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 GroboClone
Anthropomorphic Elephant
Registered: 01/07/11
Posts: 332
Loc: Canada 
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 New Year, New Projects (Update March 29) 
#15698797 - 01/21/12 05:42 PM (4 months, 5 days ago) |
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After a bit of a hiatus over the Christmas break I'm back and ready to start some new projects.
The first step in any project is to get the culture out of cold storage and growing, so it's time to make some plates.
I used up the last of my agar on my previous batch of plates.
It was a 20+ year old can of DIFCO Bacto-Agar.
Fortunately I was able to stock up on some excellent supplies.
Here we have ultra pure water, a mix of yeast/malt/dextrose/peptone, and some agarose (refined from agar)
  
Code:
Lab Grade Water Sigma Yeast Malt Broth SeaKem LE Agarose
Distilled Usage: 21 g/L Analytical Specifications
Deionized
Ingredients (g/L): Gelling temperature (1.5%) 36°C ±1.5°C
Delivered on tap Peptone A, 5.00 Melting temperature (1.5%) ≥90°C
via glass pipes Yeast extract, 3.00 Gel strength (1%) ≥1,200 g/cm²
Malt extract, 3.00
Dextrose, 10.00
Procedure is pretty straight forward. My container holds 500 mL so I weigh out
10.5 g YMDP powder, 10 g Agarose, then pour in 500 mL water.
The YMDP powder will dissolve in the water, the agarose won't.
Not a problem, the agarose will dissolve during sterilization.
For small batches I use this nifty bottle made by MacBick.
When it starts to cool the lid sucks down onto a gasket and seals.
It holds a vacuum so you don't have to worry about dirty air getting in.
 
Sterilize @ 15 PSI for 30 minutes.
I like to fill the pressure cooker with 3-4" of water.
The large thermal mass cools down slowly and keeps the broth from boiling over.
Once the pressure cooker returns to normal atmospheric pressure I move it from the kitchen to the laminar flow hood.
I wipe the outside of the pressure cooker with alcohol. I then remove the bottle and let it sit until cool enough to handle.
From 500 mL I can pour 20-25 plates, I went with 20 this time.
Stack the plates and work from the bottom to top by lifting the lid of the plate you pouring into along with the rest of the stack.
Cover the stacks with plastic bags and allow to cool to room temperature and solidify.
Store the plates you don't use right away in individual ziplock bags.
Edited by GroboClone (03/29/12 04:37 PM)
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 DrCrumbs
Alpha Blue
Registered: 10/25/11
Posts: 1,169
Loc: Gili Trawangan
Last seen: 16 hours, 51 minutes
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Re: New Year, New Projects - Making Some Plates [Re: GroboClone]
#15698867 - 01/21/12 05:56 PM (4 months, 5 days ago) |
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Cool, thanks for the tek!
I like your gifs!
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total
Post Office Tyvek Advocator
Registered: 10/03/04
Posts: 8,353
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Re: New Year, New Projects - Making Some Plates [Re: DrCrumbs]
#15699831 - 01/21/12 09:38 PM (4 months, 5 days ago) |
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Could i get some more info on your agar flask Grobo?
And i really dig the way you made this...
Animated gifs and all! 
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GroboClone
Anthropomorphic Elephant
Registered: 01/07/11
Posts: 332
Loc: Canada
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Re: New Year, New Projects - Making Some Plates [Re: total]
#15699887 - 01/21/12 09:50 PM (4 months, 5 days ago) |
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Sure, it's a vintage bottle specifically designed to autoclave liquids.
It's made by MacBick in the USA and made from borosilicate glass.
I've seen them in 500 mL and 1 L sizes, they come up on Ebay every so often.
They are often listed as apothecary bottles or scientific beakers.
Very nice piece of kit, even has a specially designed lip
so the agar doesn't drip down the side after you pour.
