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Uhurungus
Avoiderer of Bullshit


Registered: 04/20/13
Posts: 838
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A2G vs. G2G (attn: FrankH)
#18626034 - 07/28/13 09:43 PM (10 years, 6 months ago) |
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edit: for clarity sake, i'm interested in all input, not just from Frank.
been a busy few days but i wanted to continue this from a thread a few days ago where we were discussing agar. i would have taken this straight to PM but i figured some people might benefit from it.
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FrankHorrigan said:
Quote:
Uhurungus said: i always keep regulars on hand though too cause i innoc 1 full tub from a dish.
I don't follow you 
I use 3-section dishes, each section can be used for 1-2 master jars comfortably...grain to grain that into 6-8 quarts and you've got yourself 3-6 tubs per dish 
If you're doing agar, you should certainly do grain to grain as well.
so i've been working over the last 3 months to get some isolates. still not 100% convinced that what i have now are true isolates but... i have 5 or 6 full size dishes of 5th and 6th gen. vigorous agar transfers of various varieties. ex: 5 x 6th gen PE, 5 x 6th gen cambo, 5 x 6th gen PESH etc...
my train of thought was to test these "iso's" in tubs. i work in a SAB. i've been taking a full dish with what i consider 1 iso, and innoculating 6 quarts of rye spawn with wedges from the same dish. i rewrap the dishes after innocing and put in fridge well labeled, leaving enough in the dish so i can make a master slant if the mono preforms well.
one benefit to doing this as far as i can tell is quicker colonization time. i dont have to wait for a master jar to colonize then wait again for my grain transfers to colonize before i spawn to bulk.
i possibly see 2 errors in this way of doing things:
the first is negligible, in that i'm using more agar to inoculate this way than i would if i made 1 master jar then G2G?
the second is that the physical act of doing G2G in a SAB is less cumbersome and quicker than A2G, thus possibly lowering chances of contam?
i just found a contam in my most current mono. that makes 4/4 contams for me. it's frustrating but i want to get this figured out and i'm starting to think my spawn is partly to blame.
why is agar 2 master 2 grain better than agar 2 grain. i'm looking for input on where the holes are in what i'm doing. where can i improve.
-------------------- I'm never 100% on anything, just close enough to risk looking foolish.
Edited by Uhurungus (07/28/13 09:52 PM)
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MonkeyJesusFresco
am i suspended in agar?



Registered: 10/09/12
Posts: 3,306
Loc: South East USA
Last seen: 1 day, 12 hours
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Re: A2G vs. G2G (attn: FrankH) [Re: Uhurungus]
#18626191 - 07/28/13 10:17 PM (10 years, 6 months ago) |
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There's a thousand ways to skin a cat :/ and everybody skins cats slightly differntly...
For example, one method that has shown success for "high-failure-rate" people is
1) multispore 2) clone best fruits, making 4-5 agar slants from the tissue of one fruit (that way, hopefully at LEAST ONE will not contam  and if you're lucky you now have multiple master backup culture slants) 3) agar-slant to master grain jar (ah see here, with multiple master agar slants/petris, you CAN inoculate 7 jars, spawn six and now you have a master grain jar to use on your next grow 
plus, if you portion how much spawn from a master grain jar you use, say 2 tblspoons to each jar, you can make one grain jar into MANY...
Quote:
i just found a contam in my most current mono. that makes 4/4 contams for me. it's frustrating but i want to get this figured out and i'm starting to think my spawn is partly to blame.
What makes you think it's not the substrate you're spawning to?
-------------------- LAGM v 2.024 - endo cabendo
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Uhurungus
Avoiderer of Bullshit


Registered: 04/20/13
Posts: 838
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hunch more than anything. it could be the sub. but i was pretty anal during the last pasteurization i did. although... it might have picked up contam from the quart jars i used. i never sterilized them in PC after last tubs and some of them were used for grain i'm sure.
so many reasons why it could have gone wrong. i need to buckle down. need to get into a habit of cleaning monos and jars better before using them.
-------------------- I'm never 100% on anything, just close enough to risk looking foolish.
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Psilicon
Really Nice Guy


