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Invisibletripchip
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pouring petri dishes in a glovebox.
    #8961304 - 09/21/08 12:57 AM (15 years, 5 months ago)

hey everyone. hope everyone is having a relaxing weekend. its question time!

ok, i seen RR's video. he uses a flowhood. much different than trying to work in a glove box. its always hard for me to see through the plastic and i gotta bend forward all cocky and my back always hurts and gets cramped.

RR slides his sleeve back over the stack of dishes. I had left over dishes so i just left them stacked and put the unused ones back in the sleeve gently and made a knott on the end.

everything is "as i left it".
once poured i aborted the operation.
i am keeping the glovebox sealed so everything is still in the sterile environment. i mean sterile. bleach spray, oust, alcohal bottle, the works!

ok guessing now i wait for the dishes to cool.
once completely cool open the holes (arm holes have plastic over them) place the rest of my items in the glovebox needed to inoculate, spray a mist of oust in there, let everything settle, and go to work on innoculating the petri dishes.

i will make sure i have some parafilm in the glove box and ill also make sure i dont get it wet by spraying oust on it.

anyways, go in there inoculate everything, wrap parafilm, and then they are ok so come on out into the clean but not sterile environment. since the parafilm is on the dishes its cool.

hmm i guess my question is does anyone see anything wrong with my procedures? any concerns or questions?

i sprayed the inside of the glovebox down till it was soaking wet.
i even dusted off the sleeve of petri dishes and then bleach sprayed the shit out of the outside plastic before i put it in the glove box. once all tools were in the glovebox i sprayed oust in again. i let everything settle for about an hour before going to work in the glovebox.

so far i think everything went smooth. i had a drop of PDA go on one of the sides of te petri dish but that was my only fuck up. i dont htink its a huge deal but it wasnt in the plan. i mean that glove box was so sterile i think i would have died breathing all that shit in if i had to spend 10 minutes in here.

my concern now is where to store the dishes after i inoculate them? should i build something similar to a glovebox, sterilize the inside and store them in there? i just feel like these dishes are going to be really easy to contaminate. ok ill shut up now lol. someone with experience ONLY send some comments my way.
thanks ya'll


--------------------
Psilocybe Cubensis

Psilocybe comes from the Greek root "psilos" meaning bald head and Cubensis because it was first recognized as a new species from specimens collected in Cuba.

                                    :mushroom2:CHIP:mushroom2:

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Invisibletripchip
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Re: pouring petri dishes in a glovebox. [Re: tripchip]
    #8961315 - 09/21/08 12:59 AM (15 years, 5 months ago)

oh yeah, i popped my agar cherry today.


--------------------
Psilocybe Cubensis

Psilocybe comes from the Greek root "psilos" meaning bald head and Cubensis because it was first recognized as a new species from specimens collected in Cuba.

                                    :mushroom2:CHIP:mushroom2:

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OfflineRogerRabbitM
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Re: pouring petri dishes in a glovebox. [Re: tripchip]
    #8961338 - 09/21/08 01:06 AM (15 years, 5 months ago)

The plastic sleeve is the best place to store petri dishes.  Once inoculated, wrap with parafilm.  If you want an extra layer of protection, slip them after wrapping into a large zip lock bag while the mycelium grows.

Oust isn't really necessary in a glovebox, still air is.  Flame your scalpel and/or inoculating loop before use and after each transfer.

Expect contaminants.  Transfer healthy mycelium away from the contamination, not the other way around.  Never allow dishes to fully colonize.
RR


--------------------
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Re: pouring petri dishes in a glovebox. [Re: RogerRabbit]
    #8961352 - 09/21/08 01:11 AM (15 years, 5 months ago)

Hehe funny thing is yesterday i did my first cardboard experiment in the most unsanitary conditions :smile:

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InvisibleKrez
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Re: pouring petri dishes in a glovebox. [Re: omu_negru]
    #8961365 - 09/21/08 01:16 AM (15 years, 5 months ago)

Good luck Trip!  :thumbup:


--------------------
Kids, you tried your best and you failed miserably. The lesson is, never try.  (Homer)

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Invisibletripchip
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Re: pouring petri dishes in a glovebox. [Re: RogerRabbit]
    #8961369 - 09/21/08 01:17 AM (15 years, 5 months ago)

i always appreciate getting advice from you. thanks.

yes the oust might not have been neccessary. this was my first time and i knew i wasnt going to be as organized as a professional so i took the extra precaution and used the oust. i understand the "still air" concept but like i said i just took the extra step.
now that i can see what gave me problems and so on and can be more prepred for future transfers and dish pouring.

