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Offlinemoshe1

Registered: 08/29/15
Posts: 259
Last seen: 3 hours, 18 minutes
Help me understand my agar
    #27325787 - 05/27/21 10:01 PM (18 days, 3 hours ago)

Here are some of my agar dishes currently growing out. This is my first ever batches of agar, as in i've done germ plates, transfers 1 + 2s.  i'm yet to have put any agar to grain yet.

I'll label them respectively and leave it open ended- because i'm trying to understand whats going on and make decisions based on some knowledge and im having a hard time. My confusion is coming from having sporadic growth, the myc is all over the place and makes it hard for me to understand what im looking at. Theres aerial growths, thin and almost transparent growths, fuzzy areas, flat areas. Spots that seem to be growing out in an even fashion, and some that look like a jungle. Also note, I've had no discolorations, even my germ plates from a month ago are still white although unusual looking.


trying not to ask too many questions with new threads but my search isn't yielding any help that im looking for- rather confusing me more with all the mixed answers i am reading on every thread.
https://www.shroomery.org/forums/showflat.php/Number/26988516#26988516 ;  this thread/response from RR is the only thing that really looks similar to mine that i could possibly take advice for my situation without uncertainties

Using bods LME for my agar.  About 400 ML water, 10 grams AGAR to roughly 7.5 grams LME for every batch of 20 plates. 

First sign of rhizo growth anywhere on any plate. Showed up on a T1 plate way later, regrowing out of an area in the center.


Mazy germ, t1 and t2. A large portion of my plates grew out thin and translucent like this before getting any kind of thickness



A couple LGT Germs and their T1s, took over the whole plate already. Had too many plates to transfer so i let these grow out, got handful more that look like them.


BBM germ, t1 and t2


BBM T1 plates that are almost grown out completely


BBM Germ t1 and t2


Close up of a T1 plate fully grown out. Still no signs of "strong rhizo growth" anywhere to be found- anywhere



most organized / thick myc from the bunch across all varieties, but still very fuzzy


Edited by moshe1 (05/28/21 01:46 AM)


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Offlineverum subsequentis
seeker of truth
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Registered: 03/22/16
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Re: Help me understand my agar [Re: moshe1]
    #27325792 - 05/27/21 10:09 PM (18 days, 3 hours ago)

Spore syringe?


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InvisibleFeasoghorm

Registered: 10/24/18
Posts: 3,884
Re: Help me understand my agar [Re: moshe1]
    #27325799 - 05/27/21 10:13 PM (18 days, 3 hours ago)

Yeah pretty much all them plates cept for the germ plates look odd. Well, germ plates look odd in general, but i thnk that you have a contaminant fungus/mold blended in with your myc. I used to say "seamless", but it aint really seamless if you can tell it's fucked haha.


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Offlinemoshe1

Registered: 08/29/15
Posts: 259
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Re: Help me understand my agar [Re: verum subsequentis]
    #27325804 - 05/27/21 10:15 PM (18 days, 3 hours ago)

Quote:

verum subsequentis said:
Spore syringe?




all of these are off print/swab

i do have a couple from spore solution that are a little behind- but more-or-less still hard to read like these are.




Edited by moshe1 (05/27/21 10:22 PM)


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Offlineverum subsequentis
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Re: Help me understand my agar [Re: moshe1] * 1
    #27325807 - 05/27/21 10:18 PM (18 days, 3 hours ago)

Middle plate in second pic.
Bottom two in last pic.

These are your best chances at getting a clean culture.


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InvisibleFeasoghorm

Registered: 10/24/18
Posts: 3,884
Re: Help me understand my agar [Re: verum subsequentis]
    #27325812 - 05/27/21 10:21 PM (18 days, 3 hours ago)

I've also seen odd plates like this from improperly nuted grain soak agar. However i dnt thnk there is anything wrong with your mix.

Looks like he transferred from middle plate 2nd pic to 3rd plate 2nd pic.
Bottom right in last pic doesnt look too bad.


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Offlinemoshe1

Registered: 08/29/15
Posts: 259
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Re: Help me understand my agar [Re: verum subsequentis]
    #27325814 - 05/27/21 10:23 PM (18 days, 3 hours ago)

Quote:

Feasoghorm said:
I've also seen odd plates like this from improperly nuted grain soak agar. However i dnt thnk there is anything wrong with your mix.

Looks like he transferred from middle plate 2nd pic to 3rd plate 2nd pic.
Bottom right in last pic doesnt look too bad.





what are the odds of this happening on every batch? I mean, im only like 4 batches of agar in sure- but whats riding on every single plate i germ or transfer?  I let my flowhood in the clean-room run with the door shut for like, 3 hours, sometimes up to 5 to scrub the air before i work. Mind you this room is only for mixing/spawning my tubs and doing anything that requires the flowhood- and i do my best to keep sterile technique when i work.  of course im still a noob with agar- but i still need to identify problems to fix em.

if all is fucked in these pics than im pretty concerned- cause that would indicate that my whole shelf of agar plates are contaminated with mold?


