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EntheoGod
Entheo



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First agar to agar attempt! :) Colonizing in hot temps(hot temps fixed)😅Advice Appreciated! *with pics and updates over time*
#26816004 - 07/10/20 08:20 PM (3 years, 6 months ago) |
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Just did my first agar to agar transfer here’s the aftermath I do know there was bacteria on this in the spot just ahead of one of the transferred. The seed I used to inoculate the agar was bacterial and I accidentally bounced it around the plate before it settled in the spots you see. I did a few other transfers of a other plates but didnt take pics of those 
Also the room I keep most things in has been getting really high temp, like 90 at the highest and hottest point of the day. So I tossed a cold water soaked towel in an old tub and sat my plates on it. The temp stays about 82-84 in the hottest part of the day. Do you think the plates will colonize okay in this weather and would the colonize grains decently once I get healthy growth in agar, if it stays around that temp or lower? I also have a fan blowing in the room and over the wet towel that the plates are sitting on. I plan on getting a window ac for the room very soon but for a few days would you think this will work to give me time to get an ac (they sure ain’t cheap around this time of year)
I could also put them inside an old fridge that’s off but has an ice pack and wet cool towel inside? Im afraid if I did that, there would be too much condensation to buildup inside the pastyplates. It seems to be a LOT cooler inside the fridge once I place the ice pack and towel in. I checked the temp when it was 90F in the room. Inside the fridge with an ice pack and wet towel its 80F. I have an ac unit coming for the room this coming friday but whats your opinion on temporary storage during colonization?
Heres the fridge with some plates inside. Shroomery tends to reorient my pics even if I choose not to change anything during the uploads, ive tried both ways sorry for the tilted pic.

Also, i will post updates as things progress. I tried posting in the all things agar thread but didnt seem to receive an answer because other interesting things were being discussed in there at the time so I decided to make my own thread that shows the progress of this first timers attempt with agar transfers.
Hope all are doing wonderfully
Edited by EntheoGod (07/31/20 06:28 PM)
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EntheoGod
Entheo



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Re: First agar to agar transfer. :) Need advice on storage during colonization in hot temps. [Re: EntheoGod]
#26819398 - 07/12/20 01:03 PM (3 years, 6 months ago) |
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So I had one contaminate but the others are looking clean af to me any opinions on the transfers. Here’s a pic of the one that molded and an example of what the others look like.

The black dot one is the only one with a contam present so far. I believe one more had bacteria but it wasn’t any in these pics. Still transferring away from the contams to get clean agar. Shouldn’t take more than another two transfers until I have a useable plate to make a master grain jar with. 😅 I believe the other plates pictured are the best growth I have just yet. It’s only been a few days since I’ve transferred them from bacterial plates. I have like 20 plates going atm. Some with spore swabs and some were agar to agar transfers that started with a grain to agar plate. The grains I had colonized were a bit bacterial so I put a couple on agar to clean it up and finally manage to have clean spawn. I gave up making grain jars until I’m sure I can get clean spawn. Tired of wasting my time
Edited by EntheoGod (07/12/20 01:05 PM)
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Trichodingus
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Re: First agar to agar transfer. :) Need advice on storage during colonization in hot temps. [Re: EntheoGod]
#26819576 - 07/12/20 02:31 PM (3 years, 6 months ago) |
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Quote:
EntheoGod said: So I had one contaminate but the others are looking clean af to me any opinions on the transfers. Here’s a pic of the one that molded and an example of what the others look like.