I thinking about building or looking for something rather than a regualat tuppaware because the one i used stood barely taller than a sleeve of petri dishes. i planned on using them all but i guess i poured too much in each one?? i stopped pouring right after the bottome was no longer visable. maybe a tad more after bottom was covered. i definately did not go overboard. i used a full whisky bottle. hmmm?? i think i only hit up 13 or 14 dishes with it. whats considered average? or does it depend on the dish size?
these were not partitioned dishes either although transfers will go into partitioned dishes.


--------------------
Psilocybe Cubensis

Psilocybe comes from the Greek root "psilos" meaning bald head and Cubensis because it was first recognized as a new species from specimens collected in Cuba.

                                    :mushroom2:CHIP:mushroom2:

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Invisibletripchip
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Re: pouring petri dishes in a glovebox. [Re: tripchip]
    #8961393 - 09/21/08 01:25 AM (15 years, 5 months ago)

thanks krez! i think it all went well. we'll see. i took notes on agar preperation so that i can back track if anything went wrong but im pretty confident since i watched rr's video in the kitchen while doing it.

one quicky question: how accurate does the water to pda ratio have to be? rr used less media in his video. i think 6 grams is about i tablespoon. for just over 500ml of water he used 3.5 tbl sp. in his video but the recipe should have called for closer to 4 tbl sp.
when i figured out the math it seemed as if he used less media than described in the recipe.

25g per 500ml water
he said "6 grams equals about 1 tbl spoon"
6 X 4 = 24
plus he said he was making slightly more than 500ml

well i used 4 tbl of PDA.. that wont hurt will it? also i used slightly more than 500ML of water.


--------------------
Psilocybe Cubensis

Psilocybe comes from the Greek root "psilos" meaning bald head and Cubensis because it was first recognized as a new species from specimens collected in Cuba.

                                    :mushroom2:CHIP:mushroom2:

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Invisibletripchip
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Re: pouring petri dishes in a glovebox. [Re: tripchip]
    #8961402 - 09/21/08 01:29 AM (15 years, 5 months ago)

to sum the last post up i may be confusing but it just seemed like RR used slightly leess media than what the recipe called for. 

LOL also i watched him carefully and he dug the TBL spoon in his MEA 4 times but the second 2 scoops didnt look full and he said about 3.5 tbl spoons will do the job. im guessing it doesnt have to be accurate. just cant be WAY off or anything.
right?


--------------------
Psilocybe Cubensis

Psilocybe comes from the Greek root "psilos" meaning bald head and Cubensis because it was first recognized as a new species from specimens collected in Cuba.

                                    :mushroom2:CHIP:mushroom2:

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Invisiblewisp

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Re: pouring petri dishes in a glovebox. [Re: tripchip]
    #8961403 - 09/21/08 01:29 AM (15 years, 5 months ago)

Consider using these (with lids) in place of petri dishes. They're PP, so are autoclavable, meaning you can sterilise the agar inside them, they're recyclable and due to being autoclavable, they are fully reusable too.

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OfflineThe shroomy 1
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Re: pouring petri dishes in a glovebox. [Re: tripchip]
    #8961409 - 09/21/08 01:30 AM (15 years, 5 months ago)

Congratulations!  POP!  Everything as far as the procedures seemed solid.  The only information that is not known is "HOW" you prepped the agar.  Most importantly, did you use a pressure cooker.  Then, did you let the agar cool to the point that it was "almost at room temp.  That would eliminate the condensation that tends to form in the petri's.  As far as where to store them while they colonize...  somewhere cool and dark.  I have an old TV stand with "cabinets" that I got at the Salvation Army for like 50.00.  Here are the pics of it and how I use it.  You'll get the idea.




I like the fact that it's smooth non porous and can be sterilized at will.  Keep the area closed and make sure there are no drafts when you do open up the chamber.  Of course still follow all other sterile procedures.. gloves, mask, etc. etc.

The Shroomy 1


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


AMU Q&A thread.

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Invisibletripchip
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Re: pouring petri dishes in a glovebox. [Re: wisp]
    #8961415 - 09/21/08 01:34 AM (15 years, 5 months ago)

damn those are expnsive.. lol.. thanks im sure they are good but im going to stick to petri dishes. i found a site where u get a sleeve of 25 for $4.50. also they are partitioned (3 way)
i bought some from a sponsor. i actaully have almost 300 dishes from 3 different companies lol. 4 compartment ones, 3 compartment ones, and single. i figured i would start the inoculation on full size dishes then transfer to the partitioned ones.


