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Pastywhyte
Say hello to my little friend



Registered: 09/15/12
Posts: 37,808
Loc: Canada
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Pastywhyte's EzLC tek. 40
#23740857 - 10/15/16 09:09 PM (7 years, 3 months ago) |
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Pastywhyte's EzLC
Like many people I am a closet LC enthusiast. I find it great to inoculate bags and the speed is nice as well. But of course LC is a method that carries high contam vectors and needs a little extra care. With my EzLC we can visually inspect the culture before we introduce the liquid meaning as long as your confident in the liquid phase you can be sure of a clean LC (test it at first to be sure you have your movements etc down). We also are able to make LC as easy as transferring agar wedges, with the same action as blenderless, but an aggressive colonizing culture with plenty of stored energy to recover. I find that with this LC I often see recovery in 48 hours with fast aggressive growth. This EzLC is as simple as making no pour agar and requires little in the way of special equipment. Super EzLC doesn't even require the use of Whatman filters or SHIP's. This is to LC what blenderless LI is to LI.
The best part of the method is the elimination of sediment. You can make brf LC. Or potato flakes. All the sediment is trapped in the agar puck. These more complex media's result in quicker recovery after inoculation. I always found karo or SITR to make for sluggish recovery. I have seen 3 day recovery and explosive growth consistently with some of these recipes you normally would avoid for LC.
Materials needed:
2 half pint or pint jars. Fancy Jelly Jars also work well. 2 LC lid with whatman and SHIP 1 60cc Syringe. Smaller will work but more will be needed. 1 PC
You will also need agar supplies. I like powdered food grade agar and really any nutes that you like works for this. PDA, MEA, GW, DFA are all good and kick ass for this.
So the first thing you need to do is mix up some agar. First you need to decide how much LC you plan to make. 120 ml is a nice amount, more than enough to knock up a pile of grains or bags, and if you do this in widemouth half pints, 120 cc fills it pretty good.
So for 120 ml final LC I want to be using about 2-4 grams worth of nutes, be it potato or malt etc. I then make up about 50 ml of water with 2-4 grams nutrition and 1 gram agar. This puts the agar at 2% and nutrient rich.
I have tailored the above recipe for use of one LC. You will probably want more than that, especially if you plan to use this to test cultures or get bored of growing the same thing all the time.
At the end of the day I wouldn't stress to much over ratios etc. Make up some agar, use lots of nutes, make up the jar.
Quick example:

Bam! Its that damn easy. So now ya pressure cook it up. Once PCd and cool we can now knock these up with a clean culture. Take a clean wedge and put it too the plate same as you would make transfers. Let it grow out to the size of a quarter or so. Do to the high nutes it may grow a touch slower but that's okay cause we don't need it to grow out much.

Once I have a clean culture I can visually inspect it for mold or bacteria or other contams. Once I am happy that its clean I then sterilize a jar of water with a lid outfitted with a SHIP. Have a sterilized syringe ready and then using proper sterile procedure aspirate the water and inject into the culture jar.
Sterile water and syringe:

You will soon notice that the nutes from the agar puck leach into the water (as will the food coloring if you use it) but any sediment or solid matter remain trapped in the agar puck. Give it some time to colonize, usually will take a week to 10 days. Swirling it aggressively will help expand the colony faster. You might dislodge the agar but that is not an issue.
Pic of colonized EzLC

Once colonized you are free to aspirate and use. It makes for a pretty aggressive and fast recovering LC compared to the simple sugar recipes many people make. BRF LC is damn fast to recover and dog food agar usually provides explosive growth. Grain water, potato and of course malt extract all kick ass. It can get very thick if you let it go to long so be careful, I have had it get pretty solid. Makes it hard to aspirate. You can use any size jar for this but I find a quart to end up with more LC than I. Half pints and 120 ml LC is enough to do 60 quarts easy. More than enough. I also like the fancy Jelly jars. They are so low that often you can aspirate the liquid without tilting the jar. Makes things much easier.
Grain water EzLC. Dark but clear.

I may test this one because IME media like grain water or dog food can become susceptible to colonies blooming over time. I would never use grain water or dog food recipes for long term use due to high populations inherent to that media. But it looks very good at first glance.
Super Ez LC
Supposing you want to try this but don't have any LC lids. With taller jars its perfectly safe to use a grain jar lid with SFD and just pour the water and then the LC. You just need to be careful when swirling that you don't get the liquid up to the filter. Taller jar helps with this.
Materials needed
2 Grain jar lids with SFD 2 pint jars (I prefer reg mouth but widemouth can work too) Agar supplies.
Make your no pour agar the same as with the EzLC. Pour in pint jars and PC. Transfer the wedge as normal but the drop might cause it to bounce off to the side. It doesn't matter if its not dead center, just leave it. When sufficiently colonized sterilize a jar of water and in a SAB or in front of flow hood pour the sterilized water into the colonized no pour jar. As long as the level is kept low (less than 250 ml) its easy to swirl and keep the liquid from hitting the filter.
Potato flakes and karo with simple grain jar lids. Note how thick and healthy the colony looks. These were also mostly ignored, very little agitation was done.