The black dot one is the only one with a contam present so far. I believe one more had bacteria but it wasn’t any in these pics. Still transferring away from the contams to get clean agar. Shouldn’t take more than another two transfers until I have a useable plate to make a master grain jar with. 😅 I believe the other plates pictured are the best growth I have just yet. It’s only been a few days since I’ve transferred them from bacterial plates. I have like 20 plates going atm. Some with spore swabs and some were agar to agar transfers that started with a grain to agar plate. The grains I had colonized were a bit bacterial so I put a couple on agar to clean it up and finally manage to have clean spawn. I gave up making grain jars until I’m sure I can get clean spawn. Tired of wasting my time 
Awesome man! I feel like that's the ah-ha moment with mycology. When you say fuck the shotgun/shortcut/hammer approach and just take your time doing it right the first time.
90 is pretty warm, but the plus side is that because the environment is so conducive to bacteria you're gonna get really obvious feedback on your technique.
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EntheoGod
Entheo



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Re: First agar to agar transfer. :) Need advice on storage during colonization in hot temps. [Re: Trichodingus]
#26819767 - 07/12/20 03:50 PM (3 years, 6 months ago) |
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I just cannot wait until I get the ac delivered that I ordered for the room. Temps will no longer be an issue at that point. Right now I’m trying to manage with what I have. So far I’ve not had many contams at all but only time will tell and I’ll definitely keep this updated as long as this goes. I plan to transfer the clean myc over for a second agar to agar transfer in a day or two when the myc that’s there has had time to grow more and any contams that are there have time to show their ugly little faces. I’m having about an 80% success rate right now with temps ranging from 85 on the cool days to 91 on the hot days. I’m hoping that shows how little contams I might run into when I’m able to actually keep the room a cool 72-75 degrees F.
I’m loving experimenting with agar especially since it only takes a few minutes to prep and within 4 or 5 hours you have fresh agar ready for inoculating or storage. It’s so simple as well. I cannot say enough good things about my switch from purely doing MS syringe to grain and then now only working with agar to get clean spawn. First plates I inoculated with Ms syringe were so bacterial I tossed the whole batch. It got slimy and reeked. I’ll NEVER put a syringe to agar again unless I prep it myself in front of a flow hood, which I don’t have.
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EntheoGod
Entheo



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Re: First agar to agar transfer. :) Need advice on storage during colonization in hot temps. [Re: EntheoGod]
#26826032 - 07/15/20 09:38 PM (3 years, 6 months ago) |
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Still been working on getting the technique down for agar. I have such a difficult time getting the agar wedge onto and off of the exacto knife. -___- . Something seems to always go wrong when I’m doing the transfers or inoculating. I’ll accidentally knock the seed around a few times in the plate by placing the seed in the plate at a weird angle or I’ll accidentally squirt too much solution into the plate somehow. The most recent attempt has been with a spore swab and so far 2 of those have contaminated almost fully with cobweb myc. I’ve accidentally had a lid fall face first onto the contam filled ground of the SAB.
BUT I am learning what not to do each and everytime I fuck up. I feel like the room has also been staying a bit too hot for work of any kind. That’ll be changing this Friday, I’ll have an ac unit for that room and be able to maintain the temp better. I need to practice better sterile technique and also do a few control plates with every batch.
Just an update on how things have been going. I will add pics soon!
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w00tmycelium
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Re: First agar to agar transfer. :) Need advice on storage during colonization in hot temps. [Re: EntheoGod]
#26826160 - 07/16/20 12:11 AM (3 years, 6 months ago) |
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Going to echo this... every transfer I've done I learn how to not fuck it up. You just have to get a feel for it. Get a feel for your scalpel and everything, and how the agar behaves on the tools, and it usually seems to flow from there. Take 10 practice plates and do a bunch of transfers and see from there too (with real cultures!)
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EntheoGod
Entheo



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Re: First agar to agar transfer. :) Need advice on storage during colonization in hot temps. [Re: w00tmycelium]
#26829105 - 07/17/20 02:08 PM (3 years, 6 months ago) |
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I have one plate looking great but it’s not one of the spore swab plates.
The plate that’s doing well is a transfer from a grain to agar plate. It’s one of the only that didn’t turn bacterial after transfer.
I’m really hoping it grows well enough for one more transfer with no contamination. This would mark the most success I’ve had so far with agar! I’m remaining hopeful, definitely think it’ll get much better once I’m able to maintain the temperature better.
I apologize if the pics are blurry and I can’t help them being turned sideways I’ve tried using the function in the uploads to not change the pic when uploaded but it still does. Here’s a pic of the spore swab plates that were inoculated about 4 days ago via golden teacher spore swab (I’m unsure if these are any good or not) any opinions?:

Here’s the one plate that came from grain to agar. The grain was bacterial as well so I cleaned up the myc on agar.

