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shroombab
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Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth 1
#12124877 - 03/01/10 11:25 PM (14 years, 2 months ago) |
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Hello lovely helpful friends
I have spent a long time searching google, this forum, and even studying Paul Stamets book, and given the vast range of information, I'm still having a hard time confirming if I understand agar / LC right, and choosing the best course of action.
So I would greatly appreciate anyone's feedback / very simple explanation of isolation vs. cloning and if what I'm planning to do is worthwhile or negated by any other part etc. I am only able to try this out for a specific amount of time, and can't afford to waste time making careless mistakes. I am going to do quite a few different things all side by side to record the results, and be able to see for my own personal system what works best and what I like the best.
So thanks!
1. inoculate grain jars with spore syringes, just to get some jars going for this grow and compare results in speed / flush (is this even worth comparing to agar / lc? am I wasting my spores?)
2. Make a bunch dextrose/malt agar dishes, and inoculate with 1 drop from syringes.
3. Cut strongest mycellium from dishes and repeat process until no "sectoring" occurs (if I understand correctly, "sectoring" means seeing more than 1 growth point??) and how many repetitions of agar to agar does this commonly take? And commonly how long for each repetition?
4. Now I should have a bunch of strong agar "cultures?" or "isolates" (same thing?) For each "strain" that I used to make more dishes, I should have several dishes each with strong single isolates from each individual starting strain. Those are "substrains" of that initial strain? Seeing as I haven't grown any yet, I can only speculate that if it looks like strong mycellium it will fruit well (in theory?)
5. I take my dishses and do the following: a) use wedges to inoculate grain jars b) use wedges to inoculate LC (for cloning that substrain?) c) take one? wedge for each substrain and put it in a test tube with a lid on and refrigerate for use in the future? (and if im correct, all agar is done at room temp in ambient light, however "slants" are refrigerated and can be kept for a very long time?)
6. Meanwhile do some g2g from the first spore syrings to grain jar set, to test side by side with other methods (again, is this even worth doing alongside an agar /lc tek?) and go to my bulk sub with the rest of the jars.
7. Take fully colonized jars that were inoculated with agar wedges, and do some g2g transfers, and go to bulk sub with the rest.
8. Take some LC and use to inoculate some grain jars. and if I'm brave enough and not overwhelmed at this point, use some to inoculate some bulk substrate (if I understand right, I should be able to go straight from LC to my bulk that I normally use, possibly with some added grain, and skip the jar step)
side note: I was also planning to try out several different good recipes I have for bulk subs side by side as well.
9. Finally, I plan to take spore prints, to save along with my slants, but what might I do with the flesh if I wanted to try starting from that route to? Can you "isolate" a better strain from the flesh of a good fruit, or better with spores on agar? Are flesh on agar / spores on agar producing similar results because they are both multispore?
10. Is it worth trying out some spore to LC? or is that what people refer to as why LC is a gamble? Can I ensure a strong "culture?" by going agar isolate to LC? So in other words I guess cloning the isolate?
11. When people refer to creating a contaminant free agar isolate what does that mean exactly? Because surely it can get contaminated in the process of transferring the agar to whatever the next media is - or during the colonizing of the bulk sub, or in fact any part of the process? Does this mean that some syringes are already contaminated? OR does this mean that some spores in the syringes are contaminated? Does this effect the possibility of getting contaminated during fresh air (in glove box) exposure during inoculation of grain or LC? In other words, are you more likely to NOT get contaminated at other stages in your grow, and is it related to this at all?
12. Lastly, I might have got some of my jargon words wrong (and as many people who post annoyingly do which throws me off when researching) correct anything that is wrong above, and please add - is germination just when mycellia strart to grow on media? what is "culture" referring to exactly (I don't mean LC).
Thank you so much. I appreciate the help. And I love you all. : )
Edited by shroombab (03/01/10 11:30 PM)
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teesionbear


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Re: Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth [Re: shroombab] 1
#12124999 - 03/01/10 11:44 PM (14 years, 2 months ago) |
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Whew that was a long post.
It seems your end result is to do bulk with a good isolate. Your plan could work but you would have no surefire way of knowing if you have a good fruiting isolate if you went spores-->agar.
I would highly suggest just going multispore solution to grain and complete a grow. Then you can clone for the best clusters or biggest fruit or whatever you want and have an isolate that you can use for bulk, bypassing the sectoring on agar.
Afterwards, I would maybe get several more clones from your first clone isolate grow and put them on agar. select from them the quickest to colonize.
