  
   
I like using liquid innoculant. I'm not completely convinced that it's the best way but i have my suspicions. I've been through all of the ups and downs of using LC. In a nutshell, LC is dangerous if you are slacking in any aspect of your sterile tek. Done properly though, it can be quite easy and amazing.
I've been meaning to do this for Quite some time. Thanks for the inspiration Mushboy. Here i plan on documenting various ways of doing LC. We'll see what it turns into but the only way to get something done is to dive in and get started.
To start we will be watching 2% malt vs 0.4% malt. Nocced up with the same genetics from the same TOC clone. From the same plate in the same session with nearly the same size agar transfer. The agar pieces inserted where about the size of a hit of blotter.
These have no Marble/ broken glass/ stir bar/.... NADA. I vigorously swirl them up once a day/ twice a day/ I'm out of town for a day or two.... I just swirl them whenever i think about it and am around to do it.
P.S. I started documenting these because Mushboy told me that i shouldn't offer advise when i don't know what the fuck i'm talking about (tongue in cheek. No harm no foul.) I was arguing that, to achieve a nice clear LC, one should dissolve the malt into the water by heating and stirring. He disagreed. So i decided to check myself. I didn't realize that he was only suggesting .4% malt. So i made up a few my way and a few his. I did these exactly as he recommends. I just measured the malt out, put it in the jar, added the appropriate amount of cold water (tap), put a unmodded two piece lid on and shook the hell out of them. Then i switched to plastic lids with an little SFD and PCed them (30 minutes) and nocced them up.
I made up a 1000 cc and a 500 cc of each nutrient ratio. Here They are right after getting impregnated. You can see the agar wedge in the 0.4% pic.
 
 
Turns out that i use a lot more malt and without heating and stirring prior to PCing the 2% ended up with a shitload of sediment. This is not a problem, it just isn't as pretty.
So, that turned out pretty much how i thought it would. But now, I wonder, which will grow faster and end in a thicker/ snottier LC? I like super thick LC. Maybe they won't differ much at all. If this be the case, I've been wasting malt.
Here they are 3 days in. Looks like the 2% is a little ahead on growth but i'm not convinced. We'll see though.
 
  
Here they are on the 24th. It's still looking like the 2% are in the lead.
 
 
They are looking good to me. I always test LC on agar before noccing up a bunch of bags. It's not entirely necessary but i highly advise it to anyone learning LC and sterile practices.
Edit 8/26/19
Here they are today. I took a picture of each before swirling and after swirling. Every single one went from sitting on the bottom to floating. That was a couple hours ago and they are all sitting on the bottom again. I've heard people say all kinds of things about floating and sinking LC. My own experience suggests that it is simply a matter of how much oxygen is in the myc. I really don't know but that's what makes sense to me.
 
 
 