The jars above are crystal clear and look strong. They could be tested if you had any concerns and I recommend testing or simply tossing anything that looks off but, these I personally am confident would be fine.
Once colonized you can simply pour it into your grain jars same as G2G. The colony with this method recovers really fast. Buy a dozen reg mouth pints and keep them rolling real easy. Fast easy spawn.
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nobody83
stranger danger


Registered: 03/15/14
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Loc: around town
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Re: Pastywhyte's EzLC tek. [Re: Pastywhyte]
#23742488 - 10/16/16 01:02 PM (7 years, 3 months ago) |
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This method would probably work very well with pint pp5s as well if you have them at your disposal vs. The jelly jars etc. (Threw all mine away. I'll have to give it a try and see the results
Edit:
 Looks like i can put these old whatmans to work...never would have thought
Edited by nobody83 (10/16/16 02:33 PM)
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Edmunter
Mr



Registered: 05/01/13
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Re: Pastywhyte's EzLC tek. [Re: Edmunter]
#23827958 - 11/13/16 05:16 AM (7 years, 2 months ago) |
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This AA+ ms is looking nice and clean. Nearly time to add water but thought I might let it grow to the edges just in case.
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Edmunter
Mr



Registered: 05/01/13
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Last seen: 17 days, 13 hours
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Edmunter
Mr



Registered: 05/01/13
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Last seen: 17 days, 13 hours
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Re: Pastywhyte's EzLC tek. [Re: Edmunter]
#23848980 - 11/19/16 03:25 PM (7 years, 2 months ago) |
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A nice looking sector from my 2nd PE transfer

Using a 10:10 ratio DF to Agar to 500ml water to see how lower nutes responds
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Edmunter
Mr



Registered: 05/01/13
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Re: Pastywhyte's EzLC tek. [Re: Edmunter]
#23850911 - 11/20/16 09:36 AM (7 years, 2 months ago) |
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My LC is a bit cloudy today. The jar and lid im using are not a great seal and when I swished it some liquid came out. So im not sure if it is the mycellium and agar leaching into the mix or bacteria.
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Edmunter
Mr



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Re: Pastywhyte's EzLC tek. [Re: blackout]
#23853617 - 11/21/16 09:02 AM (7 years, 2 months ago) |
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Quote:
blackout said:
Quote:
Edmunter said: My LC is a bit cloudy today. The jar and lid im using are not a great seal and when I swished it some liquid came out.
Those tapered jam jars have very large lids. You had other jars, maybe dolmio or uncle bens, some of those have quite narrow lids which would be better for LC.
These jars are designed for single use only, well to last the use of the contents in the jar, not like canning jars some use. If you tighten them too much the lid will deform where it locks onto the glass thread and then it comes off. If this happens once it is hard to repair it, and the lid will always have to be only semi tight, and certainly not for use with LCs. If may not be obvious if it is damaged as it may not totally fail like that, it could have simply been overtightened too many times. I have several large jars and replace the lids from time to time with identical lids from smaller jars.
Some supermarket regular jars will have better lids than others, usually in fancier brands.
Most of these single use jars have similar threads and cap designs. Therefore if you tighten it to the same level, e.g. if it was attached to a drill and twisted on until the drill torqued out -then the smaller diameter lid would have a better seal, similar force acting over a smaller sealing area.
I had some nice big jars that I could not get replacement lids for, to repair them I put silicone in the lid.
Great point. Let it be stated that the jar you use is in the top 3 of important factors.
Clean culture Good sterile technique The correct jar for the job
Dont be lazy and get yourself a proper sealing fully closing jar and lid or this can happen
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Edmunter
Mr



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Re: Pastywhyte's EzLC tek. [Re: tombosley8]
#23877752 - 11/29/16 10:03 AM (7 years, 1 month ago) |
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So after my last attempt which finally turned into some ind of Welsh beer im back!!!!
This PE culture is looking good and a few more days should do it before adding water.
DFA ratio 10:10 pressure cooked for an hour this time. I should have made it less firm but forgot. Lets see what happens.
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Teemo 6T3
႟тнe мedιcιne мan ☼



Registered: 07/21/14
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Re: Pastywhyte's EzLC tek. [Re: Pastywhyte]
#23953828 - 12/24/16 10:11 AM (7 years, 1 month ago) |
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Can i use only this?
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ComebackKid
Multispore Enthusiast



Registered: 05/27/16
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Re: Pastywhyte's EzLC tek. [Re: Pastywhyte] 1
#24141421 - 03/06/17 02:26 PM (6 years, 10 months ago) |
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Super happy this is back!
First time I tried this I didn't have 60cc syringes so I poured water in like in the super easy method
 BRF EZLC

The growth was pretty thick and I had a little trouble with a 16g needle but it tested clean and Ive got almost full colonization in a week from 5cc It also got me thinking about unmodified lids. I have a couple going right now that are unmodified. Hoping that I can still get clean growth and be able to shake the shit out of them to help break up the culture before using my syringe.
Question: Was the grain water LC in your write up made by putting grain soak water on the agar? Or was it in the agar and stained the sterile water?
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Substrate surface conditions / Monotub prep and care
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Moabfighter
Tam Fighter


Registered: 12/13/15
Posts: 2,710
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Wouldn't have to modify anything I don't think.

