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Mr.Christmas
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Registered: 04/08/20
Posts: 117
Loc: Colombia
Last seen: 13 days, 25 minutes
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My Agar Mess Up Update
#26784724 - 06/26/20 01:04 PM (3 years, 6 months ago) |
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So... I messed up lol. 5/10 agar plates were infected this morning, and by tomorrow I think all the agar plates are going to be infected. I didn't have parafilm, I didn't ziplock them inside my glove box, so I can see how I messed up.
If one of these miraculously survive, am I able to cut little wedges of it and move it to other agar plates to clone that one? Here's a pic. My parafilm comes in next week.
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Todo lo que hagas en la vida será insignificante; pero es muy importante que lo hagas.
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ManifoldPrime
Per Ardua Ad Astra


Registered: 03/16/17
Posts: 1,313
Loc: South Africa
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I dont think a lack of parafilm caused this, the contams would be on the edges. You've gotten contams flourishging, you absolutely smeared them with spores. Some of my most successfull inoculation plates looked untouched except for the most faint traces of scoring by the loop itself. whereas those look like you sprinkled potting soil on them.
Now if some mycelium does germinate and grow enough to be visible in time before the con tams take over completely , you can definately transfer. Thats the whole point of agar - to allow contams present on the spores to germinate as well as our desired cube spores, and then physically select the cybe myc over anything else, and repeat to make sure we have a monoculture of just cubensis.
However, because the agar surface is so laden with innoculant, you are gonna have to take a very small sample, and repeatedly transfer to confirm cleanliness.
Next time, when innoculating, perform this technique:
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herbnmyco


Registered: 02/28/18
Posts: 353
Loc: West Coast
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Sounds like you know where you went wrong. If you get good growth you can transfer it. I use saran wrap for dishes. Just gotta cut one inch thick rolls out of it.
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Mr.Christmas
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Registered: 04/08/20
Posts: 117
Loc: Colombia
Last seen: 13 days, 25 minutes
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Quote:
ManifoldPrime said: I dont think a lack of parafilm caused this, the contams would be on the edges. You've gotten contams flourishging, you absolutely smeared them with spores. Some of my most successfull inoculation plates looked untouched except for the most faint traces of scoring by the loop itself. whereas those look like you sprinkled potting soil on them.
Now if some mycelium does germinate and grow enough to be visible in time before the con tams take over completely , you can definately transfer. Thats the whole point of agar - to allow contams present on the spores to germinate as well as our desired cube spores, and then physically select the cybe myc over anything else, and repeat to make sure we have a monoculture of just cubensis.
However, because the agar surface is so laden with innoculant, you are gonna have to take a very small sample, and repeatedly transfer to confirm cleanliness.
Next time, when innoculating, perform this technique:

Wait, so I'm not supposed to smear it? Lol! I totally did.. I'm confused on how to actually place the spores inside then, should I scrape some off the foil and then dump it inside, cuz that's what I did but I would then smear. The gif you posted looks like they're smearing side to side too. My biggest issue is it looked as if my loop wasn't grabbing any spores. I made the loop myself using safety wire and a precision knife, taking out the knife and putting in the piece of safety wire.
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Todo lo que hagas en la vida será insignificante; pero es muy importante que lo hagas.
Edited by Mr.Christmas (06/26/20 03:29 PM)
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CocaineBuffet
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Registered: 08/29/19
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I bought a box of 100 of these and they have lasted me over a year. I prefer them over loops/scrapping spores. So much cleaner and easy.
https://www.amazon.com/Puritan-25-806-Sterile-Applicators-Overall/dp/B003IT74FO/ref=sr_1_5?dchild=1&keywords=sterile+cotton+swab+applicator&qid=1593207289&sr=8-5
I just do a zig zag down the middle and a single spot in the empty space on the plate.
As far as your bacteria issue it sounds like your sterile technique needs some work. How did you prep your agar?
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ManifoldPrime
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Registered: 03/16/17
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Making a loop like that and doing a single small smear with a small amount of spores will put thousands of spores on the plate. You flame your loop. you cool it in fresh, untouched agar. you touch your spore print, or scrape it a little bit with the loop, just enough so that spores stick to the loop. Spores are microscopic. you can only see a strong concentration of them. you then streak a portion of the plate. then you flame, cool in agar, and streak a little bit of that initial site of spores to spread it. Like spreading fuckin peanut butter. repeat 2-5 times. only a portion of the plate recieves spores directly, and the rest of the plate is coated in less and less spread out spores.
https://www.shroomery.org/forums/showflat.php/Number/24330957
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Mr.Christmas
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Registered: 04/08/20
Posts: 117
Loc: Colombia
Last seen: 13 days, 25 minutes
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Quote:
ManifoldPrime said: Making a loop like that and doing a single small smear with a small amount of spores will put thousands of spores on the plate. You flame your loop. you cool it in fresh, untouched agar. you touch your spore print, or scrape it a little bit with the loop, just enough so that spores stick to the loop. Spores are microscopic. you can only see a strong concentration of them. you then streak a portion of the plate. then you flame, cool in agar, and streak a little bit of that initial site of spores to spread it. Like spreading fuckin peanut butter. repeat 2-5 times. only a portion of the plate recieves spores directly, and the rest of the plate is coated in less and less spread out spores.
https://www.shroomery.org/forums/showflat.php/Number/24330957
So if some of my petri shows contamination but some doesn't, I can cut the non contaminated portion and move it to a new dish? And basically repeat small transfers to have unlimited dishes?
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Todo lo que hagas en la vida será insignificante; pero es muy importante que lo hagas.
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Mr.Christmas
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Registered: 04/08/20
Posts: 117
Loc: Colombia
Last seen: 13 days, 25 minutes
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Quote:
CocaineBuffet said: I bought a box of 100 of these and they have lasted me over a year. I prefer them over loops/scrapping spores. So much cleaner and easy.
https://www.amazon.com/Puritan-25-806-Sterile-Applicators-Overall/dp/B003IT74FO/ref=sr_1_5?dchild=1&keywords=sterile+cotton+swab+applicator&qid=1593207289&sr=8-5
I just do a zig zag down the middle and a single spot in the empty space on the plate.
As far as your bacteria issue it sounds like your sterile technique needs some work. How did you prep your agar?
Oh cool, I can get those from work. I didn't prep my agar, I bought it from a professional mycologist with one of them fancy flow hoods. As for my glove box procedure, I wipe the box down with a 10% bleach solution and then I let it dry. I wipe all my dishes and tools with alcohol and then place them inside the glove box where I then spray the inside with the same bleach solution. I shower, after I shower I let my arms air dry and sit on my towel and place new latex gloves on, spray alcohol on them, turn my torch on, and begin working. Where I fucked up previously is not having ziplocks in there and also not having parafilm or saram wrap or whatever. I'll also try putting a damp towel under a cookie rack like posted somewhere here and then doing everything on top of the cookie rack. I'm going to post a video next time to see if what I'm doing is correct
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Todo lo que hagas en la vida será insignificante; pero es muy importante que lo hagas.
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ManifoldPrime
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Registered: 03/16/17
Posts: 1,313
Loc: South Africa
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Ditch the bleach. Soapy water is just as effective and wont be toxic for you. everything else sounds fine.
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So if some of my petri shows contamination but some doesn't, I can cut the non contaminated portion and move it to a new dish? And basically repeat small transfers to have unlimited dishes?
Yes. That is the magic of agar. but you have to be observant.
Pouring your own plates is cheap and relatively easy.
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mushpunx
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Registered: 04/20/14
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Get a roll of plastic cling wrap and cut a 1-1.5" section off the end. This works very well for wrapping the edge of plates like you would Parafilm.
Also fresh, unused ziplocks are sterile, you can set your freshly solid plates into them unwrapped untill inoculations time.
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Zakkery



