|
Tolldom
Noob Swede
Registered: 08/19/20
Posts: 23
Last seen: 3 years, 1 month
|
Questions about cloning, agar and sterilization.
#26952564 - 09/24/20 11:30 AM (3 years, 4 months ago) |
|
|
Heya...
I just failed/gave up hope to my first grow... Ordered a 6L pressure cooker and ingredients for agar, and a hell lot of sauce cups...
Bought 250x sauce cups, realized these may be too small for the use of agar/mushroom growth? These are 1,4" at the bottom. If i fill them half way up, maybe 1,6"... What are the recommended size? Will these be enough or am i gonna struggle in some way? Is this size enough? Recommended size? What happens if a too small amount of mycel is being but in a grain colonization jar? Will the colonization be too slow and things will start to rotten????
Yesterday I tried to clone a king oyster mushroom in my first ever agar... (Destilled water, 4-5 mineral drops, agar agar powder, nutritional yeast, dry malt extract and fine powdered coconut activated carbon) Recipe OK? Should I have skipped mineral drops, maybe too salt? Mycel doesnt need much minerals?
How long will it take before I start to see some growth in the agar cups?
Also... The reason why I make this post... I don't know if you have this problem in the US, but here in Sweden.. Its extremely difficult(or expensive) to find rubbing alcohol without dilutants(denaturants).. After I cleaned my sauce cups (chlorine bath, clean with rubbing alcohol, let it dry in my sterilized oven), I realized that the rubbing alcohol I used, that is filled with denaturant, (denatonium benzoate), which makes it taste like crap.. Thats how I realized my cups was filled with it.... Will this kill the process???
Give me all you opinions! I need to learn!
Thanks in advance // Noob swede...
|
polaritymind
relaxed attention


Registered: 10/10/16
Posts: 994
Loc: Germany
Last seen: 3 months, 8 days
|
Re: Questions about cloning, agar and sterilization. [Re: Tolldom]
#26952597 - 09/24/20 11:53 AM (3 years, 4 months ago) |
|
|
If you PC there is no need for rubbing alcohol. I cant tell you if it works because it depends on wether the denaturant is also of a similar boiling point so it could also evaporate. I assume so though, sice they often so that to prevent distilling off of the denaturant.
No; if properly sterilized in a PC rot shouldnt occur, except if contam is introduced while inoculating. An SAB (still air box) helps. If you want i can tell you very easily how to make one. There is no too little mycelium. Of course more is faster but I also usually just put one small piece and dont think its too slow (2-3 weeks if you shake at 30% colonization).
I dont know your agar recipe so cant tell you if it will work. You can try, but I would recomment doing PDYA or MEA. The first is only household items, but more steps and ingredients, the second has only 2 ingredients but you might need to buy malt extract online.
-------------------- "to affirm life is to also affirm death" -Albert hofmann
|
Kmacmo
The aborted pin



