filter disc and screw it all down with the metal band as before.

PS - The breath hole in the metal lid could be about twice as big, but I didn't have a way to drill a larger one. I don't seem to have any trouble with my liquid cultures maturing very fast even with the small hole, though.

[BuckarooBanzai]

I am quite new at this and I thank you MUCH for the excellent detail!

Having a stir plate at home would be so SWEET. It would make my little cultivation area so much lab geek cooler! Something else for the wishlist...

How do you break up those pretty little mycelia balls before you suck them up in the syringe?

And if I may be so bold, what kind of camera took those sweet closeups?

Sorry for so many questions!!!

[bluehelix]