Home | Community | Message Board


Vaposhop
Please support our sponsors.

Mushrooms, Mycology and Psychedelics >> Mushroom Cultivation >> Mushroom Cultivation Archive >> Agar and Liquid Culture

Welcome to the Shroomery Message Board! You are experiencing a small sample of what the site has to offer. Please login or register to post messages and view our exclusive members-only content. You'll gain access to additional forums, file attachments, board customizations, encrypted private messages, and much more!

Amazon Shop for: ½ Pint Jars, Agar, Magnetic Stirrer, Malt Extract, Paul Stamets, Pressure Cooker, Scales

Jump to first unread post. Pages: 1 | 2 | 3 | 4 | 5 | Next >  [ show all ]
Invisiblecreamcorn
mad scientist
Male User Gallery

Registered: 03/13/06
Posts: 2,962
Trusted Cultivator
LC Cloning - Newbie friendly pictorial tek! * 26
    #5874305 - 07/18/06 07:31 PM (8 years, 5 months ago)

Cloning with liquid culture, the easy way.

This is a tek on how to clone without the need for advanced methods, agar, gloveboxes, flowhoods, or even a pressure cooker.  Its simple enough for a beginning pf-tek'er to complete.  You'll probably also find some good LC tips here if you've been struggling with those.  As always, comments, questions, suggestions, and kudos appreciated! :smile:  I apologize if this is a little wordy, but I wanted to cover all the bases.  I've been answering newbie questions long enough to anticipate them before they've even been asked, so hopefully I got everything covered.

Materials needed:


Step 1.  Prepare a small liquid culture.

For this, I am of the thinking that SIMPLE IS BEST.  My liquid culture containers are simple jars, with a regular old metal lid and ring, with a single hole punched at the top.  The hole should be big enough to accomodate your needle, with a little room to spare so that a vaccum is not created later when aspirating solution.  The hole is covered with a piece of micropore tape, and then covered again either with a coffee filter and rubber band, or a piece of aluminum foil loosely crumpled, to act as a dust cover.


Jar with just a hole

Recipe:  Recipe is the same as any liquid culture; 4% media by weight.  You may use any of the standard LC media, honey, caro, dex/malt will all work.  Water quality is important to LC.  I find that distilled works best, and gives the clearest solution.  You may also use bottled spring water, or tap water filtered with a Brita pitcher or the like.  (Your mileage may vary depending on the quality of your locality's tap water.  In my area, I avoid it.)  Honey quality also varies widely - I've found this particular variety works fantastic for me, and doesn't make any sediment in my LC's even without filtering.  You may need to experiment with brands and types of honey to find one that works.  For a sure thing, you can definitely use karo, and if you're industrious and willing to hunt down dextrose/malt, by all means use that.  The honey I've got seems to outperform karo, so it works for me.


Measure 4 grams of media per 100ml of water

When cloning, I generally start with a small half pint container.  You'll see I've measured exactly 4 grams of honey.  Water weighs 1 gram per 1ml, so I'll fill it to the 100ml point.  I nuke this in the microwave for about 30 seconds, just to warm the water, allowing the honey to dissolve easier, and I stir it up.  Obviously, when making larger LC's, just do the math to stay at that 4% mark.

This will be my "master" culture.  From this culture, small samples are taken to start larger liquid cultures in quart jars that are my "working" culture that I use for inoculation.  The remainder of the master culture can be refrigerated, where it will keep for several months.  When I run out of working culture, it only takes a few drops of the master to start a whole quart of working culture all over again.  Its important to take from the master each time, rather than go from working culture to working culture, so that each of your working cultures are "second generation."

Step 2.  Sterilization.

In my opinion there's only two acceptable ways to sterilize LC for consistent results: steaming, and pressure cooking.  This gives those an option who don't own a PC.  Microwaving works for some, but I don't trust it.  If it works for you, fantastic, go with it.

In both cases, wrap the top of your jar in tinfoil as usual, and take a syringe about half full of water, and also wrap it in tinfoil.  We'll sterilize them both at the same time.


Syringe with some water; wrapped in foil and ready to sterilize

Steam sterilization:
Place a cloth at the bottom of a pot with a tight fitting lid.  Use an inch or two of water.  Place your jar and syringe inside.  Bring to a slow rolling boil.  Once a boil has been achieved, start your timer for 30 minutes.  At the end of 30 minutes, turn off the heat, walk away and don't even think about it for a few hours.  Let it cool.

