Caps' SAGARI Method (Soft Agar Innoculant)

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Caps McGee
Grandaddy Smurfshack

Registered: 10/28/17
Posts: 14,366
Loc: ally known as ...

Caps' SAGARI Method (Soft Agar Innoculant)

15
#25517047 - 10/06/18 12:00 PM (7 years, 11 months ago)

Due to my reluctance to use LI/LC, I've devised a way to gain the benefits of both agar, and liquids... By mixing the agar soft, and pouring one 60mm dish for each intended jar of grain, or splitting 100mm dishes between 2 jars, I've seen 100% colonization in as little as 4 days (5-7 is common)with a good, clean culture, and properly prepared grain...

Here's what I do:

Agar recipe:
I use 3.5-5g of agar, and 6.5g of powdered LME to 500ml of water... I've also started adding different colors to each prep to avoid dating plates (not doing this anymore as I find it more difficult to spot contaminants)... the nutrients aren't important, so this will work for MEA, PDA, DFA, and GSA: the important thing to get right is the agar powder, as this will result in a less gelatinous, or "softer" agar.

procedure:
Mix ingredients in a vented glass vessel of your choosing: I've got a graduated media bottle with a 1/4" hole drilled in the cap, but I've seen liquor bottles used... I put the dry ingredients in, then about 120ml cold tap water, quickly spinning it to prevent the malt from clumping, then filling it to 500ml... The bottle is covered in tyvek (cut squares from free envelopes from the post office) before screwing cap on. I like to use hot water for MEA bc it seems to dissolve the LME faster, and leaves less "sediment" in the final product.

Pressure cook the elevated agar (I use a small pot lid, a jar ring and tinfoil to hold the bottle in place (pc is too short)lol)at 15psi for 40 minutes , only filling the water about a 1/3 up the bottle, and then I go burn one... :pipesmoke:

I let it sit in the PC for an hour or so after removing from heat, remove it with gloved hands, and set it next to my SAB to keep check on every 10-15 minutes, and then I go burn one... :jah:

It's usually about an hour longer before it's cool enough to grab bare-handed, but still warm... i wipe the bottle down superficially before going into the SAB(not wiping with iso anymore, alternatively, I've been wrapping the cap down over the lip and onto the glass in foil prior to PC cycle and taking it directly into SAB from PC (after 1hr partial cool down)... it takes a little longer to cool, but I'm pretty sure that the heat trapped in the SAB is directly related to a dramatic decrease in condensation), load the agar and dishes into the SAB, and then I go burn one... :smoker:

by this time, I'm thoroughly stoned and ready to pour some plates! I wipe down the agar vessel once again, being particularly attentive to the bottom rim of the lid, and section of the bottle directly below it...(again, no longer using iso, but I'm leaving the original description bc it worked as well, and some people don't like using foil: alternatively, I reuse foil when I can, and view wiping alcohol as more a vector for the introduction of contaminant spores (i.e. wet paper towel, floating spores) as alcohol does not kill spores...

After removing the cap, I cut open my sleeve of trays with my scalpel, enough to get a handful(5-6 dishes),and tear it further back as I need more dishes... after placing the first stack of plates on my elevated work surface (i use an empty wide mouth 1/2 pint jar, or 3 old dishes wrapped together)I then remove the tyvek, basically peeling it off from the side, as to not place my hands over the mouth of the bottle. I usually get 19-21 regular 100mm plates, or 27-30 60mm plates from 500ml prepared agar... while the plates cool and set up, I go burn one... :blazed:

2 hours is usually ample time before use, though I typically pour plates the day before I intend to use them... I leave them unwrapped, but have been covering the armholes of the SAB with a 6" wide roll of glad wrap left over from cutting 1" rolls for dishwrap) I transfer wedges from a clean culture to as many 60mm dishes(or half as many 100mm dishes) of soft agar as jars I plan to use... after these grow out, I hold the petri sideways and make 3 horizontal, and 3 vertical cuts, making 16 "squares", turn my scalpel 90° and use it to flip the wedges from the plate into the jar... once the SAB session is over and all jar lids are placed back onto the jars, I remove the SAB and shake the living FUCK out of them, in an attempt to liquify and spread the culture evenly across all grains, and then I go burn one... :joint:

The theory behind it all:

Contamination is notoriously difficult to spot in liquids, and any additional use presents additional vectors: but liquids are fast because they coat each grain with culture... agar is popular due to the ease of contamination identification, on a 2D surface, which can keep desirable and undesirable cultures separate, long enough to transfer to obtain a clean culture... My SAGARI (though I'm sure I'm just the first to write it up) takes the advantages of both, in that it is faster than standard A2G, and better avoids the risk of unseen contamination than LI/LC. Give it a whirl and let us know what you think! And I'm gonna go burn one... :gethigh:

Results

day 4, before second shake:

40 hours after shake (day 6 since SAGARI drops):

Edited by Caps McGee (05/25/19 08:17 AM)

Extras:

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