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InvisibleJosex
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Josex' Poke: A different way of doing LC. * 39
    #24740168 - 10/27/17 12:01 AM (2 years, 10 months ago)

The Gangsta way of doing LC


This is my post 3000! :dancer:

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? :hehehe:
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.


Disclaimer:
This tek breaks a few well-established rules about LC's, and some may find it blasphemous.  :stonedjerk:

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! :evil: '.

:hypnotoad:




The Procedure


SETTING 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. :thumbup:

  • 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. :nono:

  • 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. :mad2:

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. :shrug:

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... :bow2:


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.

Materials:
  • 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 notes:
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. :sad:

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! :awesomenod:

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.

You either:
  • 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.


Or:
  • 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. :shrug:

Both have their pros and cons and for me it's hard to decide which one I like best.
  • 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).


MAKING LME BROTH

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:
  • 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.


MAKING AN UNFILTERED GW BROTH FROM GRAIN FLOUR, CUZ YOU'RE A NASTY FUCK...


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. :rockon:

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.

The prep:
  • 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. :evil:

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:
  • 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.



Testing a Culture for Cleanliness


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!
:mindblown:


Ok that's it for now!
Stay tuned because the biopsy method has more interesting uses, not only LC's.

:cheers:


Edited by Josex (07/04/20 04:19 AM)


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InvisibleZiranS
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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #24740190 - 10/27/17 12:14 AM (2 years, 10 months ago)

Seems like a good simple method. :thumbup: lets see some results down the road :rockon:


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InvisibleJosex
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Re: The Biopsy Method: A different way of doing LC. [Re: Ziran] * 4
    #24740212 - 10/27/17 12:26 AM (2 years, 10 months ago)

Thanks man! What do you mean you want to see some results? :lol: like I should post pictures of grows done this way? I thought that was pretty backwards, as it really adds nothing to the tek perse and it's a petty way of stroking your ego but... I could def show you some pics from grows done this way, even though I've been out of the game for months.

 


Edited by Josex (10/27/17 12:28 PM)


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Invisiblehamloaf
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Re: The Biopsy Method: A different way of doing LC. [Re: Josex] * 1
    #24740236 - 10/27/17 12:39 AM (2 years, 10 months ago)

Heck ya, Josex.  The biopsy LC method is an old technique.


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InvisibleJosex
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Re: The Biopsy Method: A different way of doing LC. [Re: hamloaf]
    #24740256 - 10/27/17 12:49 AM (2 years, 10 months ago)

Quote:

hamloaf said:
Heck ya, Josex.  The biopsy LC method is an old technique.



No way! :lmafo: You're an old hand so I'll take your word for it but I swear to god I have not come across anything similar anywhere. Actually I was surprised apparently nobody came up with such a simple and effective method.

Well heck, if it's not original at least is a safe way of doing LC for people that use SAB's. Let's "resurrect" it then :rockon:

BTW, can you post links to a similar tek? And I was here thinking I had the copyright :lol:


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Re: The Biopsy Method: A different way of doing LC. [Re: Josex] * 6
    #24740301 - 10/27/17 01:14 AM (2 years, 10 months ago)

Quote:

hamloaf said:
Heck ya, Josex.  The biopsy LC method is an old technique.




I knew of the noob method where you stab a fruit and squirt the tissue into lc...  But not josex' method.

I am currently using his method.  Should have fruits within a week! Thanks josex for the sacred knowledge:stoned:

Super easy and awesome :thaaannks:


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Re: The Biopsy Method: A different way of doing LC. [Re: mushboy]
    #24740306 - 10/27/17 01:18 AM (2 years, 10 months ago)

:splooge::vibin::highfive:


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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #24740441 - 10/27/17 03:42 AM (2 years, 10 months ago)

"Now I'm going to talk about your feelings.
After the poke, you may feel skeptical, as if you just did nothing"

I always find myself saying this

Poke meth
it's not a tek; it's a meth
I like it


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Re: The Biopsy Method: A different way of doing LC. [Re: Apples in Mono]
    #24740705 - 10/27/17 09:40 AM (2 years, 10 months ago)

Might have to give this a try this weekend. Good write up!