PC sterilization:
Use your PC as per its instructions with something to keep the items off the bottom of the pot (like a cloth, or a rack if you got one.)  Slowly bring up to pressure.  10psi is plenty if that's all you got.  15psi is fine too.  Do not exceed 15psi.  Once at desired pressure, set your timer for 15 minutes.  DO NOT exceed 15 minutes.  When the time is up, cut your heat and walk away until its cooled.

Step 3.  Select specimen.

Find a nice looking fruit!  We want to clone desirable qualities.  Many have different ideas as to which fruit to clone; ie. choosing the first one up might give you a speedy isolate to work with later, choosing a big one might give you a genetic predisposition towards large fruits; and so on.  I believe that there's just so much variation in growing methods, even though we are taking a clone, we have no idea how closely its going to resemble its "parent" when grown out later - so I take a nice looking fruit and hope for the best.  You'll find out in time if it was a good choice or not when you get your first crop of clones in, and can assess how long they took to grow, the yields you got, and of course, the potency after sampling some.  If it didn't turn out as good as you were hoping, don't fret, you can try again from a different specimen from a different multispore grow.  If it did turn out for you, treat that master culture like gold!

Step 4.  The biopsy

Here's where you'll want to practice your sterile procedures.  No need to go overboard, all I do is ensure there's no drafts, clean my work surface, wash my hands with antibacterial soap, and hit them a second time with some hand sanitizer.  If spraying Oust into the air and washing everything with bleach or whatever makes you happy, knock yourself out. 

Take your fruit, and clean the stem thoroughly with an isopropyl soaked cotton swab.  Clean it all the way around.  Do not touch any areas with your hand near the site of the biopsy.  Quickly wipe your needle with iso (flame if desired, I don't bother - we did just sterilize it after all!) and stab it straight through the stem to the other side.


Swabbing the stem; taking the biopsy.  (This is where 3 hands would come in handy, somebody had to work the camera!  You'll obviously want to hold the mushroom near the top and swab the bottom, and when taking a biopsy not allowing the mushroom to touch any surfaces.)

Remove the foil from your prepared LC jar.  Using a dry cotton swab, clean up any condensation/moisture on the lid.  Then using an alcohol soaked one, clean the lid again.  Give the needle another wipe, insert into hole, and push the plunger.  The water in the syringe will force the cross section of the stem out into your LC.  Quickly cover the hole with a piece of micropore tape (if your lid is still wet with alcohol, that's fine, the tape will stick just fine once it dries) and cover with a dust cover of your choice.


Knock 'er up!  You might need to push a little hard, but the liquid will force the piece of stem out.


You can sort of make out the piece of stem in there.  My solution really was clear I promise, this was without a flash to reduce glare and hopefully get a picture of the piece inside.  On the right, foil loosely back on as a dust cover, don't forget to label!


She's looking a little beat up after being stabbed!

Incubate, and wait.

7-10 days later you should have a nice looking cloud of mycelium.

As they do on the cooking show.... "here's one we've prepared earlier!"  When doing all the above steps, you'll want to work quickly to avoid contamination.  I obviously stopped to photograph everything so who knows if we got a clean sample here.

Here's a "master" culture, about 10 days colonizing.  You'll notice some is already missing, as I've used some of the solution to inoculate larger LCs.


The master culture.  Notice its very thick!  Obviously my little micropore'd hole provides all the gas exchange you need.  You'll also note a few dark specs, that's leftover pieces of the stem.  It kind of dissolves and breaks up to an extent.  It turns dark blue/grey, because you bruised it for sure when poking it with a needle!

Here's a working culture, this is 5 days old.  It was started with about 4cc of solution from a master, and then constantly agitated on a magnetic stir plate.  Use it (or make it) if you got it, but its not necessary.  They'll colonize great without, it just takes a little longer.  LC to LC is FAST no matter what.


Enough LC to kill a small army, from a few CC's of the master.