--------------------
Psilocybe Cubensis

Psilocybe comes from the Greek root "psilos" meaning bald head and Cubensis because it was first recognized as a new species from specimens collected in Cuba.

                                    :mushroom2:CHIP:mushroom2:

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OfflineThe shroomy 1
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Re: pouring petri dishes in a glovebox. [Re: tripchip]
    #8961421 - 09/21/08 01:37 AM (15 years, 5 months ago)

LOL!  Just trying to give you an "Idea" of the type of place you want to store them in.  Actually Just get a gray sterlite storage container and store them in there. 

The Shroomy 1


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


AMU Q&A thread.

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Invisibletripchip
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Re: pouring petri dishes in a glovebox. [Re: The shroomy 1]
    #8961437 - 09/21/08 01:45 AM (15 years, 5 months ago)

The Shroomy 1

i didnt mention all that because im more than positive i did everything correctly. here it is though:


jack daniels whisky bottle
cleaned it
drilled small hole in the lid.
mixed 4 regular size tablespoons of PDA in slightly more than 500ML of water.
i added the PDA first.
poured in "SOME" water.
swirled it around.
poured more water in the bottle.
swirled it around.
finally added the rest of the water.
gave it another swirl.

i put tyvek over the top of the bottle and twisted the lid on.
THEN i twisted the cap back off and put a small piece of polyfil inside the lid. (extra protection)

i filled the pressure cooker up with XXXTRA water so that I had more THERMAL mass and my PDA didnt "over boil" from cooling to quickly.

i put the lid on the PC and turned the heat on.
once steam was being released i put on the weight and allowed pressure to build to 15psi

once 15psi was achieved i cooked for 40 minutes and turned it off.
2 hours later it was still very hot. i opened the lid and peek inside. VERY HOT STILL.

15 minutes later i took the bottle out. sprayed it with alcohal and slipped it in the glove box.

let it sit for about 45 more minutes (maybe an hour)
WENT TO WORK!

the agar took along time too cool down. i didnt want a lot of condensation in my dishes. it came out thick, warm and smooth.. lol just like ya know :grin:


Edited by tripchip (09/21/08 01:51 AM)

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Invisibletripchip
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Re: pouring petri dishes in a glovebox. [Re: The shroomy 1]
    #8961442 - 09/21/08 01:46 AM (15 years, 5 months ago)

Quote:

The shroomy 1 said:
LOL!  Just trying to give you an "Idea" of the type of place you want to store them in.  Actually Just get a gray sterlite storage container and store them in there. 

The Shroomy 1




LOLOLOLOLOLOLOL no i was replying to the other post about the petri dish substitute. my LAST reply now was for you.


--------------------
Psilocybe Cubensis

Psilocybe comes from the Greek root "psilos" meaning bald head and Cubensis because it was first recognized as a new species from specimens collected in Cuba.

                                    :mushroom2:CHIP:mushroom2:

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OfflineThe shroomy 1
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Re: pouring petri dishes in a glovebox. [Re: tripchip]
    #8961450 - 09/21/08 01:49 AM (15 years, 5 months ago)

Your procedure seems solid!  Great job.

The Shroomy 1


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


AMU Q&A thread.

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Invisibletripchip
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Re: pouring petri dishes in a glovebox. [Re: The shroomy 1]
    #8961483 - 09/21/08 01:59 AM (15 years, 5 months ago)

i eddited the last sentance. i mean deleted it. it said "i poured the PDA in the whisky bottle" not sure if you saw that but donty know how that slipped in at the end. any ways i think it took a long time to cool because there was so much water in the PC. i was almost afraid it cooled too much but it didnt.

ALSO: I want to mention i put foil on top of the bottle before PCing. AND, i gave the bottle a good shake while it was in the glove box before pouring (before the foil came off).
the little bit of polyfill fit between the tyvek and the lid when it was closed. after i shook it i checked the polyfill and it seemed that the liquid never made it through the tyvek. i think im going to do it all the time so when i give it that shake it doesnt get contams off the filter. i doubt the filter had anything on it because it went right from the PC to the glove box with foil still on. if anyything the little chuck of polyfill would have got the contams first so i think its a good extra step to take. i had to give it the shake because too much PDA sank to the bottom. I though the stuff would have mixed a bit better than it actually did.


--------------------
Psilocybe Cubensis

Psilocybe comes from the Greek root "psilos" meaning bald head and Cubensis because it was first recognized as a new species from specimens collected in Cuba.