I gotta be honest though I’m not noticing a difference between plates stored in the manually cooled fridge or the plates sitting in the 90 degree room until sundown. I have dealt with contams more than I’d expected so maybe once I get the ac placed in that room then things will turn around and I’ll see less contams? I’m curious about that.
The room remains a steady 85 degrees during the day in the cool days and on the hot days it gets around 90-91 which is just unbearable if you ask me. You can see in an earlier post that I tried stuffing a towel that’s been soaked and ice packs in a unplugged mini fridge to keep them cooler which is definitely cooler. Like 10 degrees cooler but I’m not seeing a difference in those and the ones I leave sitting on the counter or in the sab. 🤷♂️
I’m sure there’s a huge difference in the big picture but I’m just not able to see it yet until I try things in a cooled room.
Any opinions would be very much appreciated and considered 😉
Edited by EntheoGod (07/17/20 02:23 PM)
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fiddy
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Re: First agar to agar transfer. :) Need advice on storage during colonization in hot temps. [Re: EntheoGod]
#26829157 - 07/17/20 02:35 PM (3 years, 6 months ago) |
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It's a pain in the ass but you could bury them in the ground, that would be less then your 90 degrees.
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EntheoGod
Entheo



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Re: First agar to agar transfer. :) Need advice on storage during colonization in hot temps. [Re: fiddy]
#26829159 - 07/17/20 02:35 PM (3 years, 6 months ago) |
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That would be an option if I had permission to dig in the ground where I live 😅
I should have an ac by the end of today so the room will be better maintained then. 🤞
Scratch that lol just checked the order and now it’ll be in 3 more days before I get the ac but I’m still hoping for the best!
Edited by EntheoGod (07/17/20 02:42 PM)
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EntheoGod
Entheo



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Re: First agar to agar transfer. :) Need advice on storage during colonization in hot temps. [Re: EntheoGod]
#26830450 - 07/18/20 09:37 AM (3 years, 6 months ago) |
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Here are a few more pics of two healthy pastyplates I have going.
The black dot is a marker dot on the outside of the lid lol my bad
These also originated on bacterial grain. I started this by always doing ms syringe put into pressure cooked clean wbs. Trust me they were clean. But as soon I began only getting bacterial growth and just couldn’t get them to fruit once colonizing a mono tub. I took a few steps back and started learning as much as I could about agar. So far I’ve learned pastyplates and 123 agar. I’ve learned a few lessons along the way as well obviously but you can see in earlier posts what I mean by mistakes.
Here’s a pic of the grains fully colonized and obviously bacterial.

And here’s the grain recently placed to agar to clean up.
Edited by EntheoGod (07/18/20 09:41 AM)
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EntheoGod
Entheo



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Re: First agar to agar transfer. :) Need advice on storage during colonization in hot temps. [Re: EntheoGod]
#26833998 - 07/20/20 10:36 AM (3 years, 6 months ago) |
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Update: I’ll have the ac for the room I use for all of this today! Here are the two plates I’ll be using to transfer from. This will make the 2nd transfer and I’m hoping they all stay healthy and clean so I can start a few master grain jars.
These are the same plates showed above that were really healthy imo. One of them developed some mold but nothing I can’t transfer away from. By super healthy I mean they are the best plates I have to transfer at the moment. I do have a few swab plates germinating in the meantime as well. Not sure how healthy they are though, they’re shown above.

Should be making several transfers from those two tonight if all goes well! I’m just excited to have something clean for the first time in what feels like forever.
Edited by EntheoGod (07/20/20 10:40 AM)
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EntheoGod
Entheo



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Re: First agar to agar transfer. :) Need advice on storage during colonization in hot temps. [Re: EntheoGod]
#26834010 - 07/20/20 10:53 AM (3 years, 6 months ago) |
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Here’s the swabs that are questionable to me because I’ve never done swab to agar and have no idea if this looks like what I’d expect. Doesn’t appear how I’d think and it looks kind of like cobweb almost but not nearly as fluffy and all over the place like cobweb usually is. I’m hoping these are healthy because I got the swabs from a friend who said he guarantee they are clean. He works in front of a flowhood and has the right equipment 🤷♂️.