Some additional LC tips:
-Follow the measurements carefully.  You'll get no growth or poor growth if you use too much media.  When in doubt, use a little less than 4%.
-If your myc cloud is thick and can't be sucked up easily, suck up what you can, and squirt it back down into the cloud.  It will break right up.
-To aspirate from my single-hole jars, I just tilt them on their sides, being very careful not to spill any out the hole.  The needle reaches in just fine.  I gently swirl it around and capture as much mycelium in the syringe as possible.  The thicker the better!
-People often ask, "when is my LC done?"  Well, its done when you want to use it!  If there's enough mycelium floating around that you can capture a bunch in your syringe, go for it.  The rest will continue to grow until the nutrients have run out.  When it doesn't seem to be growing any more, you can refrigerate it, and it lasts many months.  (6 or more.)
-When I aspirate, I take an alcohol soaked cotton swab, and swab right over the piece of micropore tape.  I then stick the needle right through the tape, and fill up my syringe.  I quickly cap the syringe, and quickly replace the soggy piece of micropore tape with a fresh piece.  You can certainly use any number of the fancy LC container teks out there, but it doesn't get much simpler than a hole with a piece of tape on it kids.
-LABEL EVERYTHING.  Keep track of dates, strains, generation number, anything you feel relevant.
-A perfectly prepared karo or honey LC should be nearly crystal clear.  Dex/malt will produce a yellowish solution.  If your karo or honey solution turns yellow, you may have overcooked.  Did you follow my sterilization directions to the T?  Either way, a little overcooked will still work, its not the end of the world.  Likewise, sediment from honey, or from malt/dex will not harm a thing - it just makes spotting possible contamination a little more difficult.  Some take some pretty extreme measures to filter their solutions before sterilizing to get the sediments out, I'll leave it up to you.
-Unless you want cracked jars, heed my advice and allow them to cool gradually and naturally.

Quick Cloning FAQ:

Q: Why clone?
A: Cloning ensures the most efficient use of your substrate, the best possible yields, and once you've found a clone that has a desired potency, the most consistent potency!  See, with a multispore inoculation, you end up with many many strains (each dikaryotic spore mating is technically its own strain).  These strains sometimes grow side by side, and sometimes mate a second time by a process known as anastomosis.  So in the end, you've got a cournicopia of genetic materials and strains.  Not all of these are capable of fruiting!  I think of these strains as "guy on the couch" strains - they'll gladly eat your food and drink your water, but never produce any fruits for you.  By cloning an actual mushroom, we're dealing with one single isolated strain that's already proven itself as being able to fruit.  Another benefit to cloning is nice even pinsets.  When you're dealing with all the mixed strains of an MS inoculation, some parts of your substrate are going to be ready to pin before others.  With clones, they're all going to be ready at nearly the same time, resulting in even, consistent pinsets, and more defined flushes.

Q: If I clone a large fruit, will the resulting fruits grown from it be large?
A: Maybe!  If you have identical twin humans and one eats nothing but cheeseburgers and plays Nintendo all day, while the other eats a balanced diet and exercises regularly, are they going to be the same shape and size?  Of course not.  There's genetic predisposition of course, a large fruit obviously has the genetics needed to be capable of producing more large fruits - but conditions have to be right.  Its nature AND nurture.  Nurture is very, very important, so take care of your babies.

Q: If I clone a mutant, will the resulting fruits be mutants?
A: Again, maybe!  It depends on why the mutation occurred.  If there was a genetic problem that caused the mutation, then sure, it will carry over.  You might not end up with the same exact mutations later, but you'll end up with some funky fruits.  If the mutation occurred because the fruit came in contact with a harmful chemical while growing, that's not going to carry over - the result may very well be a healthy clone.  Nature versus nurture again.  Using the human analogy, if I cloned a person with 6 toes (yeah it happens), the resulting clone will be born with 6 toes.  If I clone a human who broke their leg, of course the baby isn't going to be born with a broken leg!  Got it?

Q: Where should I take my biopsy?
A: I've found that near the base of the stem works best.  I can't tell you why, I don't know, just my experience.

Q: How mature should the mushroom be before cloning?
A: As the viel breaks, or just before.  If it starts to sporulate, it will drop spores on the stem.  You're likely to get spores mixed in with your biopsy, and guess what - its no longer going to be a clone!  You want to avoid spores.

That's all for now, if anybody has any ideas for the little mini clone FAQ above, ask away, and I'll answer to the best of my knowledge (or maybe others can chime in and help out.)  The only way I can make this any simpler is if I were to come to your house and do it for you - and unless you got boobies, and plan to pay travel expenses, it ain't happening. :smile:  Thanks for reading, and good luck.


