The Gangsta way of doing LC
This is my post 3000!
It's been a while and to celebrate, I'd like to share with the community a little something that I confess I've been keeping to myself like a pig.
It's a different way of doing LC that can give your competitive ass quite the fucking edge.
A method that is simple, effective and especially good for people who like me only own a 'lowly' SAB, as it pretty much guarantees clean as fuck inoculants... every time.
But don't take my word for it, try it and see for yourself.
This is not so much a LC tek as it is an inoculation method. You're going to be inoculating your LC with a piece of tissue so ridiculously tiny that the only way to see it would be under a scope... how about that for removing vectors?
And as an extra safety bonus, the invisible
tissue sample is going to be protected inside a thin needle untill you squirt some water from the syringe into the LC broth.
If you are a MS enthusiast, you're going to narrow down the genetics considerably by just inoculating your LC with such a tiny sample, which in my experience almost always results in homogeneity and nice canopies. Not too shabby considering you can make quite a few tubs from one single 400ml LC.
This method has few vectors:
- The agar culture:
Some experience with agar is required in order to know when a culture is clean. I recommend using cultures that look organized and preferably round. So put in some work to get that nice looking culture!
- The cultivator:
This procedure is stupid simple and fool-proof and very little goes in the way of sterile technique to perform it successfully but... the possibility of screwing it all up is there, however slim.
This tek breaks a few well-established rules about LC's, and some may find it blasphemous.
With this method, some common golden rules such as 'always test your LC on agar first' become redundant, so long as you're sure you're working with a clean agar culture.
You're going to find more 'blasphemies' being perpetrated as you keep reading. Like pouring your LC into grains (although many people see this as good practice these days) and making a nasty, cloudy and unfiltered LC broth made with ground-up grains.
As a buddy of mine once said, 'Fuck golden rules! This shit is too gangsta for rules!
'.The ProcedureSETTING UP THE SAB
From left to right:
- The agar culture. As you can see, the plate is upside-down to facilitate the operation. I look through the front of my SAB, so for me it's much easier to do it this way. If you work with your head over the box, simply leave the plate upright and open the lid to take the tissue sample.
Find yourself a nice something to put the plate on, so the plate and the LC jar are more or less at the same level. This will make the operation smoother and minimize the movements you make. Pictured is a plastic bottle, again upside-down and without the lid, covered with a paper towel soaked in alcohol, which is not really needed but me likes it for stability.
- The LC jar. I use a regular mouth mason jar and a whole SFD covering the lid, in the spirit of keeping things simple stupid. Although you can use whatever as long as you know what you're doing. I just dislike the idea of having to silicone a filter to the lid.
The jar was pc'd placing the lid with the rubber side up in order to facilitate the operation. Leaving the rubber side down will create a nasty vacuum and the lid will be a bitch to open.
- A 10ml syringe with some sterile water. More details about this later.
All has been properly sanitized, the lid of the container has been 'pre-opened' and the metal ring of the LC jar has been removed very carefully. Remember, we sterilized the LC jar placing the lid with the rubber side up, so when you take off the ring do it slowly and carefully while holding down the SFD with one finger, otherwise the ring might catch the SFD. It goes without saying you want the lid to stay in place and without moving a bit while you take off the ring.THE 'POKE'
"Poke once, poke fast, poke smart..."
The poke is the crux of the whole procedure.
- In the SAB, take your freshly sterilized syringe and squirt some water to break the vacuum that the little of air in the syringe may have created during sterilization, so the plunger can be a little hard to push.
- Now, flame the needle red hot. If you're using a torch 2 seconds will suffice, and always be careful not to melt the plastic hub at the base of the needle.
- In the SAB, squirt a little of water from the syringe to cool the needle, just a quick squirt. If you squirt more than needed, water can start dripping from the needle like crazy.
- Quickly choose the spot of the culture that looks the prettiest and poke it with the needle until you touch the bottom of the plate with the tip. Done.
To inoculate with the biopsy you have to squirt some water from the syringe into the broth (see next step).
You can repeat this procedure to inoculate several LC's during the same session and using the same needle.
Now I'd like to talk a little bit about the 'poke'.
By poking I mean just that, poking a spot of the agar culture with the needle, fast and clean, no fucking around needed.
There's absolutely no need to poke twice, or to do it slowly or to scrape the surface of the culture with the needle and of course, no need to squirt water into the plate AT ALL
. This is not some shitty LI tek we're doing here.
Now I'm going to talk about your feelings.
After the poke, you may feel skeptical, as if you just did nothing, cuz no way in hell this is going to work...
You may feel an uncontrollable urge to stab the culture to death, cuz no way in hell this is going to work...