Registered: 08/14/19
Posts: 1,675
Loc: Central hemisphere
Last seen: 6 hours, 36 minutes
|
Re: Questions about cloning, agar and sterilization. [Re: Tolldom]
#26952600 - 09/24/20 11:54 AM (3 years, 4 months ago) |
|
|
The standard petri dishes are 90-100mm or you can get 60mm ones, you could use any size but too small it will be hard to work with. You can put the tinyest piece of mycelium into a grain jar it will be slower to colonise but it won't start to mold if your jars are made properly and pressure cooked properly.
Ive no experience with oysters so can't help you specifically with those, but do you pressure cook your agar? If so you don't need to clean the sauce cups like that just wash them in soapy water and the PC will kill anything anyway.
You tasted the rubbing alcohol? Your cups are filled with it?
--------------------
|
Tolldom
Noob Swede
Registered: 08/19/20
Posts: 23
Last seen: 3 years, 1 month
|
Re: Questions about cloning, agar and sterilization. [Re: Kmacmo]
#26952717 - 09/24/20 01:01 PM (3 years, 4 months ago) |
|
|
Quote:
polaritymind said: If you PC there is no need for rubbing alcohol.
Well, I PC the agar... The sauce cups are made of plasting, so i used a plastic sterilization technique.. Can I put plastic cups filled with agar in a pressure cooker?? If so, would be amazing! I PC the agar in a glass bottle first and then filled the recently sterilized cups..
Quote:
polaritymind said: I cant tell you if it works because it depends on wether the denaturant is also of a similar boiling point so it could also evaporate. I assume so though, sice they often so that to prevent distilling off of the denaturant.
I looked up denatonium benzoate + mycel and I found a thread here on shroomery.. Seems like the mycelium won't care. Good news...
Quote:
polaritymind said: No; if properly sterilized in a PC rot shouldnt occur, except if contam is introduced while inoculating. An SAB (still air box) helps. If you want i can tell you very easily how to make one. There is no too little mycelium. Of course more is faster but I also usually just put one small piece and dont think its too slow (2-3 weeks if you shake at 30% colonization).
I dont know your agar recipe so cant tell you if it will work. You can try, but I would recomment doing PDYA or MEA. The first is only household items, but more steps and ingredients, the second has only 2 ingredients but you might need to buy malt extract online.
Thanks for the tips!
Quote:
Kmacmo said: ***
***
You tasted the rubbing alcohol? Your cups are filled with it?
Alright, thanks for info/tips!
Lol, yeah, drank alot when I was a teenager, lol. I know the taste... makes your body smell like glue for a few days after...
Now at this time, I'd just happen to touch my finger in my mouth, recently touching a cup... Had to lick the cup all way around to be sure, lol... Did taste awful and it made me worry..
Edited by Tolldom (09/24/20 01:02 PM)
|
verum subsequentis
seeker of truth



Registered: 03/22/16
Posts: 8,732
Last seen: 1 year, 7 months
|
Re: Questions about cloning, agar and sterilization. [Re: Tolldom]
#26952793 - 09/24/20 01:41 PM (3 years, 4 months ago) |
|
|
Get some glad mini round, holy grail plates, small mason jelly jars, real petris or something else that can either be PCed or come sterile. Trying to sterilize plastic without heat is a dangerous game and will result in many many failures and headaches.
You don't need very much colonized agar to get a master jar done.
|
Tolldom
Noob Swede
Registered: 08/19/20
Posts: 23
Last seen: 3 years, 1 month
|
|
Alright, thank you for the info...
I got 2 agar dishes with cubensis cambodia mycel in the post yesterday, so I tried put some in my agar cups and filled the rest in a PCed grain jar that I accidentally boiled dry lol. I think its OK, some grains looked a little burned though...
Also starting to see some growth on my king oyster clones! One of them might be contaminated though..
If all of this will be a total failure I'll probably try to buy some sterilized plastic petris to start off with... Maybe even pre-filled lol.
I also start to see something in some of my dishes that might be contamination?

Do air bubbles necessarily have to be contam? Like in pic no.3.. Is 1 and 2 contaminated?
I cooked some black agar since i read about it had a few benefits.. But maybe is this a not so beginner friendly method?
Is it more difficult to spot contam in a black agar?
Thanks
Edit:
Looks like some bug or something?
Can i save this some way?
Edited by Tolldom (09/26/20 04:33 AM)
|
Tolldom
Noob Swede
Registered: 08/19/20
Posts: 23
Last seen: 3 years, 1 month
|
Re: Questions about cloning, agar and sterilization. [Re: Tolldom]
#26955629 - 09/26/20 06:23 AM (3 years, 4 months ago) |
|
|
Is it recommended to wait a few days using the agar plates to see if there was some kind of contam? If so, how long is it recommended to wait?
Should the plates be stored in the fridge?
|
polaritymind
relaxed attention