Should I take a small transfer from the center area to clean this up or just toss em and start new plates up.
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EntheoGod
Entheo



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Re: First agar to agar transfer. :) Need advice on storage during colonization in hot temps. [Re: EntheoGod]
#26840905 - 07/23/20 03:32 PM (3 years, 6 months ago) |
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UPDATE:
I finally have an ac to keep the room a lovely 74-75f degrees. I also made 7 transfers from the 2 healthy plates seen earlier in the thread. One plate had a contam but was growing super clean so I transferred 3 from it into 3 different agar plates. I used the completely clean agar to transfer into 4 different agar plates. I’m hoping these all grow clean and I’m able to make master grain jars 🤓 I’m so excited. I believe I did much better with the tek this time so I’m a bit more confident in the outcome. I still have a lot of other stuff going at the moment too so i don’t have all my eggs in one basket.
I will post pics in a few days when the agar begins to grow significantly. I can’t wait to make grain jars again and attempt to get clean spawn!
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EntheoGod
Entheo



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Re: First agar to agar transfer. :) Need advice on storage during colonization in hot temps. [Re: EntheoGod]
#26849982 - 07/28/20 01:51 PM (3 years, 5 months ago) |
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Update:
Several of the transfers still had bacteria except for a couple that look wonderful so far. Just hoping they keep up the healthy growth. I also decided to make a few trays and a mono tub with the bacterial seeds I had left. They colonized in 90 degree f weather. I now keep it 74-75 at all times in the room where everything is located. Here’s a few pics for updates 

The trays were each made with a 2:1 ratio of spawn to substrate. The sub is CVG coir verm and gypsum as usual. Hoping it pulls through and produces some fruits. I’m not too hopeful considering the grains were mostly bacterial and colonization was weak. I’m fairly sure there’s a pic further up in the thread of one of the grain jars and what it looks like. Although the growth was incredible after I changed the temps to cooler ones. I noticed all bacterial jars, that were taking ages to colonize, took off with rhyzomorphic growth. Too bad I didn’t snap a picture. I shook those jars and that’s what made these trays and the tub. Each were made yesterday morning. The tub seems to be recovering really well and looks how I’d expect a normal healthy spawn tub to appear.
If the agar you see above colonizes fully or mostly and I don’t see any contams or bacteria, I’ll be making a couple master grain jars to get some stuff going with! I’m excited. The temp change definitely helped things along and slowed my contam rates down by almost 100%. I think my bacteria is simply coming from poor technique when doing the transfers. I’m still learning but discovered that I can pour my receiving plates thicker so it’s easier to transfer an agar slice onto it.
Any tips or advice is much appreciated! 😛
Btw I can’t keep the photos from changing orientation unless I upload the full size iPhone pic and that’s huge so sorry.
Edited by EntheoGod (07/28/20 01:52 PM)
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EntheoGod
Entheo



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Re: First agar to agar transfer. :) Need advice on storage during colonization in hot temps. [Re: EntheoGod]
#26855535 - 07/31/20 01:12 PM (3 years, 5 months ago) |
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Update:
This will be the last new post to this thread, instead I will simply continue updating this post.
This is what I have going so far. There are 3 healthy agar plates that are turning out nicely. I believe I will make those 3 into 3 master grain jars and go from there. The pics are those agar plates as well as a tub I made with bacterial spawn I had.
   