Edited by creamcorn (07/18/06 08:51 PM)


Post Extras: Print Post  Remind Me! Notify Moderator
OfflineOatman2000
-=Outa Space=-
Male User Gallery

Folding@home Statistics
Registered: 05/10/05
Posts: 2,863
Loc: Planetary Nebula
Last seen: 2 months, 10 days
Trusted Cultivator
Re: LC Cloning - Newbie friendly pictorial tek! [Re: creamcorn]
    #5874429 - 07/18/06 08:07 PM (8 years, 5 months ago)

I like it!

straight and to the point... having a Magnetic stirer when making lc's, is prolly the best thing i have bought for this hobby... i have 4 different strains of master lc on hand at all times....

sticking the fruit is a very clean way of cloning.... no contact with human hands..

as for the micopore tape for your injection port.. not a big fan of saturating the micopore tape. I go with AGAR's idea of using a Self Healing Injector Port , and a piece of winshield washer hose from auto zone as the filter... they are cheap as hell and no worries of saturation of your filter material.


--------------------
Spawning to COIR
:thumbup:  My Chocolate Recipe
WBS QUART SPAWN JAR PREPERATION
----------------------------

4-PO-DMT; 4-phosphoryloxy-N,N-dimethltryptamine


Post Extras: Print Post  Remind Me! Notify Moderator
InvisibleRoadkill
Retired Shroomery Mod
Male User Gallery

Registered: 12/11/01
Posts: 22,598
Loc: Snoqualmie, Wa.
Trusted Cultivator
Re: LC Cloning - Newbie friendly pictorial tek! [Re: creamcorn]
    #5874447 - 07/18/06 08:11 PM (8 years, 5 months ago)

Nice write up!~

:thumbup: :thumbup:



:popcorn: <--waiting for a newb to ask what an LC is.


--------------------
Laterz, Road

Who the hell you callin crazy?
You wouldn't know what crazy was if Charles Manson was eating froot loops on your front porch!


Brainiac said:
PM the names with on there names, that means they have mushrooms for sale.



Post Extras: Print Post  Remind Me! Notify Moderator
Invisiblecreamcorn
mad scientist
Male User Gallery

Registered: 03/13/06
Posts: 2,962
Trusted Cultivator
Re: LC Cloning - Newbie friendly pictorial tek! [Re: Oatman2000]
    #5874454 - 07/18/06 08:12 PM (8 years, 5 months ago)

my tape never gets wet with LC.  jars aren't filled to the brim for that reason.  and like i mentioned, i replace the piece of tape with a fresh one anyway each time i draw out solution.  i've done countless LC's and only had the "privlege" of seeing a contamination once or twice.

i'm sure self healing ports and air filters and all that jazz do work spectacular, but like i said i'm a fan of keeping it simple. :smile:  i encourage everyone to do it however it makes them comfortable!


Post Extras: Print Post  Remind Me! Notify Moderator
InvisibleOmnicracker
Crusted Trolltivator

Registered: 09/18/05
Posts: 1,421
Loc: Wal-Merica
Trusted Cultivator
Re: LC Cloning - Newbie friendly pictorial tek! [Re: Oatman2000]
    #5874477 - 07/18/06 08:17 PM (8 years, 5 months ago)

excellent! i love to see a proper write up.


--------------------




xxx..Learn Something..xxx


Post Extras: Print Post  Remind Me! Notify Moderator
Invisiblefastfred
Old Hand
Male User Gallery

Folding@home Statistics
Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
Re: LC Cloning - Newbie friendly pictorial tek! [Re: Omnicracker]
    #5876068 - 07/19/06 01:57 AM (8 years, 5 months ago)

Awesome CC! This is the best LC writeup I've seen, BY FAR! I was going to compliment you on several specific points you make, but there's too many! Every paragraph has at least one nugget of gold in it!

That said, I have some suggestions, which I offer because I think this should become THE LC tek of choice.

1. I would split it into two sections. One for the step-by-step procedure and another for discussion, alternatives, suggestions, and Q&A. That might make it easier for noobs to follow when they're actually performing the procedure. Easier to print the procedure also.

2. As far as 4% by weight, that's awesome that you point this out. I hate seeing "a dab" or "a squirt" that's a recipe for failure if you're not familiar with the procedure.

I would point out that the optimal range is 2-4%, you might mention that. Also honey and karo are partly water, so that should be accounted for if you want to be precise. Here are some values I have in my notes...

Honey = 17.6% water
corn syrup = 19.7% water
High fructose corn syrup = 29% water

Using these figures, with some rounding, it takes about 5g (or ml) of karo to equal 4%. Almost the same for honey.