Registered: 10/15/18
Posts: 293
Loc: UK
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Something to note also, are you using an actual glove box? or a still air box?
A box with gloves attached to the holes will create air currents inside and possibly stir up contams. If you have a still air box the contams will mostly stay on the bottom of the box and out of your dish.
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Mr.Christmas
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Registered: 04/08/20
Posts: 117
Loc: Colombia
Last seen: 13 days, 25 minutes
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Re: My Agar Mess Up Update [Re: Zakkery]
#26787018 - 06/27/20 10:42 AM (3 years, 6 months ago) |
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Quote:
Zakkery said: Something to note also, are you using an actual glove box? or a still air box?
A box with gloves attached to the holes will create air currents inside and possibly stir up contams. If you have a still air box the contams will mostly stay on the bottom of the box and out of your dish.
I cut arm holes in a plastic tub, put PVC pipe on the holes, siliconed it shut, then used hose clamps to put gloves with the hands cut off. I've heard that these type of glove boxes do more bad than good as well but I don't see much of a difference from a still air box, to be honest.
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ManifoldPrime said: Ditch the bleach. Soapy water is just as effective and wont be toxic for you. everything else sounds fine.
Do you just squirt a little bit of soap in the water and then mix it?
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Todo lo que hagas en la vida será insignificante; pero es muy importante que lo hagas.
Edited by Mr.Christmas (06/27/20 10:44 AM)
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Roger Clemency
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Registered: 03/23/20
Posts: 2,005
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I just use a tub with two 4" arm holes, spray the inside of the tub with water before working. I've found making it soapy doesnt make any difference just more difficult to clean up.
You can definitely transfer away from contams on agar but if you're consistently getting other stuff on your plates you have a hole in your game somewhere. You probably won't have much luck going to grain until you're getting consistently clean plates.
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ManifoldPrime
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Registered: 03/16/17
Posts: 1,313
Loc: South Africa
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Quote:
I've heard that these type of glove boxes do more bad than good as well but I don't see much of a difference from a still air box, to be honest.
Gloveboxes create a piston/bellows action that makes the air not still. you want still air.
Every tek for making an SAB I've ever seen on this site and others mentions this. Please read the tek in full so you actually know why you are doing the things you are doing. Don't skim. read more.
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Do you just squirt a little bit of soap in the water and then mix it?
Yes, use dishwashing liquid or liquid hand soap.
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Zakkery



Registered: 10/15/18
Posts: 293
Loc: UK
Last seen: 6 days, 18 hours
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Quote:
Sucio said:
Quote:
Zakkery said: Something to note also, are you using an actual glove box? or a still air box?
A box with gloves attached to the holes will create air currents inside and possibly stir up contams. If you have a still air box the contams will mostly stay on the bottom of the box and out of your dish.
I cut arm holes in a plastic tub, put PVC pipe on the holes, siliconed it shut, then used hose clamps to put gloves with the hands cut off. I've heard that these type of glove boxes do more bad than good as well but I don't see much of a difference from a still air box, to be honest.
There's quite an important difference. You can't sterilise the inside of the box so there will be contamts present. You want to use still air so they stay to the bottom of the box. Having gloves attached creates air currents and disturbs the contams, potentially landing them in ur petris. I'd say its something worth changing.
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Mr.Christmas
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Registered: 04/08/20
Posts: 117
Loc: Colombia
Last seen: 13 days, 25 minutes
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Thanks everyone for the advice and the pointers. Much appreciated, I'll make those changes and see how things go.
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Todo lo que hagas en la vida será insignificante; pero es muy importante que lo hagas.
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