And finally I can keep the room a comfortably temp for everything 
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EntheoGod
Entheo



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Re: First agar to agar transfer. :) Need advice on storage during colonization in hot temps. [Re: EntheoGod]
#26855833 - 07/31/20 04:54 PM (3 years, 5 months ago) |
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I actually do need advice now!
When would you put these plates into a master grain jar? How much of the agar should be colonized? Sorry, I’ve never done this 😳.
I feel like it should be all colonized or the agar could get contaminated while in the grain jars.
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Roger Clemency
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Re: First agar to agar transfer. :) Need advice on storage during colonization in hot temps. [Re: EntheoGod]
#26855883 - 07/31/20 05:12 PM (3 years, 5 months ago) |
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it’s alright if some of the wedge isn’t colonized, all the grain in there is already susceptible anyway.
If you’re satisfied that the plates are clean I’d just criss cross them like a pizza and toss them in any time between like 3/4 and fully colonized.
-------------------- Sour grapes, sweet revenge Heaven starts right where hell ends
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EntheoGod
Entheo



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Re: First agar to agar transfer. :) Need advice on storage during colonization in hot temps. [Re: Roger Clemency]
#26855895 - 07/31/20 05:19 PM (3 years, 5 months ago) |
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Okay sounds good. 👌 I’ll be making a few jars for master jars tomorrow or Sunday night. I believe these are clean and healthy. Took a couple transfers but they look good to me now. I would want to use one plate per jar correct? Or do I split it and use one plate for a couple master jars? I believe I will do the tiger drop and only make 3 master jars. I’m just wanting to be 100% sure. I know I can’t just keep using the same genetics after a few g2g transfers but I’ll have more going by then I’m sure. Thanks for the assistance!
I look forward to seeing if the grain jars finally succeed for me. Hoping for better than ms syringe to grains...🤞
Edited by EntheoGod (07/31/20 09:09 PM)
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EntheoGod
Entheo



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Re: First agar to agar transfer. :) Need advice on storage during colonization in hot temps. [Re: EntheoGod]
#26859172 - 08/02/20 03:40 PM (3 years, 5 months ago) |
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Today I will be doing the Tiger Drop tek to inoculate 4 different master grain jars.
I have 3 healthy plates.
  
At least I hope these are healthy enough. I will only post updates on this thread post and no longer "bump" this to the top with just an update.
Anyways, I am going to be using 2 of the plates and doing the tiger drop as normally done. But I worry about whether the uncolonized agar will lead to any problems somehow so I also plan to use one of the plates and split it in two with a flame sterilized xacto knife making sure to slice out only the mycelium (no uncolonized agar) and attempting to drop a half in one master jar each. Im unaware if it will even make a difference but to satisfy my paranoia I will try both of these methods.
Feel free to chime in with how you inoculate grains with agar. I will update this with more pics after the process has been done.
No more bumping this with my own updates. 😅
UPDATE: it’s 1:30am the next morning from the last post and I have pressure cooked and allowed 4 grain jars to cool. I then proceeded to do the tiger drop with them. Here is the aftermath. Two of them have a whole plate in them and two of them have half of one plate that has been cut away from uncolonized agar.

3 days later here is what we have on the most impressive and visible ones. No contamination just yet, so far so good. The others look basically the same just more hidden behind the seeds.

5 days later, I hope these are colonizing at a healthy rate! They still look all clean I’m very hopeful for em. I need healthy clean spawn!

7 days later, the two jars that were made with a split agar plate have both contaminated with cobweb. The two solid plates are doing great still, likely shaking them up soon!
Edited by EntheoGod (08/09/20 06:56 PM)
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EntheoGod
Entheo



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Re: First agar to agar transfer. :) Need advice on storage during colonization in hot temps. [Re: EntheoGod]
#26883658 - 08/16/20 03:48 PM (3 years, 5 months ago) |
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So, all of these failed sadly and it was likely due to my poor technique but I will improve in the future! The only success I had was in making trays with the bacterial grains I was using to inoculate the agar. It was a great success actually. Here’s what that did..

Swabbed a few plates yesterday with fresh new spores instead of starting the culture using bacterial grains and will likely start up a new post about all that so this is likely the last one on this thread. Any tips for doing agar to grain I’d appreciate!
I have been reading as much as I can about agar and agar to grain on the forums so I have an understanding but I suppose practice will make me much better with this. I believe I simply had a very dirty inoculant as well.
HERES TO THE NEXT ATTEMPT!
Edited by EntheoGod (08/16/20 03:57 PM)
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