Registered: 08/26/12
Posts: 7,057
Last seen: 3 years, 1 month
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Re: A2G vs. G2G (attn: FrankH) [Re: Uhurungus]
#18626568 - 07/28/13 11:34 PM (10 years, 6 months ago) |
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Have you tried shaking your spawn jars and seeing if they recover?
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Uhurungus
Avoiderer of Bullshit


Registered: 04/20/13
Posts: 838
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Re: A2G vs. G2G (attn: FrankH) [Re: Psilicon]
#18627596 - 07/29/13 08:08 AM (10 years, 6 months ago) |
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i haven't in the past but am going to start
-------------------- I'm never 100% on anything, just close enough to risk looking foolish.
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FrankHorrigan
The Inquisition



Registered: 01/04/11
Posts: 10,573
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Re: A2G vs. G2G (attn: FrankH) [Re: Uhurungus]
#18627673 - 07/29/13 08:30 AM (10 years, 6 months ago) |
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It sounds like you should definitely shake your spawn at 100% and see if it recovers properly. Give it about 48 hours post-shake. If it stalls out anywhere in the jar, you should not use that jar.
I don't know about you but when I inoculate my masters, I remove the lid from my petri, set it down, and make all my cuts after one flame sterilization of my blade.
I then inoculate each jar with one wedge each. I stick the middle of the wedge on my scalpel, move it carefully to my jar while removing the jar's lid with my other hand. I knock it into the jar and close the lid as quickly as possible.
I don't like leaving the petri dish open while moving around to inoculate each jar but it is unavoidable. I certainly would not trust setting down the lid and placing it back on the dish between inoculations either. It is very easy to picture tiny spores or bacteria working its way on to the culture during this time.
So agar inoculations can be a pain in the ass, and I've always had very high success rates doing G2G in my SAB. G2G is simple- hold master jar in one hand, manipulate the lids of the receiving jars with the other.
I live in a high risk environment and have to be very precise with my clean work or everything goes to shit 
So grain to grain is the safest bet for expanding my mycelium. With the right amount of inoculant those G2G jars will colonize in a week too.
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Uhurungus
Avoiderer of Bullshit


Registered: 04/20/13
Posts: 838
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i do the same with my dishes but mine are exposed much longer than yours. 6-8 jars instead of 2. and i've been putting 2 smaller wedges in each jar instead of 1 large one.
looking back, there are a few loose ends i can tie up with a little more attention and effort.
it's no big deal to throw away a bad mono. $5-10 in material and a few weeks, but a small part of me dies each time i've had to do it.
-------------------- I'm never 100% on anything, just close enough to risk looking foolish.
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FrankHorrigan
The Inquisition



Registered: 01/04/11
Posts: 10,573
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Re: A2G vs. G2G (attn: FrankH) [Re: Uhurungus]
#18627892 - 07/29/13 09:36 AM (10 years, 6 months ago) |
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Try minimal cuts, minimal master jars. G2G is your best friend.
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Uhurungus
Avoiderer of Bullshit


Registered: 04/20/13
Posts: 838
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after the final shake and recovery, is there a need to break the grains up again before spawning or is that too much trauma?
-------------------- I'm never 100% on anything, just close enough to risk looking foolish.
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FrankHorrigan
The Inquisition



Registered: 01/04/11
Posts: 10,573
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Re: A2G vs. G2G (attn: FrankH) [Re: Uhurungus]
#18627960 - 07/29/13 09:52 AM (10 years, 6 months ago) |
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You will need to shake it again. And give it a drink too 
I don't buy into that "too much trauma" crap.
I've shaken some jars three, four, five times before full colonization...not to mention the "trauma" inflicted by successive G2Gs.
I've never seen problems with performance due to shaking too many times.
I think a lot of people just had bacterial contams and misidentified the cause as shaking too many times.
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Uhurungus
Avoiderer of Bullshit


Registered: 04/20/13
Posts: 838
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word
-------------------- I'm never 100% on anything, just close enough to risk looking foolish.
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