Registered: 10/10/16
Posts: 994
Loc: Germany
Last seen: 3 months, 8 days
|
Re: Questions about cloning, agar and sterilization. [Re: Tolldom]
#26955664 - 09/26/20 07:15 AM (3 years, 4 months ago) |
|
|
Well isnt pastywhite's agar recipe also with plastic cups? Thats not my personal preference either, I like small glass jars with regular Polyfill filters (same lids since same size as my large grain jars) better, but as I gather from his quite popular tek its possible, as long as the cups are made from PP (Polypropylene).
And yeah thats contam, proving the point made by another poster, that doing agar wihtout PC will only lead to headache.
I dont know about the air bubble, as such it could be fine, but you say there is a fly in there? As long as it doesnt grow bacteria or mold there it doesnt matter, even though its not exactly a super clean style.
Im not sure I understand your question about waiting with the agar plates? If you mean before inoculating then I could say yes this would be a good idea here to test your sterilization techniques. If it contams then you know the method doesnt work, since contams arent introduced while inoculating but are even there before. I dont generally do this every time but only when testing a new method or troubleshooting where there might be a problem in my workflow when I have a spike of contam rates.
As far as fridge goes, after inoc I would nt recommend it since it slows all growth, including mycelium, and therefore you will have the plate in there for weeks increasing the chance of a contam finding its way past your filter/lid seal with every day. Also the fridge, with all that food in there, is usually not a very clean place contam wise (In the air there). So therefore before I dont store them there either, again as with the contam test if the sterilization and seal are proper, plates can be stored and stay good for weeks. Which is why I always premake a larger amount of plates and then slowly use them up instead of making fresh batches every time I need them.
This is pastys tek https://www.shroomery.org/forums/showflat.php/Number/19208976#19208976
and this is a collection of other possibly useful teks https://www.shroomery.org/forums/showflat.php/Number/16453495
Edited by polaritymind (09/26/20 07:45 AM)
|
polaritymind
relaxed attention


Registered: 10/10/16
Posts: 994
Loc: Germany
Last seen: 3 months, 8 days
|
Re: Questions about cloning, agar and sterilization. [Re: polaritymind]
#26955682 - 09/26/20 07:33 AM (3 years, 4 months ago) |
|
|
-------------------- "to affirm life is to also affirm death" -Albert hofmann
|
verum subsequentis
seeker of truth



Registered: 03/22/16
Posts: 8,732
Last seen: 1 year, 7 months
|
Re: Questions about cloning, agar and sterilization. [Re: polaritymind]
#26956212 - 09/26/20 02:30 PM (3 years, 4 months ago) |
|
|
1 contam (yeast or bacteria). Transfer away from it. 2 I can't tell 3 air bubbles are air bubbles. Not contams. 4 looks like a bug.
I don't like black agar because it is hard to tell what's going on. I prefer blue.
|
Tolldom
Noob Swede
Registered: 08/19/20
Posts: 23
Last seen: 3 years, 1 month
|
Re: Questions about cloning, agar and sterilization. [Re: polaritymind]
#26957245 - 09/27/20 10:12 AM (3 years, 3 months ago) |
|
|
Quote:
polaritymind said: ...
And yeah thats contam, proving the point made by another poster, that doing agar wihtout PC will only lead to headache.
I dont know about the air bubble, as such it could be fine, but you say there is a fly in there? As long as it doesnt grow bacteria or mold there it doesnt matter, even though its not exactly a super clean style.
Im not sure I understand your question about waiting with the agar plates? If you mean before inoculating then I could say yes this would be a good idea here to test your sterilization techniques. If it contams then you know the method doesnt work, since contams arent introduced while inoculating but are even there before. I dont generally do this every time but only when testing a new method or troubleshooting where there might be a problem in my workflow when I have a spike of contam rates.
As far as fridge goes, after inoc I would nt recommend it since it slows all growth, including mycelium, and therefore you will have the plate in there for weeks increasing the chance of a contam finding its way past your filter/lid seal with every day. Also the fridge, with all that food in there, is usually not a very clean place contam wise (In the air there). So therefore before I dont store them there either, again as with the contam test if the sterilization and seal are proper, plates can be stored and stay good for weeks. Which is why I always premake a larger amount of plates and then slowly use them up instead of making fresh batches every time I need them.
This is pastys tek https://www.shroomery.org/forums/showflat.php/Number/19208976#19208976
and this is a collection of other possibly useful teks https://www.shroomery.org/forums/showflat.php/Number/16453495
Thanks for good input and info!
What I can see so far, 4 days from doing the agar plates, only like 4/20 seems to have some kind of contamination... Did let them soak in chlorine for like 6 hours, then clean/dip it off in a alcohol bath - let dry in sterilized and slightly hot oven... But yeah, maybe to early to say..
This fly/bug/something, looks like some kind of larva at the moment but may be just some undissolved activated carbon, maybe.. I'll see how it evolves and maybe gonna have to cut it off...
I think you understood my question fairly! My use of english ain't the best :p ... But yeah, what I meant to ask was if it was a good idea to let the agar plates sit for a time, how long and in what temperature, to see if there's any contam before inoculating.
What i've learned so far from my own experience that contamination can develop at a very different time frame. Some plates appeared to be contamined almost in just a few hours while some I started to see 3 days in.
So how long is recommended to wait? Think I didn't got that clear yet... 1 week, room temperature will be OK/enough?
Reading this pastrys tek, made me realize I might improvizing way too much considering im a total beginner... Improvizing from things i've read and videos i've looked though and mashing things I think might work, will work. Lol. But I think it will be for no waste.. Im learning and yet i havent screwed something totally up..
..Except my last (also first) grow... I think my impatience fucked that up totally though..
Also, I want to ask... This contam in red, is just to cut away right? This marked in green, that is spreading on a larger area faster, how to deal with it?
Lets say we have a plate with healthy mycelium. This contam marked in green is there also. Will the mycelium struggle with it? Lets also say that the mycelium is growing straight over this green marked contam and fully colonizing the top layer of the agar plate.
Will this be a problem later on??
(Experiment/test agar plate.. Dipped a used face mask on the top layer to see what happened)