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Re: The Biopsy Method: A different way of doing LC. [Re: Buddha19er]
    #24740722 - 10/27/17 09:55 AM (2 years, 10 months ago)

:neat:


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Re: The Biopsy Method: A different way of doing LC. [Re: van hatton]
    #24740755 - 10/27/17 10:21 AM (2 years, 10 months ago)

Nice write up brutha


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Re: The Biopsy Method: A different way of doing LC. [Re: cronicr]
    #24740799 - 10/27/17 10:50 AM (2 years, 10 months ago)

:takingnotes::popcorn:


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Re: The Biopsy Method: A different way of doing LC. [Re: catnip40] * 3
    #24741106 - 10/27/17 01:25 PM (2 years, 10 months ago)

i cloned this bitch on the left.(normal methods. scalpel and 3 or 4 transfers.)


and tried the biopsy method and used it two ways:



WAY NUMBER THE FIRST
i just did the poke and shot the sample onto a plate. it basically grew out like a MS germ plate would.. only its a clone.

ive done this with a few other MS cultures. its really cool because you can have multiple
areas of growth on the plate from the same tiny ass sample. really makes identification of growth easier/quicker.(more pics later)

WAY PART SECOND
the way it was outlined. sample into grain water broth. i fucked up and used 5grams of oat powder per 500ml. ooops.

mega cloudy and sediment like you wouldnt believe:shrug:

i didnt upload pics because it was impossible to see the growth but it cleared up considerably as it colonized.


colonization took about 15days. i poured it in 3!!! three separate sessions.. all poured. no contamination
breakin josex' rule because im gangsta like that:cool:



(LC beats the wedge by weeks)

these colonized rather fast. i used little LC for each jar. the bag got maybe a shot glass worth at most.
the bag is about 90% colonized. each jar took about 5-10days to colonize. since i poured in different sessions
i waited for all the quarts(8) to colonize before spawning. i spawned a few shoeboxes and 2 20qt minis.
the bag will be a mono. i lost one jar, the last one i poured. i banged the SAB roof when pouring and it failed its shake.

the pouring an poking are about as basic as it gets. and pouring LC into a bag with a SAB is WAY EASIER than using a wedge.

ive failed with bags like crazy in my SAB. my fucking hands shake and i hate modding lids. for me? pouring LC is fucking awesome.

:congrats:


Edited by mushboy (10/27/17 02:03 PM)


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Re: The Biopsy Method: A different way of doing LC. [Re: mushboy] * 1
    #24741137 - 10/27/17 01:37 PM (2 years, 10 months ago)

this is so awsome and well written and definitely needed to be added to the shroomery.

Great contribution and great clean safe easy lc method.

This is how I will do my broths in the futur although I like pasty's ez lc tek as well.

This just seems a bit easier and less vectors.

Great idea and thanks for sharing. You F'n ROCK!:eek:


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Re: The Biopsy Method: A different way of doing LC. [Re: tombosley8]
    #24741191 - 10/27/17 02:05 PM (2 years, 10 months ago)

I may try this with pasty's ez lc as well for spreading a plate into tons of lc with barely any work/inoculant.

Seeing as mushboy got good growth on agar I should have no problem with recovery on ezlc, right?


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Re: The Biopsy Method: A different way of doing LC. [Re: tombosley8]
    #24741279 - 10/27/17 03:01 PM (2 years, 10 months ago)

Thanks guys! That's awesome Mush, can't wait to see that clone in action :rockon: Nice contribution to the thread too.