Quote:

verum subsequentis said:
Middle plate in second pic.
Bottom two in last pic.

These are your best chances at getting a clean culture.




noted.


--------------------


Edited by moshe1 (05/27/21 10:27 PM)


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InvisibleFeasoghorm

Registered: 10/24/18
Posts: 3,884
Re: Help me understand my agar [Re: moshe1]
    #27325829 - 05/27/21 10:36 PM (18 days, 3 hours ago)

Well im not for certain wats up with your plates, although i wish i cud be. It's jst my best guess.

I've never had a contaminated syringe so i dnt knw what to say bout that, but ive run into many a dirty print/swab. I expect it.
It can be difficult to differentiate freahly germinated cube spores and freshly germinated aspergilus.
Do you scrape spores off of prints with the print above the plate? This is a contam laden practice.

There cud be something happening pre-inoculation. Do you set aside a control plate from each batch? Do you wait 2wks b4 using fresh plates? If i can i will, cuz ive had shit show up 2wks from pour.

When you work in your SAB, do you wear a mask? Are you right up on it looking through the top to work? Or in front of it?

Also, are you using the same print/swab for all these? Like, are these genetics basically all from the same shroom?

Do you wear gloves?


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Offlinemoshe1

Registered: 08/29/15
Posts: 259
Last seen: 3 hours, 18 minutes
Re: Help me understand my agar [Re: Feasoghorm]
    #27325922 - 05/28/21 12:11 AM (18 days, 1 hour ago)

Quote:

Feasoghorm said:
Well im not for certain wats up with your plates, although i wish i cud be. It's jst my best guess.

I've never had a contaminated syringe so i dnt knw what to say bout that, but ive run into many a dirty print/swab. I expect it.
It can be difficult to differentiate freahly germinated cube spores and freshly germinated aspergilus.
Do you scrape spores off of prints with the print above the plate? This is a contam laden practice.

There cud be something happening pre-inoculation. Do you set aside a control plate from each batch? Do you wait 2wks b4 using fresh plates? If i can i will, cuz ive had shit show up 2wks from pour.

When you work in your SAB, do you wear a mask? Are you right up on it looking through the top to work? Or in front of it?

Also, are you using the same print/swab for all these? Like, are these genetics basically all from the same shroom?

Do you wear gloves?




My process:

After rooms air has been scrubbing for a few hours at least, i go in. remove shirt i was just wearing outside/during the day, spray arms up to shoulders up with ISO, gloves on, mask on. Then with my alchohol towel i wipe down all surfaces, and mist ISO on the filter too.  Then ring unopened sleeve of plates over, tools, and whatever else.

then i go pick out what im going to transfer or germ, bring those as well.  As for technique, always wiping with ISO before bringing in flow, always flame sterilizing tools when needed. I always focus on not letting my hands get in-between any flow whatever it is im working on.


I've been prepping my agar and pouring it the day of, or next day- and usually inoculate the next day after pouring, unless i've been at the office long enough for it to cool, but thats only happened once so far.

i have not set aside any control plates on purpose- but i have had extra plates that have been poured i didnt use because of condensation- and those have stayed clean even without being wrapped with parafilm and moved around carelessly. They look spotless, and thats with almost zero care, they're just sitting in the lab rn still clean-  my plates i've worked with are exponentially handled with more carefulness.

i've never waited longer than a day or two after pouring my plates-  after i pour i just put the sleeve back over the stack without sealing them until i work with them again the next day or two. I have a bottle of agar in the fridge now waiting to be poured, maybe ill pour early and give it more time. At this point i'm not sure what to do.

also, they are all different! thats why- its so confusing when they all show oddness.  I got Blue meanies from print/swab, Mazy from print/swab, LGT from print/swab which are all pictured above.
They are all similar in "oddness" lol...

I really need another friend in this hobby to come and look at my stuff in person and be able to conversate with lol


Edited by moshe1 (05/28/21 12:31 AM)


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InvisibleFeasoghorm

Registered: 10/24/18
Posts: 3,884
Re: Help me understand my agar [Re: moshe1]
    #27326013 - 05/28/21 12:46 AM (18 days, 58 minutes ago)

Shit dude...idk


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Offlinemoshe1

Registered: 08/29/15
Posts: 259
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Re: Help me understand my agar [Re: Feasoghorm]
    #27326053 - 05/28/21 01:30 AM (18 days, 14 minutes ago)

all good man- when i figure out whats happening i'll definitely post it. Like i said- no one has given me definitive answers yet and searching on the forums will yield you in so much mixed feedback you will catch yourself going back and forth on what you think.

when i get a chance to test a couple plates the answer will be clear on the contam side of things.

bump for more eyes - here are some close ups of germ plates and transfer ones as clear as i could get them.  you could really zoom in on em if you open in a new tab and see.

https://www.shroomery.org/forums/showflat.php/Number/26988516#26988516 this response from RR to someone with the closest lookign things to my plates is all thats giving me hope lmao



--------------------


Edited by moshe1 (05/28/21 01:42 AM)


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Offlineverum subsequentis
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Re: Help me understand my agar [Re: moshe1]
    #27326324 - 05/28/21 09:01 AM (17 days, 16 hours ago)

Yowzers. You've got some wild shit going on. I'd start some new spores.  Use a loop and streak a few plates. Update here with pics as you go and we can help you understand where things are going wrong.