Thanks <3
|
Tolldom
Noob Swede
Registered: 08/19/20
Posts: 23
Last seen: 3 years, 1 month
|
|
Quote:
verum subsequentis said:
1 contam (yeast or bacteria). Transfer away from it. ...
I don't like black agar because it is hard to tell what's going on. I prefer blue.
Best way to transfer away? Transfer the bad (contam) or the good (mush) part?
Blue agar? Do you want to share a good recipe?.. =) Googled but didnt find more than pics..
|
verum subsequentis
seeker of truth



Registered: 03/22/16
Posts: 8,732
Last seen: 1 year, 7 months
|
Re: Questions about cloning, agar and sterilization. [Re: Tolldom]
#26957409 - 09/27/20 12:38 PM (3 years, 3 months ago) |
|
|
Always transfer a tiny piece of cube myc from as far away from visible contams as possible to a fresh plate.
Just make agar and put a little blue food coloring in it. I use malt extract as my nute source.
|
Tolldom
Noob Swede
Registered: 08/19/20
Posts: 23
Last seen: 3 years, 1 month
|
|
Quote:
verum subsequentis said: Always transfer a tiny piece of cube myc from as far away from visible contams as possible to a fresh plate.
Just make agar and put a little blue food coloring in it. I use malt extract as my nute source.
allright, thanks! =)
I'll try it out!
|
Tolldom
Noob Swede
Registered: 08/19/20
Posts: 23
Last seen: 3 years, 1 month
|
Re: Questions about cloning, agar and sterilization. [Re: Tolldom]
#26958848 - 09/28/20 01:36 PM (3 years, 3 months ago) |
|
|
I just have one more question i really want answer from now...
How long can it take to see contamination in a agar plate?
How long is recommended to wait after making it - to using it?
I made 20 plates, only one looks contaminated without opening it, and 4 others looks like they have a contam after using it (cloning attempts and one mycelium agar - agar transfer)
Having 3 unused plates left now...
Whatever but, how long is recommended to wait?
Guess i will learn with experience, but i've tried to look for info a a few times/days without success, and im kind of curious =)
Edited by Tolldom (09/28/20 01:36 PM)
|
verum subsequentis
seeker of truth



Registered: 03/22/16
Posts: 8,732
Last seen: 1 year, 7 months
|
Re: Questions about cloning, agar and sterilization. [Re: Tolldom]
#26959597 - 09/29/20 12:09 AM (3 years, 3 months ago) |
|
|
a week is more than enough
|
|