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SombraGris

Registered: 11/17/19
Posts: 20
Loc: Argentina
Last seen: 1 month, 21 days
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{agar} mold help
#27290143 - 05/02/21 08:52 AM (3 years, 8 months ago) |
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Hi Guys, be patient with my english am from Argentina
i had great sucess with Agar in the past
But now am not able to keep the culture clean
Although there was growth and I observe an improvement in the way the mycelium looks, I am not being able to prevent the growth of mold on the plates, around the mycelium as you can see in the following image

some dishes grow without contaminants but they are the great minority, this is the fourth generation of mycelium. At this point, in the past, all dishes were in perfect condition to be used
I always work with the pastyplates technique, with SAB and as many hygienic measures as possible. Even in this last opportunity, leave the sterilized agar plates for 5 days to observe whether or not contaminants grow in it before making the transfers.
The problem occurs when I make the transfers, in principle the mycelium grows without problem but eventually I see germination of mold spores somewhere on the plate and I have to discard or isolate it.
I ask you please if you can help me determine what you think is the source of the contamination, as well as some useful advice.
of course I sterilize the agar 30min at 15psi, I wear gloves, I thoroughly clean the SAB, I work in the bathroom,flame the scapel, and I slpash water in the SAB just before work

many thanks for reading me
regards!!
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Benson
The Kidd ⭐


Registered: 09/29/20
Posts: 898
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Based on what you said about leaving the cups for 5 days without contam, and how the contam seems to be scattered on the surface, I would guess something is getting in when you open the cup.
Do you have a fan running in the room while you work? Do you let the air settle in the room for a while before you work? Bathrooms aren't the cleanest places... when you flush the toilet bacteria gets thrown all over the place.
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TheStallionMang
I nvr said I was afraid of dying


Registered: 10/18/17
Posts: 5,591
Loc:
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Re: {agar} mold help [Re: Benson]
#27290189 - 05/02/21 09:43 AM (3 years, 8 months ago) |
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Most importantly, keep all sources of air movement off or blocked, spray a fine mist of water with 1 drop of liquid soap in the SAB 2 minutes before starting work, move slowly and never move anything above your pastyplates when they are open
Good luck, you'll get it!
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TheStallionMang
I nvr said I was afraid of dying


Registered: 10/18/17
Posts: 5,591
Loc:
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What do you mean by "splash water" in the SAB?
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SombraGris

Registered: 11/17/19
Posts: 20
Loc: Argentina
Last seen: 1 month, 21 days
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Quote:
TheStallionMang said: What do you mean by "splash water" in the SAB?
Spray sorry
i change the work space from my office to the bathroom to determine if my office was the problem but the mold keep growin in agar plates
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TheStallionMang
I nvr said I was afraid of dying


Registered: 10/18/17
Posts: 5,591
Loc:
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I thought you might have meant spray but wanted to make sure
Check out the pastywhyte link in my signature. I've seen great success with it
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TheStallionMang
I nvr said I was afraid of dying


Registered: 10/18/17
Posts: 5,591
Loc:
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Generally the office would make a better work place than the bathroom
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SombraGris

Registered: 11/17/19
Posts: 20
Loc: Argentina
Last seen: 1 month, 21 days
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The new 12 plates all contaminated in the same way as seen in the image. With mold germinating outside the mycelium. I don't know how to avoid it. I understand that there is a flaw in my technique with the SAB but I have made changes and taken precautionary measures without success
Is it possible that the place where I store them is the problem?
Am using pastywhyte tek
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Ashtray161
SettledNomad



Registered: 03/21/21
Posts: 4,503
Loc: Rugby, England
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Quote:
SombraGris said: The new 12 plates all contaminated in the same way as seen in the image. With mold germinating outside the mycelium. I don't know how to avoid it. I understand that there is a flaw in my technique with the SAB but I have made changes and taken precautionary measures without success
Is it possible that the place where I store them is the problem?
Am using pastywhyte tek
Are you wrapping your plates? I was having super bad problems when I started w agar as well, lost every single plate to stupid amounts of contams. I built a new SAB and moved my workspace to somewhere less drafty Are u making sure no air is blowing into ur SAB while ur working?
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(You Know What Time It Is)
Major Issues in the Psychedelic Movement: https://www.shroomery.org/forums/showflat.php/Number/27677086
"You never have to prove the fool a fool, just let them speak."
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LadysKnight
Hello Ladies


Registered: 10/09/15
Posts: 2,329
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Try with a dry sab
-------------------- Don't follow leaders
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SombraGris

Registered: 11/17/19
Posts: 20
Loc: Argentina
Last seen: 1 month, 21 days
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going to try a dry SAB
Quote:
Are you wrapping your plates? I was having super bad problems when I started w agar as well, lost every single plate to stupid amounts of contams. I built a new SAB and moved my workspace to somewhere less drafty Are u making sure no air is blowing into ur SAB while ur working?
I am using pp5 plastic containers similar to those of pasty white plates, a little harder. In the past I was successful, they always worked well for me. I honestly don't think they are the problem
I changed the work location from my office to the bathroom to eliminate a possible factor of contamination since my office has a window and I thought it could be influencing
I will work with the dry SAB and try to clean it better ... Is it possible that the mold is already inside the SAB?
Edited by SombraGris (05/03/21 11:20 AM)
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Ashtray161
SettledNomad