You may feel the impulse of scraping the culture after poking it, cuz no way in hell this is going to work...This is all a crock of horseshit
and the sooner you learn this the better.
Another interesting fact is that the biopsy you take with the poke is literally invisible to the naked eye, so don't bother looking for it, not even with the help of a magnifying glass. There's just a clean needle, no agar, no myc, just a clean pristine needle.
It is by no means an exaggeration to say that we're dealing here with microscopic pieces of tissue... bitches.
The first time you do it you'll have to take a leap of faith this is going to work, and it will, every single time.
You just have to trust the mushroom gods... INOCULATION
The picture says it all.
Crack the lid and squirt some water from the syringe into the broth.
A quick squirt will be more than enough to dislodge and expel the myc cells that got stuck inside the needle.
If you expand the pic you can see the jet of water hitting the broth.
This operation should be done in less than 2 seconds, the needle should not touch anything and the jet of water has to hit the liquid, never the glass.
You may want to practice this before doing the real thing first time around, just to gain some confidence. But it's really easy and even a crackhead going cold turkey could do it.
Screw the ring back on and call it done.
Now the waiting game. You should see the first signs of growth after 2 or 3 days, like a minute little dot of myc fuzzing up to your heart's content. After that all goes fast and the LC should be colonized in 2 weeks, 3 weeks tops, depending on factors such as the amount of water you used to make the LC and the vigor of the culture.
For explosive and fast growth I recommend using the easy grain recipe you will see later.ALTERNATIVE WAY OF INOCULATING
We're going to be inoculating through the hole in the lid by poking an sterile
makeshift inoculation port with the tip of the needle, so you can pc the LC jar placing the lid with the rubber side down no problem, since we're not going to open the lid.
Put the SFD on top of the lid when you're done and screw the ring back on.
This is another way of inoculating which is the one I used when I started doing this and still do sometimes, but to be honest, it's just easier to lift the lid to inoculate as mentioned above.
This way is especially good for people who feel a little paranoid about exposing the sterile broth to the air of the SAB, peace of mind is priceless...
I was loath to include it in the 'official' version of this tek because it's a little embarrassing to admit how much of a paranoid fuck one can be.
I also did not include it as the main option because for this to work you will need a whole SFD covering the lid.
I'm aware not everybody can find SFD's easily (people from the EU need to import them from the US), so the first option I presented is kind of the standard way of inoculating that can be done with any type of lid for your LC jar, so you don't necessarily have to use SFD's.
- Metal lid and ring for mason jar, preferably regular mouth.
- Whole synthetic filter disc.
- A piece of foil, scissors and micropore tape.
- A drill.
- A thin drill bit to make a pilot hole, a thicker one to make a bigger hole and a grinding stone bit to make the final hole (~2/5 inches in diameter or 1cm).
- A scrap piece of wood.
Here's what you need to do:
- Drill a big hole through the lid with the grinding stone bit (~1cm in diameter or 2/5 inches). It's better if you drill the hole next to the edge of the lid, unlike in the picture. This will facilitate the procedure later in the SAB.
We used the smaller drill bits to make a pilot hole for the grinding stone bit. In the 2nd pic you can see the remainings of MP tape from previous LC's, shit won't quit no matter what, never mind.
- Cut out a small square of foil but big enough to cover the hole.
- Place the piece of foil over the hole and cover the foil with one layer of MP tape (you can use 2 layers but it's not necessary).
- Mark the center of the hole with a sharpie. Trust me, it's going to be useful when it's time to inoculate.
Some may think that this way entails some risks, like for instance, mold spores or bacteria can stick to the needle during the procedure and by poking the foil we might introduce them in the broth... To be honest, that ain't gonna happen even if you do this a thousand times and opening the lid to inoculate is also a risk, everything is a risk in this hobby.
Drilling the hole next to the edge will make the procedure much easier for you because you would only need to barely lift the SFD at a sharp angle to inoculate through the hole.
A useful tip if you're going to be doing this:
Before pc'ing the LC jar, align the hole with any distinguishable feature in the jar, be it the front of the jar where is says "Ball" or whatever.
This will allow you to know where the hole is before you inoculate.
This is still a pour tek, as you will see later. Never, ever use this makeshift inoculation port to aspirate the LC.Prepping the Syringe
I use a 10ml plastic syringe and a sharp 21G needle. Needles deteriorate very quickly after flaming them, so I'd recommend always using a new one per session.
I was recently informed that blunt needles also work for this but the sample taken with it appears to be bigger, which in my opinion is a disadvantage. I like the sample to be as tiny as possible, in order to considerably narrow down the genetics while at the same time reducing the chances of contamination.
Sharp thin needle for the win!