Thats a way to get isolates that probably favor the good genes in your multispore quickly. you are not gonna be creating any new strains this way though.
Cakes are especially good for testing your isolates/strains. You can make a couple batches of jars and be able to grow all your isos out without having bother with making tons of trays.
have fun!
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Re: Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth [Re: shroombab]
#12125041 - 03/01/10 11:50 PM (14 years, 2 months ago) |
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im not advanced at all. you are very well educated. bravo to you miss. all of your stuff sounds good, as long as you get fruits, i believe there is no wasted spores. ill explain what i am starting to slowly do on my minimal budget. if i am correct, when you isolate your agar to have 1 sector, you have that "strain" but you do not know if that strain is a mutant strain or all short fruits or maybe its a strain that is inactive, since youre starting from MS. i would shoot MS into some grain jars, fruit most of them, keep some for myc if need be, then once you test some fruits and make sure they are active, you pick what traits you want, maybe you want large fruits, or small fruits, its up to you, this is when you take some tissue ( i found the idea of putting it in a fresh ziplock baggie for a few days with the air pushed out to work well. the fruit flesh will start to grow fuzzy myc) then you put it on agar. this way not only do you know they are active, but it narrows your strains down from millions of possibilities, to maybe a few, idk how many can actually be present to produce fruit but ive read there can be more than one. once it grows on agar, this is where i would start to look at sectors. im thinking there should only be one or two since MS only forms a few from what ive seen. so you get your best sector and grow it out on a few dishes to get it nice and strong and alone. then use it to start a dish and then use another part in lc and do some on grain. fruit the grain to see what you have "created" then you can use your lc (once you know its clean after a few test jars) to inoc more lc. you can store it that way, do a few grain jars, then g2g and it seems like you want this for awhile so you can make your master slant and keep it for quite some time. ive read that after awhile you dont wanna use the same "iso thingy" for awhile, im sure you could use this for 6 months atleast, but maybe around then youre gonna wanna make some agar then use some tissue from a recent batch to start that process over: fruit tissue-agar-sector(s)-sector-master slant-lc-lc-lc-grain x1000- fruit x _00 lbs
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Re: Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth [Re: oh_you_know]
#12125053 - 03/01/10 11:52 PM (14 years, 2 months ago) |
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teesionbear said basically what i said, only alot shorter haha
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shroombab
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Re: Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth [Re: teesionbear]
#12125065 - 03/01/10 11:54 PM (14 years, 2 months ago) |
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Thanks
So multispore solution refers to any spore syringe or spore print scraping etc. (of course solution means in liquid)
If a spore print from a single fruit has multiple strains, ie. multispore? then how can you clone from a fruit?
What you're saying is, go spores > jars > bulk > fruit and then take a fruit and then take a flesh sample from one good fruit and go straight to agar, which immediately makes a strong isolate?
Again, I guess I'm not understanding the meaning of "sectoring" exactly, I don't understand how you can skip this step by using a fruit?
Thanks again
edit: oh you know sort of answered the question about isolating from flesh. However, RR does say that he starts agar from spore syringes, I guess that's slower and less accurate than isolating from the best fruits? Because you're not seeing the end result until you fruit, and then lots of other factors come into play.
Do you think its worth doing some g2g with my MS to grain jars? Or waiting until I have some strong isolates? Can people get strong pinsets just from MS to grain? I've not had that happen yet but I haven't used nearly enough grain to sub in the past.
Edited by shroombab (03/02/10 12:04 AM)
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Re: Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth [Re: shroombab]
#12125117 - 03/02/10 12:06 AM (14 years, 2 months ago) |
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you would use the inside of the stem of the fruit. youre not skipping the step of "sectoring". starting agar from a fruit just skips alot of the chances of random strains and sectors. i assume youve seen this video. you can see theres lots of sectors so taking fruit tissue will help slim down how many sectors you have. cutting down time, which you have little of, getting you to the end result you want faster.
it doesnt immediately make a strong isolate. it immediately gives you a few strong possible isolates. then you sector and get your strong, single sector, single strain iso.
just keep asking questions as they come, ill help the best i can, being a noob and never doing this, just knowing this from reading makes me feel pretty good!
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teesionbear


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Re: Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth [Re: shroombab] 1
#12125166 - 03/02/10 12:17 AM (14 years, 2 months ago) |
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Quote:
shroombab said: Thanks
So multispore solution refers to any spore syringe or spore print scraping etc. (of course solution means in liquid)
Yes
Quote:
If a spore print from a single fruit has multiple strains, ie. multispore? then how can you clone from a fruit?
Directly from the tissue.