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GroboClone
Anthropomorphic Elephant
Registered: 01/07/11
Posts: 332
Loc: Canada
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Re: New Year, New Projects - Making Some Plates [Re: GroboClone]
#15699902 - 01/21/12 09:52 PM (4 months, 5 days ago) |
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The plates have cooled so now it's time to get the cultures from the fridge.
All my cultures are stored in agar master slants along with a thin piece of wood.
I use a small bar fridge controlled by a digital thermostat set to 35°F.
The tubes are stored in Styrofoam boxes to keep the temperature stable.
Once I start working in front of the hood I try to keep a good work flow.
I'm being very conscious of sterile procedure and my orientation relative to the air flow.
I make sure to never get between the airflow and sterile material or directly above it.
I like to use a small wire rack that gives me two levels to work on
along with raising the work surface up into the laminar flow.
What I'm working with:
Two King Oyster strains, Lion's Mane, Reishi, and Shiitake 75
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NeuroFunk
Stranger
Registered: 02/07/11
Posts: 81
Loc: EU
Last seen: 11 days, 15 hours
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Re: New Year, New Projects - Making Some Plates [Re: GroboClone]
#15701059 - 01/22/12 08:20 AM (4 months, 5 days ago) |
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I always read your posts with great interest, GroboClone 
--------------------
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GroboClone
Anthropomorphic Elephant
Registered: 01/07/11
Posts: 332
Loc: Canada
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Re: New Year, New Projects - Expanding To Grain [Re: NeuroFunk]
#15707533 - 01/23/12 02:39 PM (4 months, 3 days ago) |
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The mycelium from the test tubes has been on the plates for around 40 hours now.
The plates are all showing good signs of growth at 24°C.
Reishi is the fastest having already grown over 5mm.
The next step in the expansion of mycelium is to transfer agar wedges to grain.
While waiting for the plates to colonize I'll prepare and sterilize some grain.
I like to use 1L jars for my first transfers from agar.
Subsequent transfers are made to 4L spawn bags.
I have four "All American" pressure cooker/canners for sterilizing grain and substrate.
This gives me a capacity of 40 jars or 16 bags per cycle, working out to 24/48 KG of spawn.
Pictured are three AA921's and a AA925
To start off I'll make 20 jars for grain masters.
This requires around 7 L or 5 KG of dry grain.
Rye has been my grain of choice. I buy directly from a local producer.
I picked up 750 lbs. to get through the winter, it cost me $50, that's 6.6¢/lb.
Rye is very cheap around here, I've seen prices from $80-240 / ton.
I'll soak the rye overnight and cook tomorrow morning.
Edited by GroboClone (01/23/12 02:42 PM)
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john-hai
oyster lover
Registered: 05/11/11
Posts: 168
Loc: san jose ca
Last seen: 22 minutes, 18 seconds
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Re: New Year, New Projects - Making Some Plates [Re: GroboClone]
#15707546 - 01/23/12 02:42 PM (4 months, 3 days ago) |
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that is a good price for rye.
john
--------------------
3 rules i use to grow mushroom
#1 local material
#2 recycle when possible
#3 make it simple
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GroboClone
Anthropomorphic Elephant
Registered: 01/07/11
Posts: 332
Loc: Canada
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Re: New Year, New Projects - Making Some Plates [Re: john-hai]
#15712270 - 01/24/12 02:56 PM (4 months, 2 days ago) |
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Last night I cleaned the rye and got it soaking.
I do this part in the bathtub since it can get kind of messy.
Here are the steps:
Add warm water to cover the rye.
Stir, a bunch of crap will float to the top. Pour off the floaters.
Use a canning rack to hold back the rye and drain completely.
Fill again with warm water, if you want pour off floaters again.
Add 1/4 cup gypsum, stir and let sit for ~12 hours.
Make sure to use warm water and not scalding hot water.
I've used hot water before and it hydrated the rye to the point of bursting.
 
This morning I split the rye into two stock pots, added hot water and set on high heat.
I cook the rye for about 10 minutes after it starts boiling, I don't go by time though.
I taste test the rye periodically until cooked "al dente".
