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#21 Dimitri2teachme

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Posted 08 April 2019 - 01:29 PM

Is the loss of permeability due to moisture hitting the tape? i am gonna cover the lid with paper towel, underneath the tin foil, just in case any moisture makes its way in there.

Edited by Dimitri2teachme, 08 April 2019 - 01:30 PM.


#22 raymycoto

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Posted 08 April 2019 - 01:46 PM

Not sure. Or the heat perhaps. Or perhaps I was wrong all together but I'm pretty sure that is what happened. I checked the CO2 level with a monitor I have and it was off the charts, like 40%.



#23 Dimitri2teachme

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Posted 08 April 2019 - 02:46 PM

I suppose we will find out

I have actually decided polyfil, just because it is what I have on hand. Just picked up an old stuffed toy from a mantle from a christmas decoration that shouldnt have been out still. Going to boil it and then dry it out in the oven for 2 hours at 210°f. I know some say this is excessive, but eh, peace of mind is the route ill take.

Edited by Dimitri2teachme, 08 April 2019 - 04:05 PM.


#24 Billcoz

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Posted 08 April 2019 - 04:55 PM

Hey, i read the coffe can post! A while back while I was lurking. I'm actually going to use a pretzel bucket too haha and a 25qt sterilite. Plan on a 1:1 ratio for spawn to sub just because i want it to recover quickly to beat contam. Going to use coco coir, alone. I will sterilize the coir for one tub, and pasturize the other. For research!

My jars, i wanted to post about. They arent jars. I figure some will laugh, or smite me, and ive never seen any real success had with them. By that i mean I havent seen them used. I have seen sterilized grain sold in them though.. so i figured why not. They hold a pint and a half. I can always still get jars, this is just what I had around. Also these are not permanent.


Btw coz that was an awesome write up

Those plastic containers will work fine as long as they are pp5 plastic, people do have success with them all the time. Good luck man.



#25 Dimitri2teachme

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Posted 10 April 2019 - 02:23 PM

Those plastic containers will work fine as long as they are pp5 plastic, people do have success with them all the time. Good luck man.


I actually just went out and got some jars. I kindof bought the pp5 in a panick haha. Thinking "oh god I dont have jars gotta get some jars" I went to the hobby store, and they over priced them, so I bought pp5. Then I saw walmart had the jars, for half the price, and picked some up today. The SHIPs are curing now and the other holes are waiting for micro pore... Agar is arriving this evening, and I will test the viability of these spores that were frozen. Then I will post results ASAP

Edited by Dimitri2teachme, 10 April 2019 - 02:25 PM.


#26 Billcoz

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Posted 10 April 2019 - 06:47 PM

Cool.



#27 Dimitri2teachme

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Posted 10 April 2019 - 09:38 PM

Ok guys so first off, got a big problem with moisture build up in my agar dishes. Is this even a problem at all or is it ok? Also (this ones funny to tell you the truth) while innoculating the dish I accidentally shot a good tea spoon of spore solution. Just kinda shot out because I applied to much pressure. I took it back to the glove box when I noticed and poured out the excess. This stuff takes finesse... Wow

#28 raymycoto

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Posted 10 April 2019 - 11:37 PM

Maybe tell us first how you made your plates re the moisture.

 

Re xs solution from a syringe. Takes practice but here is my recommendation. One drop is plenty for a plate. Have a sterile instrument of some sort. I use a mini lab spoon that's about 6mm diameter. I flame it, let it cool, balance it over the edge of an extra petri dish. Meanwhile place your spore solution drop. Hold the syringe vertical with the tip up. With one hand and with the syringe top off, use your thumb and forefinger on the barrel and 4th and 5th fingers against your palm to gently aspirate about 1 or so cc of air. Now shake the syringe. The air is needed to break up any settled spores. They gravite downward.

 

Now, the other reason to suck the air is to move the plunger. This is to unstick it. Next, still in one hand, move the plunger back and watch for the column of liquid to make it to the absolute syringe tip. No need to have a needle on it. You might even allow the solution to build up a meniscus over the syringe tip. Next, open your plate and either touch the bulging microdrop onto the plate or carefully with one hand squeeze out just a drop and touch the syringe to the agar if you want half a drop before it drops. A large drop is 100 microliters and that may be too much.

 

Now replace the syringe tip or needle. Take your now (hopefully) cool micro spoon and rub it onto the periphery of the agar to make sure it is not hot (no sizzles or melting agar). Next use a circular motion and distribute the drop starting at the center.

 

I like to have the surface totally dry (thus really spread your drop well to dry it quickly). Gelled agar really does not absorb water like you might think it would. A flow hood will do this in a minute or two. Not sure how long it will take in a SAB. Spores and myc both do best on a dry surface plump with moisture like a good agar prep or good grain prep. Spores on wet agar or wet grain may either take forever or not germinate promptly or may allow contams to present.