 
edit 8/29/19
Aloha Nui
0.4% pre swirl
Post swirl
2% pre swirl
Post swirl
Edit 8/31/19
The time has come to test these suckers on agar.
Typically i just flame sterilize a loop, dip it in the LC and streak a plate. But I've decided to do things as tippy top as possible for this write up for the sake of SCIENCE. I'll also be going rather overboard on the wrapping and labeling. I don't usually do things this detailed and carefully but i want to set a good example for people just learning the ropes and show the safest and "best" way of doing certain things. Like individually wrapping all the needles in foil and what not.
Lately I've been free pouring the LC into bags but here i will fill 60cc syringes for the sake of accurately dosing the bags. I'll take a drip from the syringe, put it on the sterile loop and then streak the plate.
So, now to sterilize everything necessary.
4 60cc syringes
4 16 G. needles
One long ass aspiration needle
Loop
Scalpel (not needed for this but I'll be using it for other things in the session.)
It's good to individually wrap everything you'll need so that you can leave things safely wrapped in foil until you need them.
When wrapping i like to be careful to wrap in such a way that they are protected but will also be easy to unwrap without contaminating. It can also be helpful to label things with a sharpie. You can be as detailed as you want but i just like to know what is what and what end of that thing matters most. This can be done by drawing and arrow, a star, a drawing of the item... Bottom line is your gloves are not sterile. They are only pretty clean. Some items don't matter as much as others. Like the scalpel and loop. They can be taken out all willy nilly and then flame sterilized later. The long ass needle and syringes are a different story though. Those are labeled in such a way to show me what end should be opened first and held with my hand while the other end is carefully and cleanly removed from the foil and dealt with appropriately. Point in case, the syringes show me what end the needle goes in. This is the important end. If you pull the sterile syringe out of the foil and then allow some bacteria or nasty ass spores to make there way in to the end.... You are Fucked.
It's also worth noting that I'll be drawing four different LCs into four different syringes using the same aspiration needle. I'm only doing this because i'm choosing to be all detailed and use syringes. I don't want to aspirate with the 16 G needles because that puts my hand closer to the LC. With the long needle I can aspirate without having to have my hand over the LC. Two of these are in half gallon masons and pretty deep. It's easy enough to safely switch out the needle and move on. But I'd highly recommend just testing with a loop and then free pouring.
Then it's good to wrap all your random shite in foil for a few reasons. This pouch holds everything together in the cooker and allows an extra layer of protection while moving the shite out of the cooker and into the flow or SAB. The pouch can be opened and the items removed safely once everything is ready.
Then we cook. This cooker also has four new LCs in it. I also have two jars of nothing with lids on them that i use to safely hold delicate things (like the lid of the LC i'm testing) up in the flow and away from the nasty table. Additionally there are a couple jars in there that are simply there to keep my foil pouches from falling in the water.
Edit 9/1/19
Last night i filled the syringes and tested these LCs on agar. I tried my best to tape my phone to the ceiling and leave it dangling at just the right angle to get some pics. I filmed the whole session and then went back and took screen shots of some pertinent moments. I tried to crop in and get good examples. You'll probably need to expand the photos and look closely if you want to understand what's going on. Hopefully this is helpful to some people.
I like to move everything to one side of the flow, iso my hands and arms, put on gloves and iso them, get a new paper towel soaking wet and use it to clean off the empty side of the flow table. I'll then clean everything one by one and move it to the clean side and iso the now empty dirty side. I'll then spread everything out and do a mental run through of what I'm about to do. In this picture everything is cleaned with iso and I'm getting my game plan straight.
I can't stress enough, to those of you who are new to sterile sessions, it is very important to know what you want to do and how you want to do it. I've done thousands of sterile sessions and i still run through my procedure in my mind before i begin. I then organize all items in the best way I can think of to facilitate smooth maneuvering throughout the session. This pic is post intentional and strategic organization of the items.
Now I'll start taking my shizzle out of the foil pouches. Always being mindful of what is most important, what is least important and what doesn't fucking matter at all.
Syringes
Scalpel needles and loop
All individually wrapped as to remain nice and sterile until it is their time
Here's where the rubber meets the road. Unwrapping things that must remain sterile until done, one must be mindful of every move.
The stainless aspiration needle will be removed in such a way that the luer lock end remains in the safest position possible. It should always remain in "first air" (i believe is the term). This means nothing should ever touch it or be between it and the filter. This is one reason labeling your foil can help. I know which end is which without having to fuck around.
  
Then i go about unwrapping a syringe. Keeping in mind that the luer lock end of the syringe is of upmost importance and must remain in "first air". All the while keeping the luer end of the needle in a safe position.
 
Now, as i connect the needle to the syringe i keep both luer ends in first air.
Flame the needle. I was careful not to touch the needle too close to the luer end because i didn't want to have to flame it all the way down. Doing this can melt the end of the syringe and cause all manner of problems.
Then i swirl the LC, remove the lid and set it upside down on the lid of one of the sterile jars that just came out of the cooker. Then tilt the LC and aspirate.
Then i set the syringe on the rack in such a way that every part that matters is floating in first air. The open LC is also very vulnerable and must be respected. Now we retrieve the lid and carefully place it back on the LC.
 
Now we streak. It isn't entirely necessary to re flame the needle but is definitely a good idea. You'll have to flame both the needle and the loop and then allow both to cool without compromising either. It's a little odd, learning how to hold things at first but gets easier with a little practice.
Once cooled, place a single drip on the loop. If you hear any sizzling, it wasn't cool enough. But it will be after the sizzle. Just put another drop on and keep rolling. You'll learn how long it takes to cool. You can also cool it in a sterile agar plate but I don't like to.
Safely set your syringe down without compromising your precious innoculant that is suspended in the loop.
Remove the lid of the petri without contaminating it. Finger placement is important. Then streak the LC across the plate. I just zig and zag from top to bottom. Probably 2 zigs and 3 zags.
 
Then i wrap the petri and LC lid with glad cling wrap. Some use parafilm but i like glad.
 
Now I remove the stainless needle and replace with sterile 16 gauge needle. Always remember what matters and what doesn't. Right now the business end of the needle and syringe are of utmost importance.
 