Once cooked the rye gets dumped into colanders to drain, each pot fills two.
After they drain I dump the colanders into tubs to steam dry.
When the rye has cooled down to room temperature I load it into jars.
Using a funnel and 1L container I can fill 20 jars in under 5 minutes.
Cap the jars and cover lids with aluminum foil.
The jars are loaded into pressure canners and sterilized for 70 minutes @ 20 PSI.
Once the PC's depressurize I move them to the clean room and let cool to room temperature.
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GroboClone
Anthropomorphic Elephant
Registered: 01/07/11
Posts: 332
Loc: Canada
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Re: New Year, New Projects - Reishi [Re: GroboClone]
#15715607 - 01/25/12 09:37 AM (4 months, 2 days ago) |
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Last night the jars were taken out of the pressure canners and wiped down with alcohol.
Since I'm only making 4 jars this Reishi plate is ready to go.
Under 4 days from cold storage to grain, pretty damn quick.
Here's how I setup the bench when transferring agar to grain.
Following mycoelf's advice I've added a layer of Parafilm over the jar lids to limit gas exchange
and hopefully prevent the Reishi from growing so thick as to be unusable.
Here you can see the agar wedges sitting on top of the grain.
I shake the jars before I make the transfers then place the wedges mycelium side down onto the grain.
I let the mycelium establish itself in the grain before I shake again.
Quote:
mycoelf said:
Quote:
GroboClone said:
Does anyone have any tricks to manage Reishi spawn? It grows so thick I can barely tear it apart. It also turns the rye to mush.
When you do your G1s do not allow FAE. I use a regular canning lid flipped upside down so fae is minimal, in-oxic conditions causes Reishi to run, yet not form the dense rubber growth that makes knocking the grain apart impossible before transfer. Reishi done in this manner is easy to shake apart and the colonization will be fast and thorough yet not thick. Using this technique my G1 colonize in 4 days, and can sit on the shelf for weeks in this condition. If you open the jar and then put the lid back on, then watch out, cause you will cause enough FAE for the condition that you despise to occur.
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HorizonSpawn
Gettin' my grow on :)
Registered: 05/14/11
Posts: 1,738
Loc: Myco-Tek.org 
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Re: New Year, New Projects - Reishi [Re: GroboClone]
#15722942 - 01/26/12 09:06 PM (4 months, 16 hours ago) |
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Lovin' this thread!
--------------------
Please assume any and all prints exchanged are "WILD" in nature; and thus, should NOT be considered ASEPTIC...
NOTE: Please excuse my brevity, as it is a bitch 'n' a half and slow as hell to type on this here phone 
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Cid2112
Newb
Registered: 09/24/11
Posts: 56
Loc: Ohio
Last seen: 2 months, 6 days
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Re: New Year, New Projects - Reishi [Re: HorizonSpawn]
#15723862 - 01/27/12 04:33 AM (4 months, 9 hours ago) |
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Great post as always, GroboClone, thanks for sharing this with us.
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GroboClone
Anthropomorphic Elephant
Registered: 01/07/11
Posts: 332
Loc: Canada
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Re: New Year, New Projects - Reishi [Re: Cid2112]
#15725695 - 01/27/12 03:04 PM (3 months, 30 days ago) |
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While I will be growing King Oysters for sale at local restaurants and markets
the Lion's Mane, Reishi, and Shiitake will only be grown invitro using "solid-state fermentation".
Solid-state fermentation (SSF) means growing the mycelium directly on grain
allowing as much of the grain as possible to be converted to mycelium.
The mycelium is then ground, dried, powdered and used medicinally.
Although rye is an excellent grain for mushroom spawn it isn't suitable for SSF.
Rye is susceptible to ergot, a fungi that produces poisonous alkaloids.
When growing mushrooms conventionally the substrate is never eaten
so the small amount of ergot found in rye isn't a problem.
With SSF the substrate is ground and consumed directly.
Instead of rye I will be trying out two types of sorghum (milo) , red and white.