For anything myco and in general, luer lock syringes are always superior than luer slip syringes (google it up).
The difference is that the luer lock syringe allows a needle to be twisted onto the tip and then locked in place. This provides a secure connection and prevents the accidental removal of the needle.
Truth is, I've only used luer slip syringes for this because I can't find luer lock syringes locally. They work just fine but I feel I need to tell you what is best.
Fill the syringe with distilled water and try to get as little air in as possible (for the pic I filled the syringe carelessly and you can see a huge air pocket).
Tap water works just fine but I just like to use distilled water for LC's and agar. Your call really.
With 10ml of water you can inoculate lots of LC's, so you don't really need to fill up the syringe till the 10ml mark unless you're planning on making gallons of LC.
Always use freshly sterilized syringes. Don't cut corners here and never store syringes for later use.
Cap the needle.
Now you can do 2 different things before pc'ing the syringes for 5 minutes at 15 psi
- Wrap the syringe in foil and put it in the PC always making sure the syringe is standing upright (needle up) or the water will boil out during the pc cycle. To achieve this, you can prop the syringe against two jars or anything that does the job.
- Find a bottle tall enough, filter it and put the syringes inside.
You can also sterilize the syringes along with your LC's, this is much more convenient. The syringes will always lose some water during the PC cycle and the longer the cycle the more water they'll lose. 30 minutes is fine, though. Even one hour is still fine.Prepping the Broth
I've tried the most common agar recipes for making LC broth, just for shit, giggles and some science. If it works for agar it works for LC.
In my opinion, out of all the recipes I tried the two that really kick ass are grain water (GW) LC and light malt extract (LME) LC. Nothing new here.
Both have their pros and cons and for me it's hard to decide which one I like best.
MAKING LME BROTH
- LME LC is a pleasure to work with just because it's sooo clear and overall awesome. Imposible not to love it. So I would recommend this if you like your LC nice and clear. It's also good for people who are just getting their feet wet in LC's and don't feel yet like being a nasty fuck (keep reading).
- LC's from GW colonize the broth more aggressively and show slighty faster recovery on grains, but it is a cloudier broth than the one made with LME. It also has sediment and lots of people don't like that. All I can say about sediment in LC's is that is pretty badass and quite useful actually (keep reading).
The standard ratio of LME to water is 1g LME for 500ml of water.
The broth will come out very clear using that ratio which is nice, but I personally prefer to pack in more nutrition when using LME, so I add 2 grams instead of 1. The broth will be darker but that's not a problem for me. You need to experiment to see which ratio you like.
The prep couldn't be simpler:
MAKING AN UNFILTERED GW BROTH FROM GRAIN FLOUR, CUZ YOU'RE A NASTY FUCK...
- Microwave the water until hot or use a pot. Boilling the water is not necessary.
- Add the LME.
- Stir well until dissolved.
- PC for ~25-30 minutes at 15 PSI.
I'm well aware the following recipe is not for everyone's liking, but I encourage you to try it at least once, see how it goes.
It is very cloudy and has lots of sediment, although the broth will clear up eventually as the myc colonizes it, revealing all its magnificence.
The best part of this recipe is actually the addition of sediment.
The sediment will act as a nice growing platform for the colony, allowing it to form a stronger network from which it can colonize the rest of the broth more vigorously.
Forget about using magnetic stirrers if you're going to be doing this, that would defeat the purpose of this method. Besides, I strongly recommend not using magnetic stirrers at all for LC's or anything to break up the myc, like marbles.
I used to use a stirrer and also blender balls, but through experience I've learned the best you can do is leave the colony in peace doing its thing, and once in a while give the jar a gentle swirl, being careful not to get the filter wet.
- Take a small handful of grains and grind that shit up into a powder. I use a coffee grinder.
- Weigh out enough flour for the amount of water you want to use for one single LC. The ratio I use is 1 gram of flour for 500ml of water. Adjust accordingly.
- Throw the powder into the LC jar and add the water (cold is fine).
- Sterilize for 1 hour at 15 psi. Some may find this overkill but I prefer to be safe than sorry when dealing with grains. Extended pc times for this does not hurt anything at all.
If you dislike sediment you can still do the same recipe but instead, filter out the flour using a cheese cloth. You could also do the old standard way of doing GW LC, which I personally don't like for a series of reasons.Pour that bitch!
So now your LC is colonized and it's time to do some pouring.
As with almost any tek, this one is also open to some tweaking based on personal preference, so if you want to noob-SHIP your LC, be my fucking guest. I just don't like to add an unnecessary vector if it can be helped and pouring LC is actually easier than G2G'ing, anyone can do it.
I have two simple rules for pouring LC:
- ONE LC, ONE USE.