                                    :mushroom2:CHIP:mushroom2:

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Invisiblewisp

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Re: pouring petri dishes in a glovebox. [Re: tripchip]
    #8961519 - 09/21/08 02:09 AM (15 years, 5 months ago)

Quote:

tripchip said:
damn those are expnsive.. lol.. thanks im sure they are good but im going to stick to petri dishes. i found a site where u get a sleeve of 25 for $4.50. also they are partitioned (3 way)
i bought some from a sponsor. i actaully have almost 300 dishes from 3 different companies lol. 4 compartment ones, 3 compartment ones, and single. i figured i would start the inoculation on full size dishes then transfer to the partitioned ones.




How is AU$4.27 for 50 "dishes" expensive in comparison to US$4.50 for 25?

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Invisibletripchip
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Re: pouring petri dishes in a glovebox. [Re: wisp]
    #8961542 - 09/21/08 02:18 AM (15 years, 5 months ago)

oh. i thought it was for 1 dish lol.. they come with lids??
maybe i outta go back and ACTUALLY read.


--------------------
Psilocybe Cubensis

Psilocybe comes from the Greek root "psilos" meaning bald head and Cubensis because it was first recognized as a new species from specimens collected in Cuba.

                                    :mushroom2:CHIP:mushroom2:

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Invisibletripchip
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Re: pouring petri dishes in a glovebox. [Re: tripchip]
    #8961550 - 09/21/08 02:21 AM (15 years, 5 months ago)

do regualr wide mouth quart jar lids fot on those? the listing doesnt give too much info on them.


--------------------
Psilocybe Cubensis

Psilocybe comes from the Greek root "psilos" meaning bald head and Cubensis because it was first recognized as a new species from specimens collected in Cuba.

                                    :mushroom2:CHIP:mushroom2:

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Invisiblewisp

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Re: pouring petri dishes in a glovebox. [Re: tripchip]
    #8961558 - 09/21/08 02:26 AM (15 years, 5 months ago)

No, you buy the lids separately. From that site they're another $3.50 for 50 lids. You'd be better off buying from an American supplier if that is where you are.

I don't like petri dishes for the fact that they are single use and not recyclable. Glass is an alternative, but expensive and breaks easily.

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Invisibletripchip
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Re: pouring petri dishes in a glovebox. [Re: wisp]
    #8961569 - 09/21/08 02:30 AM (15 years, 5 months ago)

Quote:

tripsis said:
No, you buy the lids separately. From that site they're another $3.50 for 50 lids. You'd be better off buying from an American supplier if that is where you are.

I don't like petri dishes for the fact that they are single use and not recyclable. Glass is an alternative, but expensive and breaks easily.




thanks for the tip. i may invest in some of these in the future. seems it will make the process a bit easier. 1 less step in the process. plus re-usable.


--------------------
Psilocybe Cubensis

Psilocybe comes from the Greek root "psilos" meaning bald head and Cubensis because it was first recognized as a new species from specimens collected in Cuba.

                                    :mushroom2:CHIP:mushroom2:

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Invisiblewisp

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Re: pouring petri dishes in a glovebox. [Re: tripchip]
    #8961597 - 09/21/08 02:39 AM (15 years, 5 months ago)

Yeah, they are handy. Being able to sterilise the agar inside them is advantageous in reducing the chance of contamintion, although without a flowhood you'll find H2O2 agar to be of great use. As H2O2 is not autoclavable, you'll need to pour the agar after sterilisation. These PP containers are much cheaper in the long-run too.

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OfflineRogerRabbitM
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Re: pouring petri dishes in a glovebox. [Re: wisp]
    #8962533 - 09/21/08 09:52 AM (15 years, 5 months ago)

What a pain in the ass to sterilize agar in a petri dish or other small container.  In addition, instead of one potential path of contamination(whiskey bottle with a filtered lid) as the agar mix cools and contracts(shrinks-thus drawing in air to replace the volume of agar), you have 20 or more.  Not a good idea if you're looking for the highest rate of success.  Besides the above, you need vented lids.  Mycelium MUST breathe.

Glass petri dishes are total crap for our use, because we go through so many dishes very rapidly when isolating strains, or isolating healthy mycelium away from contaminants.  I donated hundreds of dollars worth of Pyrex glass petri dishes to a local school years ago.  They're environmentally unsound because you use polluting soap to wash them and LOT'S of fossil fuel energy to re-sterilize them.  Plastic petri dishes can be re-used for whatever you want such as keeping spore prints, or you can recycle them at your local recycling center.  I've given hundreds away to boys to keep their bug collections in, and girls like them for beads and other jewelry.