Quote:
What you're saying is, go spores > jars > bulk > fruit and then take a fruit and then take a flesh sample from one good fruit and go straight to agar, which immediately makes a strong isolate?
I'd go spores>pf cakes>tissue clone>agar or spores>grain jars>bulk>take several prints and tissue samples.
Use a blade and get an inner part of the stem near the bottom, size of a grain of rice.
Quote:
Again, I guess I'm not understanding the meaning of "sectoring" exactly, I don't understand how you can skip this step by using a fruit? Thanks again
You seem to understand it well enough, the mycelium groups form colonies. pick from a strong growing colony or pick the most rhizomatic and take a small piece to a new dish. it should bring your different mycelium groups down to a much more manageable level. you keep transferring until your dish is one huge colony. then you can cut sections of that dish and transfer to grain or even toss it in an LC.
Quote:
edit: oh you know sort of answered the question about isolating from flesh. However, RR does say that he starts agar from spore syringes, I guess that's slower and less accurate than isolating from the best fruits?
I believe he also fruits every isolate afterwards and continues on with the prolific fruiters. His way would enable him to have a greater range of genetics represented in his petri dishes. I would imagine several cloned fruits from the same try would have less genetic diversity than several dishes grown out from random spores.
But that would depend on how many different mycelium strains grow out in an individual jar, and how many would have fruit representatives in the final substrate.
Cloning from fruit is simpler because IME half of the clones are immediately isolated and other half require only 1 more dish to get an iso.
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Re: Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth [Re: oh_you_know]
#12125175 - 03/02/10 12:19 AM (14 years, 2 months ago) |
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Quote:
oh_you_know said:
this time i was slower!
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shroombab
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Re: Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth [Re: oh_you_know]
#12125223 - 03/02/10 12:28 AM (14 years, 2 months ago) |
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Thanks a lot everyone, this is really making it more clear for me. Sometimes without actually doing it, its hard to grasp.
I'm going to do some MS to grain to g2g to bulk anyways but alongside I will now do as suggested: spores to cakes to tissue clone (from cakes or bulk) to agar with which I will go to; LC and grain, and grain to bulk, LC to bulk whilst continuing to isolate again if I have the time.
Now when people refer to master grain? or master culture? that means where they have an isolate in 1 grain jar which they use to to g2g? and master culture means having an isolate on agar? or an isolate in LC?
I probably won't go this far this time, but here's where it gets really muddy for me - if I take a slant from a strong agar isolate and save it, how many times can I use that over and over (by doing agar to agar to LC to grain etc.) surely there is a point at which it stops? or doesn't work anymore? What is the best way to sustain an isolate for use over and over again, negating the need to buy more spores of that strain.
Thanks so much everyone is so awesomely helpful!!!
EDIT: Also this has always confused me: why if you buy a spore syringe of print of one particular "strain" does it actually have thousands of strains? how can a strain ever be a strain if each mushroom produces thousands of different strains? How can people name strains and sell them to you as those strains if they are created from prints and not isolates?
Edited by shroombab (03/02/10 12:33 AM)
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teesionbear


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Re: Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth [Re: shroombab] 1
#12125288 - 03/02/10 12:47 AM (14 years, 2 months ago) |
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Quote:
shroombab said:
Now when people refer to master grain? or master culture? that means where they have an isolate in 1 grain jar which they use to to g2g? and master culture means having an isolate on agar? or an isolate in LC?
Yes. Master grain is the jar of isolate to be G2G transferred to other jars.
Quote:
I probably won't go this far this time, but here's where it gets really muddy for me - if I take a slant from a strong agar isolate and save it, how many times can I use that over and over (by doing agar to agar to LC to grain etc.) surely there is a point at which it stops? or doesn't work anymore? What is the best way to sustain an isolate for use over and over again, negating the need to buy more spores of that strain.
Thanks so much everyone is so awesomely helpful!!!
If you have a master agar culture, on a plate or in LC, I believe you can use that to make new grain jars or new LCs indefinitely. Well, until the mycelium dies anyways.
Its when you go from grain1>grain2>grain3>grain4 that you run into problems with mycelium vitality.
Quote:
EDIT: Also this has always confused me: why if you buy a spore syringe of print of one particular "strain" does it actually have thousands of strains? how can a strain ever be a strain if each mushroom produces thousands of different strains? How can people name strains and sell them to you as those strains if they are created from prints and not isolates?
I like to think of Strains (GT, PE, amazon, etc) as kind of like a race. they tend to have certain characteristics, but not all shrooms will possess those characteristics, and some will possess characteristics that are uncharacteristic of the strain.