Always label as you go or you'll get corn fused later on.... Fo Sho
 
I like to put syringes into brand new ziplock bags. Just to be safe and sound until ready to use.
Generally, within two days i can tell if the plate is clean but the longer i wait the more obvious it is.
Edit 9/3/19
It's been about two days (i think) and i feel comfortable declaring these plates, and subsequently these LCs, clean.
Here are the LCs pre swirl.
At this point they are just over 2 weeks old.
Post swirl
(shown in same order as above)
1000cc 0.4%, 500cc 0.4%, 500cc 2%, 1000cc 2%
   
The plates, two pics of each, in the same order.
1000cc 0.4%
 
500cc 0.4%
 
500cc 2%
 
1000cc 2%
 
The pics must be expanded and studied closely to see whats going on. Even in person i have to look closely to be sure of what i'm seeing. The two things i like to really look at are the obvious growth spots and the streak pattern. Any time you can see good portions of the streak not showing any growth, it's a good thing. This because the myc doesn't spread throughout the entirety of the liquid the way that bacteria or yeast does. If these LCs had bacterial infections you'd be able to see the slimy snotty bacterial growth all along the streak pattern.
You'll notice heavily concentrated growth, that doesn't really stick with the streak pattern, at the beginning of each streak. This is because the drip is pulled off the loop and forms a small puddle when the loop hits the agar. This distributes tiny myc nuggets all over the area. You then pull the loop through it and grab whatever you grab. Then, as you streak the plate, little nuggets fall off here and there becoming less and less concentrated as you streak.
Another thing to notice, on the "2A" plate, there are two noticeably bigger growth spots. These might be confused as being a problem by the untrained eye. The fact that they are so fast (compared to areas around them) and that they seem puffy and disorganized might cause some concern. But not I. It is my opinion that these are the result of slightly larger mycelial nuggets that more or less land in a pile... like a spaghetti noodle that's been folded over and over on itself. This growth on top of growth on top of growth can cause some abnormal appearances but will clear itself up in a few days.
It's appropriate to note that this is precisely why I prefer to leave my LCs on a stir plate. Not only does it exponentially accelerate growth, it also keeps larger myc nuggets from forming and that's how i like my LCs simply packed full of tiny tiny tiny magical mycelial fragments. It's like G2Ging with grass seed or wbs compared to oats. Simply more inoculation points per cc.
I'm going to go make up some WBS. I'll upload a few more pics in a day or two to show the growth on the plates.
edit 9\7\19
Almighty mush cult fam, the show must go on! I was busy for a couple days but got around to noccing up a bag or two with these here liquids. I'm a little bummed though because I took the time to tape my camera to the ceiling and get it dangling at just the right angle to give ya'll a good view. Set the fucker to video the session and then got to work, being sure to give ya'll a nice good look at each syringe and how i position the bags and..... all that jazz. The video didn't make it beyond me getting set up to roll. The low battery warning came on and apparently ends the video when it does. What a stupid design.
Anywho. I did a 10cc, a 20cc. and a 35cc bag with the 0.4% and the same with the 2%. I figure that'll be a fun show. Then, mid session, I decided to free pour a bag or two to get a better understanding of free pouring vs shooting from a syringe. It's definitely nice to be able to spray it all over with a syringe. The free pour doesn't get spread around as nicely. I'll update when some growth becomes obvious.
Edit 9\15\09
I let these sit a bit longer than needed before shaking just to give a little better understanding of where the myc took off on each bag. Here they are today. Every side of every bag. Except the 10cc from the 2%. I accidentally forgot to photograph it before mixing.
0.4% 35cc
   
0.4% 20cc
   
0.4% 10cc
   
2% 35cc
  
2% 20cc
   
2% free pour 20ish cc
On this one i tried to make a nice streak across the middle of the bag but it's a little hard to aim. I only took pics of the sides showing growth on these guys.
  
This one got two streaks 30 - 40ish cc
  
I often hear people asking if it's ok to leave standing LC at the bottom of jars or bags. You'll notice that most of these (specially the free pour and 35cc bags) have a ton of colonized grain on the bottom. This was standing/ pooled LC. I like to prepare my grain dry enough to handle lots of LC. I've run into no problems with standing LC as long as the grain isn't too wet.
All bags were shaken well and placed back on the shelf.
Edit 9/21/19
Here they are folks
0.4% left to right; 35cc, 20cc, 10cc
2% in the same order
Free pour
The left got one quick streak and the right got two
It's looking like the growth of the 0.4% and 2% are pretty much the same. And, as one would assume, the bags that got more are farther along. I don't think it'll make a huge difference in the end though, maybe a couple days. I haven't decided whether or not to shake them again. The ones that got less would definitely benefit from a shake but that'd kind of throw off the experiment so.... Thoughts? I'll leave it up to you.
Welcome aboard.
Buckle up and enjoy the show
Edited by verum subsequentis (09/21/19 08:06 PM)
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