I could make the LC recipe in unmodified blender bottle, cook, shake. Then in SAB swap the blender bottle lid for a filtered lid when I add my agar plate. Let it colonize. Swirl, not shake it. Sterilize the bottle lid in foil, then swap the lids again in SAB when time to pour.
Would that be correct, incorrect?
Thanks for any help. Hope to try this soon
-------------------- KSSS And PE WBS.
Edited by Moabfighter (04/06/17 12:42 PM)
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hamloaf
Loaf of Fam.

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Re: Pastywhyte's EzLC tek. [Re: Pastywhyte]
#24223256 - 04/06/17 04:04 PM (6 years, 9 months ago) |
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Yea, I do mine in front of a flow hood.
I don't do anything fancy either. I experimented with all that, and didn't care for it, so went back to what worked for me 100% of the time.
All I use is a pint wide mouth with a couple of pieces of broken glass inserted into the medium and a plastic lid with only a SFD.
I let the culture colonize the medium just before it begins the mat on the surface.

The jar is then shaken the crap out of directly into the sterile airflow of the hood, then place the jar in front of the hood, then suck the culture into syringes.

The filter gets wet, but isn't a vector unless you remove the wet filter from inside of the sterile flow of the hood.
Quote:
Pastywhyte said: Peace of mind is a good thing.
This is one of the top 2 reasons I switched from an SAB to a flow hood.
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Pastywhyte
Say hello to my little friend



Registered: 09/15/12
Posts: 37,808
Loc: Canada
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Quote:
Moabfighter said: Trying to wrap my head around the super easy LC.
I still need agar in my LC water? But not enough to make it hard, just to give it some thicker texture than just water?
Anyone got a simple potato flake/honey or Karo recipe?
You make agar and put it in a pint jar with a grain lid. Sterilize.

Then transfer a wedge. Do this in a SAB. Let it grow out a bit to be sure the colony is clean and healthy.

Then add sterile water. Let it colonize. When done pour the colonized water to inoculate.

The super easy part is it works without the need for a ship or whatman filters or syringes.
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mynakedrat
The phantom hourglass


Registered: 02/16/17
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Re: Pastywhyte's EzLC tek. [Re: Pastywhyte]
#24235209 - 04/11/17 03:42 PM (6 years, 9 months ago) |
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Quote:
Pastywhyte said:
Quote:
Moabfighter said: Trying to wrap my head around the super easy LC.
I still need agar in my LC water? But not enough to make it hard, just to give it some thicker texture than just water?
Anyone got a simple potato flake/honey or Karo recipe?
You make agar and put it in a pint jar with a grain lid. Sterilize.

Then transfer a wedge. Do this in a SAB. Let it grow out a bit to be sure the colony is clean and healthy.

Then add sterile water. Let it colonize. When done pour the colonized water to inoculate.

The super easy part is it works without the need for a ship or whatman filters or syringes.
Wow! I needed that pasty thanks.
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Moabfighter
Tam Fighter


Registered: 12/13/15
Posts: 2,710
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Thanks everyone for the help. Giving it a whirl.
Moab's pasty plate recipe LC
-------------------- KSSS And PE WBS.
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Moabfighter
Tam Fighter



Registered: 12/13/15
Posts: 2,710
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I think it's time to add the water.
-------------------- KSSS And PE WBS.
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Moabfighter
Tam Fighter



Registered: 12/13/15
Posts: 2,710
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Does this look correct?
Pastys super ezLC roughly 2 days after adding water. I've been rolling it twice a day.
-------------------- KSSS And PE WBS.
Edited by Moabfighter (05/05/17 10:01 AM)
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tombosley8
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Re: Pastywhyte's EzLC tek. [Re: Pastywhyte]
#24305412 - 05/08/17 06:21 PM (6 years, 8 months ago) |
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I did 30 g brf to 12.5 g agar to 625ml water for these 5
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Germs
Space Force


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Re: Pastywhyte's EzLC tek. [Re: mos619]
#24510329 - 07/26/17 07:46 PM (6 years, 6 months ago) |
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 This is the first stage before injecting water, I know the tek says quarter sized but I set these on a shelf and forgot about them, they're like two inches wide are they still usable? It's my last live culture of martinique so if I don't have to chunk em that'd be nice. not sure what the cons would be
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JHOVA
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Re: Pastywhyte's EzLC tek. [Re: Pastywhyte]
#24901927 - 01/09/18 09:22 PM (6 years, 19 days ago) |
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3 EZLC. 2 TYC agar wedges nocced up 1/2. 1 PE Biopsy nocced up 12/31. They are a little smaller than a quarter. Do you think I should wait or add sterilized water now?
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