3. You have karo spelled "caro" in one place.

4. You might mention that dextrose is corn sugar and found in most grocery stores. Also that malt is "spray malt extract" and is available wherever brewing supplies are sold. And that "light" or "extra light" malt extract is best for clarity.

5. You can also boil an LC rather than steam it. I usually boil water and then add ingredients.

6. As far as 15 minutes sterilization it's better to exceed that than go under it. The 10 psi equivalent to 15@15 is 22.5 min.

7. "Enough LC to kill a small army, from a few CC's of the master." Is maybe not the best line, what with search engines and bioterrorism scares and all.

8. You should mention to add the stirbar before PCing. If you don't have a stirrer you should add some sort of agitator, like a crushed marble or shard of glass.

9. I would change "strain" to "sub-strain". I think strain implies more of a genetic difference than you are referring to.

10. You might want to add a link to "FastFred's Media Cookbook"...
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/3077006


Excellent writeup, it's the best one I've seen so far. Make sure to submit it to the FAQ submission forum.


-FF


Post Extras: Print Post  Remind Me! Notify Moderator
InvisibleTippinthru
contented

Registered: 04/07/05
Posts: 1,131
Loc: "The Garden"...
Re: LC Cloning - Newbie friendly pictorial tek! [Re: creamcorn]
    #5876131 - 07/19/06 02:16 AM (8 years, 5 months ago)


Simple is GOOD. :wink:


--------------------
Perfection is attained by slow degrees; it requires the hand of time...
[


Post Extras: Print Post  Remind Me! Notify Moderator
OfflineYamidude
Stranger

Registered: 06/15/06
Posts: 957
Last seen: 6 years, 3 months
Re: LC Cloning - Newbie friendly pictorial tek! [Re: fastfred]
    #5876133 - 07/19/06 02:17 AM (8 years, 5 months ago)

just bookmarked this bad boy. Great write up CC!! 5 for you..


Post Extras: Print Post  Remind Me! Notify Moderator
InvisibleBlutjager
Inhuman

Registered: 06/12/06
Posts: 9,220
Re: LC Cloning - Newbie friendly pictorial tek! [Re: Yamidude]
    #5876416 - 07/19/06 04:58 AM (8 years, 5 months ago)

Hell yes,I picked a fruit tonight that I will do this with tomarrow.Thanks Creamcorn


Edited by Blutjager (10/08/06 05:39 AM)


Post Extras: Print Post  Remind Me! Notify Moderator
Offline2FiNiTe
ConsideratlyKilling Me
Male User Gallery

Registered: 06/12/06
Posts: 1,630
Loc: New England
Last seen: 5 months, 24 days
Re: LC Cloning - Newbie friendly pictorial tek! [Re: creamcorn]
    #5876521 - 07/19/06 06:19 AM (8 years, 5 months ago)

Creamcorn, I dont think I've seen you post anything less than gold. This mini tek is no different. I'll be sure to try this method my next flush. The last one I did I had to some dissection to get my stem matter, I never even thought of stabbing the stem with a syringe. Brilliant! Your right, you can anticipate what us noobs are gonna ask before we do lol.


--------------------
"Ours is a world of nuclear giants and ethical infants. We know more about war that we know about peace, more about killing that we know about living."

General Omar N. Bradley


Post Extras: Print Post  Remind Me! Notify Moderator
Invisiblecreamcorn
mad scientist
Male User Gallery

Registered: 03/13/06
Posts: 2,962
Trusted Cultivator
Re: LC Cloning - Newbie friendly pictorial tek! [Re: fastfred]
    #5876889 - 07/19/06 10:07 AM (8 years, 5 months ago)

>> That said, I have some suggestions, which I offer because I think this should become THE LC tek of choice.

I never intended on it being an LC tek, it was more or less supposed to be a cloning tek for the LC-familiar, I just got carried away with the LC advice.  I can definitely make a few changes to make the LC aspects more organized/complete and it can serve as both then.

>> 1. I would split it into two sections.  One for the step-by-step procedure and another for discussion, alternatives, suggestions, and Q&A.  That might make it easier for noobs to follow when they're actually performing the procedure.  Easier to print the procedure also.

Will see what I can do...

>> I would point out that the optimal range is 2-4%, you might mention that.  Also honey and karo are partly water, so that should be accounted for if you want to be precise.  Here are some values I have in my notes...