Yes Tom, you can also use the biopsy to noc your pasty's LC, but I only see it being useful as a way of narrowing down genetics, if that's what you're after. Imo the point of that tek is being able to visually inspect a culture before adding the liquid, and for that noccing with wedges would be a lot faster. Also, the biopsy takes its sweet time to show growth on agar, whereas it grows rather quickly in a nutrient liquid medium, so why not shoot the biopsy straight into the broth to shave off some time and steps? You can also inspect a culture before taking the biopsy and shooting the biopsy into the broth is not a vector at all, easy af too. You'd be hard pressed to introduce contams in your LC, and if it contams you can be nearly positve the agar culture was at fault.

Also, I'm not inclined to buy the theory you can't always spot bacteria in a LC. Shit grows fast in the broth, it grows and spreads like a fine cloud before the myc even gets a chance. Also myc growth will be ugly as hell when there's bacteria present, like it just wants to stay at the bottom of the jar like a slob.


Edited by Josex (10/27/17 03:45 PM)


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Re: The Biopsy Method: A different way of doing LC. [Re: tombosley8]
    #24741291 - 10/27/17 03:07 PM (2 years, 10 months ago)

Quote:

tombosley8 said:
I may try this with pasty's ez lc as well for spreading a plate into tons of lc with barely any work/inoculant.




:thatsayes:
I read fast I think :lol:


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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #24741337 - 10/27/17 03:34 PM (2 years, 10 months ago)

Thanks for the addition as I think I may have been over thinking it.

The main reason for the ezlc is to make sure the inoculation process and innouculant were clean before adding the liquid.

It's just a safe check but you still have to add the water after so you could potentially contam and not know at that stage.

So now that you helped me get through it I will most likely just use the broth method for this tek.

the only other thing to the ezlc is you can use better sources of nutrients without clouding up the liquid. Your grain water lc is pretty much as good as you can get except it has the cloudiness. Again that only would matter to identify a contaminated lc.

I have had a couple instances where I wouldn't have known my lc was bacterial but could see the growth before adding the liquid. Probably bad technique or possibly the culture, it just showed the bacteria better in the ezlc than the original agar plate.

I'll probably do mostly broth and just test it on agar as you said in the op.

Again thanks so much for this and it should prove to be immensely helpful


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Re: The Biopsy Method: A different way of doing LC. [Re: Josex]
    #24741348 - 10/27/17 03:41 PM (2 years, 10 months ago)

Cool stuff josex.

Blunt tips help.


When I started I did the original lc biopsy tek where the biopsy is of a fruit. 3 tried, 3 succeeded, proved to be a single step contraction of biopsy cloning and creating a powerful inoculant as per this tek.  I would agree it's a noob method though as it's preferable to hold on to isolated cultures from a source prior to such expansion, and both cloning and LCs should be learned on their own first instead of mushing them into one.


These days I'm also back into LCs, thanks to grainwater.  You may find it interesting that Ziploc twist n loc containers can apparently host LCs without modifications whatsoever. I've done a number of LCs to maturity in them that have exploded into gorgeous mycelium that cleared up thick grainwater. Somehow these containers 'breathe' without the spread of contaminants.
They're then simply poured open lid in a session as you do.  Or since I use sterile airflow I'm able to draw much of the liquid up into syringes first thing for storage and later use.


I may try a syringe biopsy for starting my next LCs from master culture. I have been growing out an agar ziploc container then squirt & scrape to create some LI for an LC and a couple ryegrass masters.  Figured more mycelium would expand to LC faster but it may not end up mattering much.

But I'm particularly interested in the agar poke method here for taking multi-spore culture samples.  Seems like an easy, instant, precise method to isolate different new cultures from germination plates into separate grows like in my grain petri culture hunt tek.  I like using different tools like tweezers and screwdrivers but I think the blunt needle and water plunger might be the perfect tool for that first step of many containers.


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Re: The Biopsy Method: A different way of doing LC. [Re: Violet]
    #24741406 - 10/27/17 04:06 PM (2 years, 10 months ago)

Nice one josex!


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