I mix agar nutrients weaker than most recipes call for.  We're not using agar to expand mycelium, but rather to clean up our cultures from contamination.  For this reason, the nutrients are not as important.  Mycelium will grow faster and more rhizomorphic on a less nutritious media.  When the time comes to expand the mycelium, use grains and grain to grain transfers.

In the DVD, I used pre-mixed agar from FP. Sporeworks also has an excellent MEA, but I was out at the time, so used some FP agar mix I had(you'll notice I turned the jar so the label didn't show :wink:. In real-life, I mix my own, but for the new growers watching the DVD, I wanted to make it as easy as possible, while still getting good results.  Once you gain a bit of experience, mix your own.  Use 20 grams of agar-agar per liter of water to solidify the mix, but use less nutrients than the recipes call for, for the reasons stated above.

Peroxide is toxic to fungi, ALL fungi, so the last thing you want is peroxidated agar.  You're supposed to transfer healthy mycelium away from contaminants, not hide or prevent the contaminants from growing temporarily.  You actually WANT the contaminants to grow on your agar so you can see them and get your mycelium away.  Antibiotics are a great addition if you're trying to isolate mushroom mycelium from an outdoor fruit that is laden with bacteria.  Antibiotics, which are made from mycelium metabolites, won't impede your mycelium, but WILL impede bacteria.
RR


--------------------
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semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
Thomas Edison

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Invisiblewisp

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Re: pouring petri dishes in a glovebox. [Re: RogerRabbit]
    #8966847 - 09/22/08 03:30 AM (15 years, 5 months ago)

Have you used the containers I'm talking about RR? They work absolutely fine, vented or not. What you say about air entering as the agar cools makes sense in theory, but in practice I have seen no contaminants form on dishes sterilised with agar inside them if they are left unopened.

Personally I think pouring 100 dishes in a glovebox in more of a pain in the arse than sterilising 100 dishes with agar inside them. Perhaps for those with a flowhood, pouring agar is a painless activity. As I only have a glovebox, I find pouring agar in large quantities to be an exercise I'd rather avoid.

As for recycling disposable plastic petri dishes, I don't know how it is in America, but here in Australia polystyrene cannot be recycled. Using 1000's of plastic dishes each year, just to throw them out, is completely environmentally irresponsible. Giving a few hundred away to be reused for other purposes doesn't justify the many hundreds more that are thrown out.

The very fact that peroxide is toxic to fungi is what makes it so useful. Mixed at the correct concentration, mushroom mycelium will adapt to its presence in agar, but it will kill any mould spores present. I don't recommend it for prolonged use or all of the time, but it is very useful in some circumstances.

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Invisibletripchip
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Re: pouring petri dishes in a glovebox. [Re: wisp]
    #8966965 - 09/22/08 05:09 AM (15 years, 5 months ago)

yep ill stick to the plastic dishes. i agree with RR.

Tripsis, get a bigger glovebox. yes its is a pain in the ass to work in one. I need to go larger myself. Ill be on the lookout for something bigger than what i got and mines pretty large. i may make my own using sheets of plexi glass. if its big enough its quick. I can pour 25 dishes in a few minutes, slip my arms out, cover the holes up where my arms whent through, and then come back to them when they are cool. Mine is a decent size but im going to go larger so i dont feel so cramped in the one i have.
If your way works for you than thats all that matters. I hate cleaning so pre-sterilezed dishes are great for me. pouring from the whisky bottle works great so far and is the easiest part. I load everything into my gloves box, spray it down with bleach water, come back in 30 minutes and then im only pouring and messing around inside the glovebox for 5 minutes. ill re-enter and do what i gotta do when they are cool.


Today i made some more PDA. I poured 20 more dishes for future transfers. Plus i want to see how easily they contam. Im kinda experimenting with them a bit since im new to it. im going to let them sit around for a week and then see how well my sterile procedures were. im betting on very little contamination in any.


ill make transfers from the first batchmto the new ones. The dishes I just made are going to sit for a bit so I can see how sterile my techniques were. Ill use the ones that show no contams when im ready for them. or as easy as it is i can make new dishes.

Is there a time frame on when these dishes have to be used after I pour them? i dont think so but a comment on it would be appreciated. they'll be stored at room temp untill use.

i was bored today and as easy as making the agar and pouring the dishes were i just did it for fun :grin:


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Psilocybe Cubensis

Psilocybe comes from the Greek root "psilos" meaning bald head and Cubensis because it was first recognized as a new species from specimens collected in Cuba.

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