Honey = 17.6% water
corn syrup = 19.7% water
High fructose corn syrup = 29% water

Yep, that's totally right that 4% of corn syrup/honey translates to less than 4% sugars... but like you also say, 2-4% is good, so that lands us somewhere in between.  Its always a safe bet then, and while precise is important, I think simplicity is too.  The "4g per 100ml" rule is just a simple one to follow, and works with any of the media.  (Except high fructose corn syrup... I was under the impression that didn't work so well?  Have never used it to know from experience.)

>> 3. You have karo spelled "caro" in one place.

Oops. :smile:

>> 4. You might mention that dextrose is corn sugar and found in most grocery stores.  Also that malt is "spray malt extract" and is available wherever brewing supplies are sold.  And that "light" or "extra light" malt extract is best for clarity.

Will mention.  I don't work with the dex/malt mix, so my knowledge of the specifics were lacking.

>> 5. You can also boil an LC rather than steam it.  I usually boil water and then add ingredients.

How is boiling then adding ingredients going to give a sterile mixture?  I do mention heating the water to dissolve the media.  Only reason I warm it after to dissolve, is because my scale doesn't have the capacity to measure a jar, with water, and honey. :smile: 

>> 6. As far as 15 minutes sterilization it's better to exceed that than go under it.  The 10 psi equivalent to [Email]15@15[/Email] is 22.5 min.

Again, true.  But when you cut the heat on your PC and let it cool naturally, it still stays at temp (thus still sterilizing) for quite a while.  I have an electric stove, and a large heavy PC, it takes almost 20 minutes just to naturally drop from 15 to 0psi... but I suppose not everyone is in that same boat.  I have personally cooked at 10psi for only 15 minutes without contam... Liquid is easy enough to sterilize, but you can overcook LCs which is whats with the strict time suggestion - but I will edit to increase the time suggestion for 10psi users.

>> 7. "Enough LC to kill a small army, from a few CC's of the master."  Is maybe not the best line, what with search engines and bioterrorism scares and all.

Haha... just a figure of speech but you're probably right given the world we live in nowadays. :smile:

>> 8. You should mention to add the stirbar before PCing.  If you don't have a stirrer you should add some sort of agitator, like a crushed marble or shard of glass.

Yup.  I purposely do not agitate my "master" culture.  That little tiny piece of mushroom flesh gets soggy and breaks up as it is, and its barely viable due to its small size and after all the isopropyl and whatnot, so I leave it undisturbed.  I should probably point that out, as well as a reminder to add your agitators before sterilizing for those who will use them.

>> 9. I would change "strain" to "sub-strain".  I think strain implies more of a genetic difference than you are referring to.

I always thought sub-strain was a bit of a misnomer?  I've honestly never looked up the actual definitions... either way you're probably right again that using the term will make it a little more clear.

>> 10. You might want to add a link to "FastFred's Media Cookbook"...
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/3077006

Will do, hadn't ever previously come across that thread and there's some interesting ideas in it.

>> Excellent writeup, it's the best one I've seen so far.  Make sure to submit it to the FAQ submission forum.

Thanks!

Will get to the editing later today, I shouldn't even be spending this much time here when I should be working as it is. :smile:


Post Extras: Print Post  Remind Me! Notify Moderator
InvisibleTippinthru
contented

Registered: 04/07/05
Posts: 1,131
Loc: "The Garden"...
Re: LC Cloning - Newbie friendly pictorial tek! [Re: 2FiNiTe]
    #5876970 - 07/19/06 10:48 AM (8 years, 5 months ago)



--------------------
Perfection is attained by slow degrees; it requires the hand of time...
[


Post Extras: Print Post  Remind Me! Notify Moderator
Invisiblefastfred
Old Hand
Male User Gallery

Folding@home Statistics
Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
Re: LC Cloning - Newbie friendly pictorial tek! [Re: creamcorn]
    #5877016 - 07/19/06 11:06 AM (8 years, 5 months ago)

> high fructose corn syrup... I was under the impression that didn't work so well?

I've never used it either. I thought it wouldn't work because of some paper I read, but I think it was actually another edible species. I later looked it up in a paper on P. baeocystis and apparently it works for that species, so I'm guessing it would work. It's not very good though. Of the sugars they tested the ranking was maltose, glucose, trehalose, fructose, galactose, sucrose, and lactose. Sucrose barely grew anything and lactose produced nothing.

> Its always a safe bet then, and while precise is important, I think simplicity is too.