When you buy multispore PE, theres no guarantee all your shrooms will look like PE. But theres a better chance they will than if you grew B+ or something.
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Re: Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth [Re: shroombab]
#12125296 - 03/02/10 12:50 AM (14 years, 2 months ago) |
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Quote:
I'm going to do some MS to grain to g2g to bulk anyways but alongside I will now do as suggested: spores to cakes to tissue clone (from cakes or bulk) to agar with which I will go to; LC and grain, and grain to bulk, LC to bulk whilst continuing to isolate again if I have the time.
looks good to me
Quote:
Now when people refer to master grain? or master culture? that means where they have an isolate in 1 grain jar which they use to to g2g? and master culture means having an isolate on agar? or an isolate in LC?
agar master slant i would think they would have a master grain jar and refrigerate it. to slow down cell division. watch the video and also read it cause that page is very informative on the master slate and storage.
Quote:
I probably won't go this far this time, but here's where it gets really muddy for me - if I take a slant from a strong agar isolate and save it, how many times can I use that over and over (by doing agar to agar to LC to grain etc.) surely there is a point at which it stops? or doesn't work anymore? What is the best way to sustain an isolate for use over and over again, negating the need to buy more spores of that strain
from reading the master slant page, i understand you can use it over and over. it says it can store for up to 2 years so it just depends how often you use it within 2 years. the best way i would say is to play it safe and after 6 months, start a new master slant from the best fruit you want from your iso, so you keep the iso going, but you keep improving it.
and to your edit- i read and understand that the "strain" company's sell have very similar characteristics. they are not isos but if you were to iso them they would clearly be different. i guess they dont really sell strains, they sell spores of a fruit that throws off fruits that look the same and can be grouped together, until you iso them. i hope that makes sense, thats harder to put in words.
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Re: Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth [Re: oh_you_know]
#12125309 - 03/02/10 12:53 AM (14 years, 2 months ago) |
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too slow this time!
Quote:
I like to think of Strains (GT, PE, amazon, etc) as kind of like a race. they tend to have certain characteristics, but not all shrooms will possess those characteristics, and some will possess characteristics that are uncharacteristic of the strain.
When you buy multispore PE, theres no guarantee all your shrooms will look like PE. But theres a better chance they will than if you grew B+ or something.
you definitely put that in words better than i did! same idea.
my first flush of PE didnt look like PE but the second flush looked alot more like PE. a few of them grew big like they were supposed to and looked like PE but one or two of them grew fat like PE and had the cap but the cap started to open more and had a nipple on top like some other "strains"
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shroombab
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Re: Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth [Re: oh_you_know]
#12125352 - 03/02/10 01:06 AM (14 years, 2 months ago) |
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Those are two very good explanations about the strains. Thank you.
I just read up on "Grain LC" and finally after finding a tek by agar, I friggin understand what that is. Seems to be to be a much better method than sugar based LC, especially as I don't have a flow hood, and anything I can do between injection ports is better.
Now if I made a Grain LC from 1 grain jar colonized from a MS inoculation, I could guarantee better chance of it knocking up more contam free jars or bulk sub than a sugar LC where contams would be impossible to detect until using it. BUT, because I made it from an MS grain jar, im still going to get a multitude of different fruits from all that LC. Its still healthy mycellia but the fruits will vary, like multiple couples having many children who are all unique.
Whereas: if I do my clone > isolate agar > grain jar > Grain LC, (which would be the same as sugar LC but better chance of being contam free) then I'm multiplying the mycellia from a single cloned "strain" (as defined by RR) and can knock up an exponential amount of jars or bulk sub with this? and therefore THIS method or simply doing agar to grain would be the best way to eventually grow the best fruits in the most economical sustainable way?
I can't wait to actually try all this and post it in a grow log.
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shroombab
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Re: Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth [Re: shroombab]
#12125361 - 03/02/10 01:08 AM (14 years, 2 months ago) |
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p.s. I can't say that I don't like cocks : ) But PE REALLY freaks me out.
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teesionbear


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Re: Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth [Re: oh_you_know]
#12125377 - 03/02/10 01:12 AM (14 years, 2 months ago) |
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Quote:
oh_you_know said: too slow this time!
Quote:
I like to think of Strains (GT, PE, amazon, etc) as kind of like a race. they tend to have certain characteristics, but not all shrooms will possess those characteristics, and some will possess characteristics that are uncharacteristic of the strain.