 

So the best spore to agar prep will look dry with maybe a hint of film on the surface if you hold it at the right angle to the light.


Edited by raymycoto, 10 April 2019 - 11:41 PM.


#29 Dimitri2teachme

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Posted 11 April 2019 - 08:14 AM

I did peace frogs agar prep, down to the last tiny detail. Peace frog even explains how to deal with condensation, so I did what they suggested and turned the container upside down for a few hours, and flung out the excess water. No matter what there is still a small amount of h2o that settles in the corners. I suppose what i will do is make my agar in a large batch rather than one by one, so i can clear the whole batch of excess moisture before pouring. Would this work?

I also turned them upside down while they germinate, to potentially run the water away from the agar. I will be doing more plates but this is my attempt at saving the two I have poured.

Edited by Dimitri2teachme, 11 April 2019 - 09:11 AM.


#30 raymycoto

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Posted 11 April 2019 - 12:13 PM

PF's recommendations are indeed good. Do you have a reference to it and I'll take a look?



#31 Dimitri2teachme

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Posted 11 April 2019 - 01:04 PM

https://mycotopia.ne...ipes/?p=1247155<--This was what I was using

#32 raymycoto

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Posted 11 April 2019 - 02:24 PM

Looks like it's a "no pour" technique. I have done that more or less. It does tend to collect moisture since the jar is sealed. Pouring plates depends somewhat on a lami flow hood to dry them out but they are inherently less wet since they are always open until they cool. And . . . with the pour technique, the plates are cool and thus the whole process cools off quickly and doesn't yield a bunch of water vapor.

 

I'm just guessing but you might be able to modify the 'pour' technique by leaving the jar open but covered with a gas permeable layer like surgical drape or Tyek, rubber banded around the top of the vial. Remove it from the sterilizer and just let it cool, then remove the cloth and cover with the metal top.

 

Maybe I'll try that if I get a chance.

 

I do prefer the pour technique. I keep 250 cc steriled bottles of agar, 1/2 full, available. Then I heat in a stock pot with 1" of water or microwave to liquefy. Then I flash the plates I need and pour and generally use right away but I like to keep 1 or 2 available pre poured.



#33 Dimitri2teachme

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Posted 11 April 2019 - 05:25 PM

I have a GE hole in my agar lid, it doesnt seem like it lets moisture out. However i am thinking maybe I should let them sit upside down longer before use. The agar plates I attempted saving are dry now on the surface. I will be attempting again tomorrow.

As for the bags that originated this thread they smell like sour milk.. Trash now.

#34 Billcoz

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Posted 11 April 2019 - 05:59 PM

I have a GE hole in my agar lid, it doesnt seem like it lets moisture out. However i am thinking maybe I should let them sit upside down longer before use. The agar plates I attempted saving are dry now on the surface. I will be attempting again tomorrow.

As for the bags that originated this thread they smell like sour milk.. Trash now.

The way I get no condensation is to let the agar cool completely, then wipe off any condensation on the sides in the jars BEFORE putting lids on, put a folded up paper towel, folded into 3" squares on the lid before adding the tinfoil over it. I never have any condensation at all, the most important part is to let em cool, and maybe add a tiny bit more agar to the mix



#35 Dimitri2teachme

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Posted 11 April 2019 - 07:22 PM


I have a GE hole in my agar lid, it doesnt seem like it lets moisture out. However i am thinking maybe I should let them sit upside down longer before use. The agar plates I attempted saving are dry now on the surface. I will be attempting again tomorrow.

As for the bags that originated this thread they smell like sour milk.. Trash now.

The way I get no condensation is to let the agar cool completely, then wipe off any condensation on the sides in the jars BEFORE putting lids on, put a folded up paper towel, folded into 3" squares on the lid before adding the tinfoil over it. I never have any condensation at all, the most important part is to let em cool, and maybe add a tiny bit more agar to the mix

Is there such a thing as too stiff? Or am I kindof just cool to play with the softness? If that is the case I will just add an extra tenth gram to my batch and see what happens.

#36 coorsmikey

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Posted 11 April 2019 - 08:22 PM

 

I have a GE hole in my agar lid, it doesnt seem like it lets moisture out. However i am thinking maybe I should let them sit upside down longer before use. The agar plates I attempted saving are dry now on the surface. I will be attempting again tomorrow.

As for the bags that originated this thread they smell like sour milk.. Trash now.

The way I get no condensation is to let the agar cool completely, then wipe off any condensation on the sides in the jars BEFORE putting lids on, put a folded up paper towel, folded into 3" squares on the lid before adding the tinfoil over it. I never have any condensation at all, the most important part is to let em cool, and maybe add a tiny bit more agar to the mix

 

Do you do this after you pour sterile agar that has cooled? Is the paper towel clean or slightly soiled? How do you get "No Condensation" then wipe off any condensation if you had "No Condensation"?


Edited by coorsmikey, 11 April 2019 - 08:31 PM.