I agree. I'm probably too much of a stickler. It's just that I couldn't possibly bring myself to write 4% on media that actually had 3.4%. I would have to bitch slap myself for that, whereas I only curse because of rounding errors.

> How is boiling then adding ingredients going to give a sterile mixture?

I guess I'm too used to making solid media. You have to boil to dissolve agar, and I figured the LC might reach a slightly higher temp with direct boiling over steaming.

> my scale doesn't have the capacity to measure a jar, with water, and honey.

Hmm... I guess it wouldn't work to well to use weighing paper or a boat to measure honey. And I suppose it dissolves easily. Malt is a little tougher. My tips for malt are to use distilled water (for sure), bring that to a low boil, preferably with mag stirring, and *slowly* add the malt *then* the agar (for solid media). Malt sometimes will clump and become a PITA to dissolve. If you make the mistake of mixing malt and dex before adding it's even worse.

> I always thought sub-strain was a bit of a misnomer?

People love to argue that point to no end. If you are of the opinion that Paul Stamets is a god, then you should flame me for blasphemy.

I just disagree with calling each individual a separate strain. Sibling spores share 50% of their DNA and 95% of the time even an expert couldn't determine any phenotypic difference between them. Calling something a separate strain implies a genetic and phenotypic difference. Sibling spores have the absolute minimum of genetic variation possible between two individuals without being clones, so I don't see any way you can call them separate strains. But some people even want to call each sector from the same myc a separate "strain" for Christ's sakes.

You can call them individuals, isolates, cultures, or substrains.

> I never intended on it being an LC tek

Just point out that you can also inject spores and it'll be a great LC tek.

Mad props for the kick-ass write-up.


-FF


Post Extras: Print Post  Remind Me! Notify Moderator
Invisiblecreamcorn
mad scientist
Male User Gallery

Registered: 03/13/06
Posts: 2,962
Trusted Cultivator
Re: LC Cloning - Newbie friendly pictorial tek! [Re: fastfred]
    #5877036 - 07/19/06 11:17 AM (8 years, 5 months ago)

Quote:

fastfred said:
> How is boiling then adding ingredients going to give a sterile mixture?

I guess I'm too used to making solid media.  You have to boil to dissolve agar, and I figured the LC might reach a slightly higher temp with direct boiling over steaming.





haha reminds me of working with the original homestead kit wayyyys back... getting it home, skimming the instructions... PC?  who needs a PC?  i'm gonna cook up this "agar stuff" in a pickle jar directly on the stove!  which... worked. :smile:  but then i got stuck with it was time to PC their little test tubes of grain, so i had to give in to "the right way"

i guess i wasnt thinking thru when i responded, i suppose it would work if you heated -> added ingredients -> boiled entire mixture direclty over burner in sealed (foil topped for steam escape) container.  from many many steam sterilized jars though i can garuntee it works with a really high success rate, and is probably safer from a jars-cracking oh-shit-there's-lc-everywhere point of view :smile:


Post Extras: Print Post  Remind Me! Notify Moderator
Invisiblefastfred
Old Hand
Male User Gallery

Folding@home Statistics
Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
Re: LC Cloning - Newbie friendly pictorial tek! [Re: creamcorn]
    #5877067 - 07/19/06 11:40 AM (8 years, 5 months ago)

> safer from a jars-cracking oh-shit-there's-lc-everywhere point of view

Hell yes! Never directly heat a glass jar on a stove! Let me tell you, it's a bitch to clean up a liter of media that's flowed into every crack and then gelled. I thought I was being all smart by heating it slowly on a hotplate/stirrer, then BAM media everywhere!

BTW what strain did you get with your homestead kit?


-FF


Post Extras: Print Post  Remind Me! Notify Moderator
Invisiblecreamcorn
mad scientist
Male User Gallery

Registered: 03/13/06
Posts: 2,962
Trusted Cultivator
Re: LC Cloning - Newbie friendly pictorial tek! [Re: fastfred]
    #5877075 - 07/19/06 11:44 AM (8 years, 5 months ago)

Quote:

fastfred said:
> safer from a jars-cracking oh-shit-there's-lc-everywhere point of view

Hell yes!  Never directly heat a glass jar on a stove!  Let me tell you, it's a bitch to clean up a liter of media that's flowed into every crack and then gelled.  I thought I was being all smart by heating it slowly on a hotplate/stirrer, then BAM media everywhere!