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OfflineBenson
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Registered: 09/29/20
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Re: Help me understand my agar [Re: verum subsequentis]
    #27326368 - 05/28/21 09:47 AM (17 days, 15 hours ago)

Not sure how helpful this is because some of your plates have thick growth of something but as for the wispy ones I encountered that a few months ago. I upped my LME and added 1g yeast and the growth got thicker/healthier. I would definitely suggest new spores in any case, cause that shit looks funky...

Wispy


Blue is more LME and +1g yeast


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OfflinemushboyM
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Re: Help me understand my agar [Re: Benson]
    #27326369 - 05/28/21 09:49 AM (17 days, 15 hours ago)

i 3rd for new spores.

or do a proper streak with the ones ya got.. but if you want quality myc relatively painlessly id just get new spores.


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Offlineverum subsequentis
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Re: Help me understand my agar [Re: mushboy]
    #27326372 - 05/28/21 09:50 AM (17 days, 15 hours ago)

Fourthed


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OfflineFeedYourMind
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Re: Help me understand my agar [Re: Benson]
    #27326514 - 05/28/21 11:56 AM (17 days, 13 hours ago)

Quote:

Benson said:
Not sure how helpful this is because some of your plates have thick growth of something but as for the wispy ones I encountered that a few months ago. I upped my LME and added 1g yeast and the growth got thicker/healthier. I would definitely suggest new spores in any case, cause that shit looks funky...

Wispy


Blue is more LME and +1g yeast





I had the same experience as you and OP. I had thin and wispy growth on agar and upped my nutes to the standard 10g of MEA per 500ml/water. I also add in 1-2g of nutritional yeast and that made ALL the difference in the world for me. Also, I started using .5g-1g of soy peptide per 500ml water and that has also improved the growth speed - especially when starting from spores.


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InvisibleFeasoghorm

Registered: 10/24/18
Posts: 3,884
Re: Help me understand my agar [Re: FeedYourMind]
    #27326617 - 05/28/21 01:49 PM (17 days, 11 hours ago)

Interesting :strokebeard:
Getting many reports of this from either too much or not enough nutes in the mix. 10A/10LME/500ml is the standard. Didnt thnk a 2.5 gram reduction in LME and 100ml reduction in water to the mix wud fux wit it that much.


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Offlinemoshe1

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Re: Help me understand my agar [Re: Feasoghorm]
    #27326844 - 05/28/21 04:58 PM (17 days, 8 hours ago)

i have some fresh melmaks that just germinated waiting for transfers. i'm gonna transfer a few of these wild looking ones and see what happens anyway and update w/ pics.

i've been on the same thought dude, some say lower nutes some say up them and they are both to remedy the same issues lmao. gotta love it.

im a little disappointed but i guess it is what it is, all my plates have that kind of vertically growing fuzz some more some less. wonder what it could be, across all the different varieties i have too. my rooms air is scrubbing as we speak to pour more agar.

interesting thing is that this stuff isnt changing color or shape or anything, just bright white like the myc. all the germ plates look like they have white cotton on them


thanks for all the feedback. any suggestions or favorites for what variety i should put to agar to catch me up?


--------------------


Edited by moshe1 (05/28/21 05:08 PM)


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OfflineBenson
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Re: Help me understand my agar [Re: Feasoghorm]
    #27327076 - 05/28/21 08:44 PM (17 days, 5 hours ago)

Quote:

Feasoghorm said:
Interesting :strokebeard:
Getting many reports of this from either too much or not enough nutes in the mix. 10A/10LME/500ml is the standard. Didnt thnk a 2.5 gram reduction in LME and 100ml reduction in water to the mix wud fux wit it that much.




I'm convinced at this point it's the nutrient level of the LME... I have nothing to base that off other than process of elimination tho. Distilled water, +10g agar (one of the two commonly used brands), +10g LME works great for some like D3, but was trash for me across multiple strains and swabs. Instant results when I upped the neuts :shrug:


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Offlinejomanda1990
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Re: Help me understand my agar [Re: Benson]
    #27327118 - 05/28/21 09:10 PM (17 days, 4 hours ago)

Hey, I've been having the exact same problem as you; almost weak, whispy growth in all my plates lately.

Before reading this thread, I was convinced my nutes were too high, and tried A BUNCH of recipes both as-is and with 25% less nutes. No good results so far.
I tried Potato flake + honey, Liquid malt Extract (alas, no dry extract here), diluted grain soak water, diluted grain boil water, and BRF.

I'll up the nutes a bit like Benson says in my next agar batch and let you know if the situation improves.

Good luck man, I understand your frustration with not getting the results you want from agar. We'll make it through :evil:


--------------------


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