#37 raymycoto

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Posted 11 April 2019 - 08:38 PM

There is indeed an optimal hardness to the agar. Too soft will slide off the plate eventually when you invert it or tilt. Too hard will not give up moisture enough for spores and myc to germinate effectively. Too hard eventually will happen with really old plates as they dry out but that takes a while, like months or more if you saran wrap them or less if you parafilm them.

 

I do think there is a reasonable range to shoot for for success so don't get too uptight about that, but I have made mistakes both ways in experimentation, particularly in the soft range. Be aware that additives to the agar may reduce its hardening effectiveness. I have found that grain water and grain and / or poop material added could soften the agar. However, I have not thoroughly evaluated, I found that I could add some extra agar to compensate. But I would not worry about this unless you note soft agar. I don't think this problem is very common.

 

 

If that is the case I will just add an extra tenth gram to my batch and see what happens.

 

I'm pretty sure that adding more agar will not help much with condensation.  Like I mentioned, I have done 'no pour' and it's a good tech to know and to master. But no pour and no lami flow to blow away condensation - I'm not sure how to handle that.

 

Perhaps a sterile airflow through a small, true hepa filter like what's used in a shop vac - into your SAB could provide safe air movement to dry your plates. It's not a SAB, then, but that may work. I have a friend who has done that. I'll ask how and why and if he does agar in the non-SAB.

 

I think that Mickey is hoping you have a really clean paper towel to wipe your jars, like sterile.

 

I would hate to have to resort to that or make that part of my agar work flow.



#38 Billcoz

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Posted 11 April 2019 - 10:43 PM

 

 

I have a GE hole in my agar lid, it doesnt seem like it lets moisture out. However i am thinking maybe I should let them sit upside down longer before use. The agar plates I attempted saving are dry now on the surface. I will be attempting again tomorrow.

As for the bags that originated this thread they smell like sour milk.. Trash now.

The way I get no condensation is to let the agar cool completely, then wipe off any condensation on the sides in the jars BEFORE putting lids on, put a folded up paper towel, folded into 3" squares on the lid before adding the tinfoil over it. I never have any condensation at all, the most important part is to let em cool, and maybe add a tiny bit more agar to the mix

 

Do you do this after you pour sterile agar that has cooled? Is the paper towel clean or slightly soiled? How do you get "No Condensation" then wipe off any condensation if you had "No Condensation"?

 

Ha, well I meant I get no excess water on the agar, I wipe any condensation off after it cools, no more forms if it's not hot. If it is hot and you put the lid on it can steam back up. The stiffer agar probably don't make much diff, your right ray.


Edited by Billcoz, 11 April 2019 - 10:45 PM.


#39 Dimitri2teachme

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Posted 12 April 2019 - 08:58 AM

I did try wiping them out with a paper towel, and then pc again, but the moisture came back. Maybe I just under estimated cooling time?? Definately should not have put spores on those plates tho. Try try again hehe


Just for the record, I did not wipe them and pc after innoculation haha. Just something I didnt really include while explaining my agar prep.

Edited by Dimitri2teachme, 12 April 2019 - 09:00 AM.


#40 Dimitri2teachme

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Posted 12 April 2019 - 09:03 AM

There is indeed an optimal hardness to the agar. Too soft will slide off the plate eventually when you invert it or tilt. Too hard will not give up moisture enough for spores and myc to germinate effectively. Too hard eventually will happen with really old plates as they dry out but that takes a while, like months or more if you saran wrap them or less if you parafilm them.

I do think there is a reasonable range to shoot for for success so don't get too uptight about that, but I have made mistakes both ways in experimentation, particularly in the soft range. Be aware that additives to the agar may reduce its hardening effectiveness. I have found that grain water and grain and / or poop material added could soften the agar. However, I have not thoroughly evaluated, I found that I could add some extra agar to compensate. But I would not worry about this unless you note soft agar. I don't think this problem is very common.


If that is the case I will just add an extra tenth gram to my batch and see what happens.

I'm pretty sure that adding more agar will not help much with condensation. Like I mentioned, I have done 'no pour' and it's a good tech to know and to master. But no pour and no lami flow to blow away condensation - I'm not sure how to handle that.

Perhaps a sterile airflow through a small, true hepa filter like what's used in a shop vac - into your SAB could provide safe air movement to dry your plates. It's not a SAB, then, but that may work. I have a friend who has done that. I'll ask how and why and if he does agar in the non-SAB.

I think that Mickey is hoping you have a really clean paper towel to wipe your jars, like sterile.

I would hate to have to resort to that or make that part of my agar work flow.

I am extremely interested in your friends not so SAB. I was going to make one myself a while ago, but could not find much solid evidence that it was effective. Are his success rates closer to lami flow, or a normal SAB? I've heard it is almost not worth it and to just use a still air box.

Edited by Dimitri2teachme, 12 April 2019 - 09:10 AM.





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