Registered: 03/21/21
Posts: 4,503
Loc: Rugby, England
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Well probably yea, I like to assume mold spores are everywhere all the time. The point of the SAB is to keep the air still enough that the spores dont get kicked up into ur sterile supplies. Some of the best advice Ive gotten was to treat the floor of ur SAB like lava. Keep all of ur sterile stuffs off of the table your SAB is on, make sure to keep ur work elevated. The goal of the SAB isnt a sterile work space, thats nearly impossible without an actual lab and proper equipment and sterile process, the inside of the SAB is meant to have such still air that all the contams sink to bottom and stay there. Thats why u want to spray some soapy water first, give it 15-20 mins to settle and then start working. Some people put down a towel to trap the contams. Most contam spores float an inch or so off of the floor when theres no air current to carry them higher.
I have a thing in mine to be sure I can work off the floor:  
I personally (and a lot of TCs will tell you this isnt needed, so id listen to them if I were you. Im a bit OCD and honestly cant help but do this) like to spray ISO with the soapy water just as an extra barrier. Every 3 days or so I flip it over and spray it and the table down with lysol, let it evap, spray it again and wipe both the inside of the SAB and the table and shelf thingy. Wiping is important in my mind because u actually remove the contams rather than just spray them.
Be sure youre not doing any of ur flame sterilizing inside the SAB either as that will create turbulent air and thus air flow. Im pretty convinced thats what was causing such high contam rates for me in the start.
--------------------
(You Know What Time It Is)
Major Issues in the Psychedelic Movement: https://www.shroomery.org/forums/showflat.php/Number/27677086
"You never have to prove the fool a fool, just let them speak."
Please, be an adult. Get vaccinated. Dont use psychedelics as an excuse. Dont come at me with some hippy dippy nonsense, GO GET VACCINATED.
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Edited by Ashtray161 (05/03/21 11:42 AM)
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give.more
Lurker


Registered: 04/30/16
Posts: 222
Loc: Copperhead Road
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Quote:
Ashtray161 said: Some of the best advice Ive gotten was to treat the floor of ur SAB like lava.
Definitely. I have a spray bottle of 10% bleach solution that I spray everywhere inside the box 30 minutes before doing any work in it. I also keep upside down jar rings just in case I do have to lay something down.
It smells lightly of bleach while I'm working but hasn't seemed to have an ill effect on anything being done.
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LadysKnight
Hello Ladies


Registered: 10/09/15
Posts: 2,329
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Re: {agar} mold help [Re: give.more]
#27292004 - 05/03/21 12:53 PM (3 years, 8 months ago) |
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Try not spraying anything. The mold is in a pattern that suggests tiny droplets made it in on the side you lifted to inoculate. The bleach isn't helping you. It's not about a clean sab but still air without raining microdroplets that can grab contams and put them on your dish.
-------------------- Don't follow leaders
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Ashtray161
SettledNomad



Registered: 03/21/21
Posts: 4,503
Loc: Rugby, England
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Quote:
LadysKnight said: Try not spraying anything. The mold is in a pattern that suggests tiny droplets made it in on the side you lifted to inoculate. The bleach isn't helping you. It's not about a clean sab but still air without raining microdroplets that can grab contams and put them on your dish.
What he said I wouldnt suggest using bleach anyways, bleach fumes are thought to be able to cause genetic mutations in the mycelium. And again like whats been said the SAB isnt meant to be clean, its meant to create a STILL AIR environment. Hence the name. Youre using physics, not chemistry.
--------------------
(You Know What Time It Is)
Major Issues in the Psychedelic Movement: https://www.shroomery.org/forums/showflat.php/Number/27677086
"You never have to prove the fool a fool, just let them speak."
Please, be an adult. Get vaccinated. Dont use psychedelics as an excuse. Dont come at me with some hippy dippy nonsense, GO GET VACCINATED.
Be Gay, Do Crime 161 1312
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Ashtray161
SettledNomad



Registered: 03/21/21
Posts: 4,503
Loc: Rugby, England
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I would either not spray anything at all or follow what TCs say and only spray a small bit of barely soapy water 25 or 30 mins b4 u work. If youre like me and absolutely (and potentially to your detriment) have to use some sort of cleaner use 70% ISO and let it evaporate completely before working. I tend to spray the ISO an hour or two before I do any work but thats again because I feel strange and overwhelming anxiety that something will go wrong if I dont. Trust the experienced ones and the teks, either dont spray anything or stick to water w a tiny bit of soap
--------------------
(You Know What Time It Is)
Major Issues in the Psychedelic Movement: https://www.shroomery.org/forums/showflat.php/Number/27677086
"You never have to prove the fool a fool, just let them speak."
Please, be an adult. Get vaccinated. Dont use psychedelics as an excuse. Dont come at me with some hippy dippy nonsense, GO GET VACCINATED.
Be Gay, Do Crime 161 1312
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SombraGris

Registered: 11/17/19
Posts: 20
Loc: Argentina
Last seen: 1 month, 21 days
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Thanks guys, today i goint to try a dry SAB
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TheStallionMang
I nvr said I was afraid of dying


Registered: 10/18/17
Posts: 5,591
Loc:
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Quote:
Ashtray161 said:
Quote:
LadysKnight said: Try not spraying anything. The mold is in a pattern that suggests tiny droplets made it in on the side you lifted to inoculate. The bleach isn't helping you. It's not about a clean sab but still air without raining microdroplets that can grab contams and put them on your dish.
What he said I wouldnt suggest using bleach anyways, bleach fumes are thought to be able to cause genetic mutations in the mycelium. And again like whats been said the SAB isnt meant to be clean, its meant to create a STILL AIR environment. Hence the name. Youre using physics, not chemistry.
The idea of spraying is that the water droplets catch what's in the air and drag it to the bottom. You just have to wait 2 min for it to all settle then get to work and more slowly enough to not stir anything up
Sorry guys but the OP is having problems getting clean agar and I don't think suggesting a dry SAB is the way to go. Dry SABs are for people that can get good results with a wet SAB
Op, keep reading and thinking. You'll figure it out
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