BTW what strain did you get with your homestead kit?


-FF




There was absolutely nothing denoting a strain anywhere in the package... just a little glassine envelope with half a print that was labelled "These are your spores." :smile:  I read somewhere on here they were amazonian in that original agar/grain/compost kit.... its unfortunately no longer with me, this was many many years ago.


Post Extras: Print Post  Remind Me! Notify Moderator
OfflineEquilibriuM
dream stalker

Registered: 07/17/05
Posts: 2,323
Last seen: 7 years, 6 months
Re: LC Cloning - Newbie friendly pictorial tek! [Re: creamcorn]
    #5877154 - 07/19/06 12:32 PM (8 years, 5 months ago)

LOL I had that kit that was 6 years ago.


--------------------
HELP!!!!!!!!!


Post Extras: Print Post  Remind Me! Notify Moderator
Offlinesplifner180
Student
 User Gallery

Registered: 03/24/06
Posts: 1,288
Loc: USA, East Coast
Last seen: 3 years, 15 days
Re: LC Cloning - Newbie friendly pictorial tek! [Re: EquilibriuM]
    #5879389 - 07/19/06 11:56 PM (8 years, 5 months ago)

What's an LC?

splif


--------------------
First Grow: Ecuador -> LC -> HPoo/Straw -> Monotub
Build a Do-It-Yourself Magnetic Stirrer in thirty minutes with no money.


Post Extras: Print Post  Remind Me! Notify Moderator
OfflineYamidude
Stranger

Registered: 06/15/06
Posts: 957
Last seen: 6 years, 3 months
Re: LC Cloning - Newbie friendly pictorial tek! [Re: splifner180]
    #5879399 - 07/19/06 11:58 PM (8 years, 5 months ago)

Quote:

splifner180 said:
What's an LC?

splif


LepreChaun


Post Extras: Print Post  Remind Me! Notify Moderator
Invisibleliamtheloser
Advanced Idiot
Male User Gallery
Folding@home Statistics
Registered: 06/08/06
Posts: 1,379
Loc: PNW
Re: LC Cloning - Newbie friendly pictorial tek! [Re: Yamidude]
    #5879604 - 07/20/06 12:55 AM (8 years, 5 months ago)

I think LC stands for Leper Choad. Which are often found to be horribly infected.


--------------------


Post Extras: Print Post  Remind Me! Notify Moderator
Jump to top. Pages: 1 | 2 | 3 | 4 | 5 | Next >  [ show all ]

Amazon Shop for: ½ Pint Jars, Agar, Magnetic Stirrer, Malt Extract, Paul Stamets, Pressure Cooker, Scales

Mushrooms, Mycology and Psychedelics >> Mushroom Cultivation >> Mushroom Cultivation Archive >> Agar and Liquid Culture

Similar ThreadsPosterViewsRepliesLast post
* Grocery store Agar Tek. Helltick 19,509 4 03/15/07 11:26 AM
by Terillius
* Super easy karo tek
( 1 2 3 4 all )
angryjslice 43,833 74 03/09/07 12:52 PM
by figgusfiddus
* G.Teacher clone on agar AnnoA 12,641 13 07/18/01 12:53 AM
by Anno
* Orissa India clone on agar AnnoA 8,712 7 08/29/01 03:02 PM
by dimitri211
* Peroxide Agar Tek (DD) Anonymous 11,131 2 11/23/01 05:51 AM
by sylo
* Agar sandwich isolation TEK... ATWAR 9,231 2 07/16/04 02:25 AM
by ATWAR
* :LIQUID CULTURE - THE EASY WAY:
( 1 2 3 4 5 6 all )
agar 90,093 104 11/02/08 10:29 AM
by Anno
* Agar for the old or finicky Suntzu 6,773 4 09/12/03 08:19 AM
by anotherdirtywop

Extra information
You cannot start new topics / You cannot reply to topics
HTML is disabled / BBCode is enabled
Moderator: Ythan, Anno, Thor, Link, Seuss, geokills, Asante, Citric, FooMan, Javadog
113,531 topic views. 0 members, 1 guests and 0 web crawlers are browsing this forum.
[ Toggle Favorite | Print Topic | Stats ]
Search this thread:
Lil Shop Of Spores
Please support our sponsors.

Copyright 1997-2014 Mind Media. Some rights reserved.

Generated in 0.04 seconds spending 0.002 seconds on 41 queries.