When you buy multispore PE, theres no guarantee all your shrooms will look like PE. But theres a better chance they will than if you grew B+ or something.
you definitely put that in words better than i did! same idea.
my first flush of PE didnt look like PE but the second flush looked alot more like PE. a few of them grew big like they were supposed to and looked like PE but one or two of them grew fat like PE and had the cap but the cap started to open more and had a nipple on top like some other "strains"
afoaf just inoculated his first PE cakes today! he's going to put some spores on agar, to preserve the 'strain', and make a MS lc. he'll save a few ml's of spore solution just in case
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Re: Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth [Re: shroombab]
#12125383 - 03/02/10 01:13 AM (14 years, 2 months ago) |
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PE is stronger IME...and what seems to be the vast majority's experience. definitely be sure to keep a grow log on all this! pics are a must.
Quote:
clone > isolate agar > grain jar > Grain LC
Yes. i did not think of grain lcs because i have not used them, i dont know how much i would trust myself with them. i just have to work "luck" with grain. it makes no sense at all. i dont understand why.
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Re: Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth [Re: teesionbear]
#12125392 - 03/02/10 01:17 AM (14 years, 2 months ago) |
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Quote:
teesionbear said:
Quote:
oh_you_know said: too slow this time!
Quote:
I like to think of Strains (GT, PE, amazon, etc) as kind of like a race. they tend to have certain characteristics, but not all shrooms will possess those characteristics, and some will possess characteristics that are uncharacteristic of the strain.
When you buy multispore PE, theres no guarantee all your shrooms will look like PE. But theres a better chance they will than if you grew B+ or something.
you definitely put that in words better than i did! same idea.
my first flush of PE didnt look like PE but the second flush looked alot more like PE. a few of them grew big like they were supposed to and looked like PE but one or two of them grew fat like PE and had the cap but the cap started to open more and had a nipple on top like some other "strains"
afoaf just inoculated his first PE cakes today! he's going to put some spores on agar, to preserve the 'strain', and make a MS lc. he'll save a few ml's of spore solution just in case 
cakes may not be the best. technically you could crumble and spawn to bulk but i believe PE does alot better as trays or bulk. cakes just dont do it. one of those mysteries. and def keep a few cc's just in case! i think youll enjoy PE. idk why someone wouldnt. i lost me PE spores and stuff, im gonna have to order some new syringes once i get some cash.
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Re: Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth [Re: shroombab]
#12125399 - 03/02/10 01:17 AM (14 years, 2 months ago) |
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Quote:
shroombab said: p.s. I can't say that I don't like cocks : ) But PE REALLY freaks me out.
all shrooms look and taste the same once you dry them and powder them in a coffee grinder plus hit alot harder and faster!
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teesionbear


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Re: Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth [Re: shroombab]
#12125400 - 03/02/10 01:18 AM (14 years, 2 months ago) |
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Quote:
shroombab said: Those are two very good explanations about the strains. Thank you.
I just read up on "Grain LC" and finally after finding a tek by agar, I friggin understand what that is. Seems to be to be a much better method than sugar based LC, especially as I don't have a flow hood, and anything I can do between injection ports is better.
Now if I made a Grain LC from 1 grain jar colonized from a MS inoculation, I could guarantee better chance of it knocking up more contam free jars or bulk sub than a sugar LC where contams would be impossible to detect until using it. BUT, because I made it from an MS grain jar, im still going to get a multitude of different fruits from all that LC. Its still healthy mycellia but the fruits will vary, like multiple couples having many children who are all unique.
yup grain lc is safer than sugar. and it will still be multispore.
Quote:
Whereas: if I do my clone > isolate agar > grain jar > Grain LC, (which would be the same as sugar LC but better chance of being contam free) then I'm multiplying the mycellia from a single cloned "strain" (as defined by RR) and can knock up an exponential amount of jars or bulk sub with this? and therefore THIS method or simply doing agar to grain would be the best way to eventually grow the best fruits in the most economical sustainable way?
I can't wait to actually try all this and post it in a grow log.
Thats right as well.
the second way, it takes longer to get your grain LC, but it will be an isolate.
in your first scenario you would have grain LC quickly, which would allow you to inoculate more grain jars and get to bulk quicker, but it would all be multispore.
you're going to end up experimenting with strains from that strain nonstop. its addicting. have fun!
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Last seen: 11 years, 2 months
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Re: Am I getting it right? Understanding Agar to Agar to Grain / LC, isolating, cloning and so forth [Re: teesionbear]
#12125406 - 03/02/10 01:20 AM (14 years, 2 months ago) |
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good luck to both of you. see ya!
-------------------- My Journal
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