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Quantus
The Eternal


Registered: 03/06/12
Posts: 8
Last seen: 10 years, 1 month
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LC, Agar and G2G method ?
#19195611 - 11/27/13 09:39 AM (10 years, 2 months ago) |
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Alright, I have been doing a butt load of reading on the forum and I have a list of questions and a rough draft for my procedure. Your input is extremely valued!
Firstly this all takes place in a room thats 10'x8' walls floors and ceilings wiped down with alcohol and bleech. There is then a true hepa air purifier 99.97% that has to run until the air in the room has been exchanged 4+ times. I turn it off and wait a few hours before I do any work.
The only tek I have ever attempted was the BRF PF tek and I did it with a lot of success.
I want to expand my mycological studies to work towards mastery. So I am attempting LC, agar and G2G.
LC Procedure
After lots of reading I still haven’t concluded which style of lid I want to use for my LC, if I want to use cybers self healing injection port lids, or CSHIPL with a tyvek piece so that when you withdraw LC you don't get a negative pressure in the jar and you also allow for some air exchange during the LC's colonization.
I'm probably going to try both unless someone recommends something better or to use one of those.I have been leaning towards MEA LC as well unless someone recommends that I try different. So I am going to 3 & 3 of those LC jars per strain and as of right now I have 3 strains so 18 LC jars total.
Agar Procedure
of those 18 LC's they are all going to be tested on No Pours Agar Agar jars. In addition to this I am also making 2 agar cultures stright from a MS syringe per strain for a total of 24 (6 MS agar jars and 18 LC test jars)
Grain Procedure
All the LC and Agar aside I am inoculating 2 rye grain jars per syringe per strain for a total of 6. I am going to inject X amount of rye grain jars with the LC that was proven on the agar to show no contams, as well as mycilium from the agar that's contam free.
Now all of this takes place in a glove box in the room that was described above. My questions are as follows;
1. Is it wise to inject this many different mediums with a single syringe even though I am using as sterile as a procedure that I can use (i.e. Glove box flame sterilization between innocs) Is there any extra measures that I can take or that you would recommend ?
2. What is the best method of incorporating this procedure into the glove box ? Do I put all the materials into the glove box including the flamed syringe spray it down with 70% iso and some oust then repeat for every innoc.
3. Given my situation with the room and glovebox do you see a method that you would think would work the best contam wise ? I know LC's are often riddled with contams. It's attractive to me because of the physical handling of LC is much less stressful than G2G and Agar.
4. Can you recommend any resources I should read prior to doing this ? Or see where appropriate resolutions can be implemented to improve my efficiency ?
Although this has the potential to create exponential yield I am only trying to get a grasp on things before Christmas. I'm not actually going to inoculate any substrate to fruit at least not until after Christmas.
Thanks for your time guys and gals!
Edited by Quantus (11/27/13 09:41 AM)
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blindingleaf
blue collar underworld



Registered: 07/19/13
Posts: 22,008
Loc: sub-surface unseen
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Re: LC, Agar and G2G method ? [Re: Quantus]
#19196256 - 11/27/13 12:31 PM (10 years, 2 months ago) |
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if u are just trying to get a grasp on things, forget the lc. just go MS> agar > grain to complete a grow. also, when u initially noc up ur petris/agar make up 5 or 6, so u can have some to practice transferring and isolating while ur grains grow. that way u get experience with agar, grains, sectoring, and the grow cycle. so u noc up 5 petris. they all show growth, no contam. transfer thickest, ropiest edges (rhyzomorphic) growth from each to new plates. now u have maybe 10-15 new plates (or less, maybe 4 more if u have limited resources) one of those (after it grows out a bit and shows no contam) use to noc up a grain jar, and the rest, watch them sector and keep transferring healthy growth till u get some isolates (which may or may not have the qualities u look for...) then u can test each iso out with BRF tek or whichever u choose.
or u can go MS to grain, fruit it and clone fresh, and repeat above process.
forget LC, its not faster than G2G. it may be faster than an agar wedge, but not by much.
-------------------- A few thoughts on cultivation MICROBIAL HUSBANDRY!!!! The whole is greater than the sum of its parts
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cronicr



Registered: 08/07/11
Posts: 61,436
Loc: Van Isle
Last seen: 2 years, 8 days
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Re: LC, Agar and G2G method ? [Re: blindingleaf] 1
#19196494 - 11/27/13 01:19 PM (10 years, 2 months ago) |
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--------------------
  It doesn't matter what i think of you...all that matters is clean spawn I'm tired do me a favor
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Quantus
The Eternal


Registered: 03/06/12
Posts: 8
Last seen: 10 years, 1 month
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Alright im going to scrap the LC idea and go with agar. Now I have a few questions about it, an isolate is taking the most outer rhizomorphic mycilium over and over from your petri dishes until you have a uniform mycillium ? also is there any relationship between lesser nutrients and more rhizomorphic mycillium ? Given my set up do you think I should use something other than just petri dishes cause wouldn't I be exposing them to an awful lot of air and possible contams even if its in a glovebox ? and I have to splice and transfer quite a bit ? and my final question how would one noc up a BRF cake using agar, would you have to make an LC first ?
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