- LESS IS MORE WITH LC, POUR WITH CAUTION!
Upon opening the lid of the colonized LC, use it in one go and throw the remaining LC down the toilet if you could not use it all in one session.
The reason for this is simple, you have no way to know if you introduced contams in your LC during a pouring session. That alone should deter you from using the LC a second time in the future.
This leads to the next piece of advice. If you don't want to waste part of your LC, adjust the amount of broth you use according to your workload.
Take into account that a 400ml LC can easily inoculate from 25 to 30 grain jars, and that's being generous with the pouring. If you can't cope with that workload, do less broth per jar.
Now, if you decide to go big, I'd recommend using caution with the way you pour. Due to the sheer number of jars a single 400-500ml LC can inoculate and the time it takes to noc them all, I personally don't feel comfortable leaving the LC jar open for that long.
A good way to handle this situation is pouring in a similar way Stareatclouds does in his vid
on how to G2G. That's the way I did it from the start and it worked great, but it feels a little awkward if you use whole SFD's like me.
What I currently do is pour the whole LC into a PP5 blender bottle like this one:
Pouring the whole thing into a bottle is not a vector if you ask me, been doing it a lot with success for months.
Pouring using this bottle is a cinch and the cap rests loosely on the spout between inoculations.
If you are going to do it this way:
Testing a Culture for Cleanliness
- Find yourself a quality pp5 blender bottle, drill a 1/8 inch hole through the lid and stuff it with poly.
- Screw the lid tight, snap the cap on the spout, foil the lid and sterilize for 15 min at 15 psi.
This is a preliminary test that should be done before using a culture to inoculate the LC. It will dispel any doubts if you are not entirely sure about its cleanliness. Skip it at your own risk.
You can be a beginner, and therefore lack the experience to determine if a culture is clean, or you can be an experienced grower that just wants to double-check for cleanliness.
Sometimes bacteria can be sneaky and difficult to spot, so it can be risky to take cleanliness at face value.
Let's do this, ain't precisely rocket science:
- Take the culture you want to test and poke it with the needle to take a biopsy.
- Squirt a little of water from the syringe into a blank agar plate.
- The biopsy tissue can take from 3 to 5 days to show growth on agar (sometimes even more). Until then, the only thing you should see on the plate is the clean water you squirted, providing the culture was clean.
- Let the test plate grow out.
- If the test proved negative, use this clean test plate to inoculate your LC.
If there was bacteria present in the culture you tested it should show rather quickly on agar. It will spread very fast with the help of the water you squirted into the plate, way before the biopsy has time to even start to show growth.
If there's bacteria you'll begin to notice after ~24-48 hours that the water you squirted is no longer clear and will usually adopt a whitish tinge that can be subtle to distinguish at first but it will become glaringly obvious if you give it more time. Also, you won't be able to move that 'dirty' water around the agar if you tilt the plate, it just won't budge.
Picture courtesy of Mushboy.
The picture shows growth on agar from a biopsy sample.
As you can see, growth looks disorganized, like a streaked plate from spores would. Don't be discouraged by this, it's perfectly normal and fine.
A biopsy sample taken from a rhizomorphic culture will also show tomentose and disorganized growth at first, but the potential for rhizo growth is still there, nothing has been lost.
Such a tiny piece of tissue needs time to regroup and establish a strong network.
A couple of transfers from this plate using a scalpel will get things back to normal, the culture will show rhizos again and you will also notice a substantial change in appearance from having narrowed down the genetics considerably.How this idea came to be
From the moment I became interested in this hobby the concept of liquid cultures has appealed to me. There's something fascinating about the easy expansion of a colony and making use of a powerful inoculant for really fast colonization.
But we all know that LC's don't come without their risks, above all if you don't own a flowhood.
I was racking my brain to find a way of doing LC that did
away with the common dissuasive aspects of working with them. Namely:
- Impecable technique is required to inoculate the broth using a wedge from a clean agar culture, as LC's are easily contaminated.
- LC's should always be tested on agar before use to check for cleanliness. This usually involves making use of a syringe to aspirate some LC through a Self Healing Injection Port (SHIP), but this procedure is not risk-free either.
- The most common LC teks tell you to build a special lid for the vessel. This usually involves using silicone to install a SHIP and a whatman filter. Aspirating LC's through a SHIP can be awkward as you have to tilt the jar with one hand and aspirate through the SHIP with the other hand.
- SHIP's can only be sanitized, they're never sterile, so poking the SHIP with a needle is a vector for contamination.
Out of the blue, the idea of poking a culture with a needle popped in my head. You can imagine the excitement when I discovered that it actually works!
Ok that's it for now!
Stay tuned because the biopsy method has more interesting uses, not only LC's.