Sorghum has some advantages over rye. Up to 98% conversion of grain to mycelium,
neutral scent and flavour, and less chance of ergot.
 
The sorghum was prepared using the same technique as for the rye.
Times have been shortened to adjust for the smaller kernel size.
I soaked for 8 hours, then boiled for 5 minutes.
For this first run I sterilized for 90 minutes @ 15 PSI.
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Javadog
Continuing along
Registered: 05/03/10
Posts: 6,055
Loc: USA
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Re: New Year, New Projects - Reishi [Re: GroboClone]
#15725802 - 01/27/12 03:32 PM (3 months, 30 days ago) |
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Great stuff Grobo.
I wanted to try milo alone as well, and bought some from Azure Standard.
I look forward to how this evolves.
JD
--------------------
Boyd Rice told my brother that life is a corny pack of freesakes
Myco-tek.org
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GroboClone
Anthropomorphic Elephant
Registered: 01/07/11
Posts: 332
Loc: Canada
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Re: New Year, New Projects - Reishi [Re: Javadog]
#15729160 - 01/28/12 11:28 AM (3 months, 30 days ago) |
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The reishi finished colonizing the plate so I transferred the rest to some sorghum jars.
The other cultures are slower but still growing nicely.
Lion's mane is the slowest, it has also been in storage the longest, around 1 year.
Transferred the reishi to 2 red and 2 white sorghum jars.
I'm trying plastic bags this time to limit the gas exchange.
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total
Post Office Tyvek Advocator
Registered: 10/03/04
Posts: 8,353
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Re: New Year, New Projects - Reishi [Re: GroboClone]
#15729245 - 01/28/12 11:55 AM (3 months, 30 days ago) |
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Love the updates Grobo!
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TripleDot
Psychedelic Artist
Registered: 07/27/10
Posts: 20
Last seen: 1 month, 18 hours
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Re: New Year, New Projects - Reishi [Re: total]
#15735701 - 01/29/12 08:05 PM (3 months, 28 days ago) |
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I really love the info/tech's in this thread. 
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GroboClone
Anthropomorphic Elephant
Registered: 01/07/11
Posts: 332
Loc: Canada
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Re: New Year, New Projects - Reishi [Re: TripleDot]
#15738165 - 01/30/12 12:38 PM (3 months, 28 days ago) |
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All except the Lion's Mane were transfered to jars today.
Here are the plates before the transfers:
King Oyster I, King Oyster II
 
Lion's Mane, Shiitake 75
 
Each plate was transfered to 8 jars: 4 rye, 2 white and 2 red sorghum.
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GroboClone
Anthropomorphic Elephant
Registered: 01/07/11
Posts: 332
Loc: Canada
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Re: New Year, New Projects - Reishi [Re: GroboClone]
#15786211 - 02/09/12 09:53 PM (3 months, 17 days ago) |
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Feb. 2
So the problem with the Lion's Mane is it decided to stop expanding and start fruiting.
Transferred what I could to some jars, hopefully they will recover.
Reishi on White Sorghum, 5 days growth.
Feb. 9
Reishi has colonized the sorghum jars in 12 days, the rye jars aren't far behind.
All the cultures are still a little slow, once they go through a couple g2g transfers
and really wake up a jar should colonize in under a week.
I'm going to start sterilizing bags tomorrow and really get this show started.
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Javadog
Continuing along
Registered: 05/03/10
Posts: 6,055
Loc: USA
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Re: New Year, New Projects - Reishi [Re: GroboClone]
#15786228 - 02/09/12 09:58 PM (3 months, 17 days ago) |
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Yes, I have seen Lion's Mane do this too.
I learned a lot about when to fruit it the first time.
I was expecting it to look way more colonized before I put it into
fruiting conditions, but it started to fruit and I had to act.
I think that this might be more of a problem if the block was more
supplemented. I used plain sawdust and did not get contams.
Good luck,
JD
--------------------
Boyd Rice told my brother that life is a corny pack of freesakes
Myco-tek.org
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