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Offlinekrypto2000
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Check my SAB/Agar procedure and a couple questions on handling petris.
    #18953602 - 10/09/13 10:24 AM (10 years, 3 months ago)

So far with agar I've had nothing but contams, but I'm going to try it again so I want to verify my entire procedure before doing so. First I run a hepa filter in the room for a few hours beforehand. Take a shower before doing the work and put on clean clothes. I'll be pressure cooking the agar in a closed jar (in the past I've used a pyrex measuring dish with foil on top). Ok, so now I'm clean, the agar is cooling.

Next I transfer the agar to the grow room where my SAB is located. Wipe down the glovebox with soapy water leaving residue all over the bottom and sides (the lid goes untouched as this can cause more problems than it solves). I have a metal rack I use to hold any utensils and petris from sitting on the ground in the soapy water so next I wipe that down with isopropyl and put it into the box. Wipe down my clean syringe for transfering agar from the jar to the dishes in iso and put that in the box (I'll be a using presterilized still in the package syringe and needle for this so I'll just wipe down the packages in this case and put those in the box. I'm a little unsure of how to transfer the sterile petris in the box, but I'll ask that in the end, lets just assume they've been put in there though for the sake of finishing this procedure.

So now everything is in the box except me. So next I turn off the hepa filter in the room and put on a nylon jacket to cover my arms, and a facemask. Then put on the gloves and tape them together. Lastly wipe down my gloves and forearms with a generous amount of isopropyl, wait for them to dry and the air in the box to settle, then in the box my hands go along with the alcohol soaked paper towel used to wipe my gloves/arms off. Set the paper towel on the rack with the syringe. Then take out the syringe and needle from the packaging and put them together, set back on rack. Pickup papertowel, wipe self healing injection port on agar jar, set papertowel down, and then use syringe to suck up some agar. Pickup dish and put it on rack, take off lid with one hand, squirt agar into it with the other, put on lid. Repeat until all dishes have been filled or agar runs out. Then I take the lid off the SAB, stack dishes (should I wipe them iso here?) and then wrap them in seran wrap to keep closed and contaminants out until use.

So first off anything wrong or missing from that procedure? Secondly is my question on how to handle the dishes. I've actually got two. Right now I have a few sleeves of presterilised dishes. Do I need to use all of them if I open the sleeve or can I simply close the sleeve and take out as many as I need? I'd prefer not to use all 20 at a time as that's quite a lot and 5-10 would suffice for now. Second, what's the proper way to then transfers these into the SAB? Wipe each dish with iso and then put them where? I don't imagine sitting them in the soap water is a good idea? How do you guys do this? Most of you don't even seem to have a metal rack like I use so is everything just being set into the soapy water? I can't imagine so.


Edited by krypto2000 (10/09/13 10:27 AM)


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Offlineskippydude
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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: krypto2000]
    #18953700 - 10/09/13 10:51 AM (10 years, 3 months ago)

SABs suck for agar because they are cramped for space.
With that said, before I bought a flow hood I would
1. Wipe kitchen table with alcohol, bleach or ammonia.
2. Mist inside of SAB & table with sanitizing fluid of choice.
3. Place sleeve of petris, vessel of agar & saran or Para Film on table.
4. Lose SABs lid and flip it upside down over supplies(table is a more stabile work surface than bottom of tub)
5. Let it sit until agar cools enough to pour
6. Turn off AC, house heater or any fans that move air around in the house
7. Wipe gloved hands with alcohol
8. Move slowly and pour dishes
9. Let cool completely and wrap dishes that won't be used right away with saran or para film. Knock up and wrap the remaining, if any.

I use a 1000ml Erlenmeyer flask or a 750ml whiskey bottle covered with foil during PC and pour, no syringe. If you seal your vessel it will suck in air when you open it, from the vacuum and possible contaminates.
When using my SAB for agar I was getting 90+ success rate


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Offlinekrypto2000
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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: skippydude]
    #18953747 - 10/09/13 11:03 AM (10 years, 3 months ago)

I wasn't planning to open the agar jar, that's what the self healing injection port is for. The reason I'm doing it that way other than it possibly being safer is because last time I attempted to do it the whole GB steamed up, I couldn't see a thing, and ended up making a mess either over or underfilling petris. This way there should be a very minimal amount of steam and I can actually measure how much solution they each recieve. Someone else had suggested that way of doing it to me when I complained of my troubles in another thread.


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OfflineOregonMushys
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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: krypto2000]
    #18953760 - 10/09/13 11:07 AM (10 years, 3 months ago)

Your problems may have come from PC'ing the agar in a pyrex dish with foil on top. Whatever you PC your agar in has to have a filtered lid. RR uses a whiskey bottle with hole drilled in the lid, screwed over a filter such as SFD or 2 layers of tyvek painter overalls. As the PC cools, contams get pulled into it slowly and if you lack the proper filtered lid, they'll get inside your agar.

Transferring the agar into the dishes with a syringe might also be where you may be inviting contams. They may have came from PC'ing your agar in a container that lacks a proper filtered lid.

I would wait for one of the pros to chime in. And ya most people use a small rack on the bottom of their SAB's. Watch RR's videos on agar http://www.mushroomvideos.com


Edited by OregonMushys (10/09/13 12:50 PM)


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Offlineskippydude
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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: OregonMushys]
    #18953788 - 10/09/13 11:15 AM (10 years, 3 months ago)

I use a 1000ml Erlenmeyer flask with foil on it with 100% success in front of my flow hood
Trust me that is not the problem
IMO the problem is the extra stuff like the syringe and the rack, you need to keep it simple
If your steaming up, then the agar hasn't cooled enough
If it's a "glove box" and not a "still air box" IMO this is the problem


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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: OregonMushys]
    #18953807 - 10/09/13 11:18 AM (10 years, 3 months ago)

hey I suck at agar too but I would suggest just pouring it into the plates. I'm experimenting with no-pour jars using ELME(spraymalt) in 1/4pint pp5 containers. I have pyrex petri dishes which are a pain to handle especially in a SAB. Like said above using the SAB without the lid helps because you can use any flat surface then. I like to use a white towel soaked in dilute bleach on the bottom of my SAB and I spray the inside with warm soapy water. Alcohol is already saturated into a few folded towels for wiping anything. I used to flame inside but I flame all tool outside of the SAB now(hasn't made a difference). IMO using the SHIP and syringe to do petris vs pouring is more contam prone than pouring since there's more movement involved. Pouring lets you do as few gestures as possible look at how RR does it in his videos it's really quite rehearsed looking and not haphazard. I think that's the real key. Every time I do it I turn into michael J fox with the dexterity of an autistic 3 year old.


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Offlinekrypto2000
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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: skippydude]
    #18953826 - 10/09/13 11:21 AM (10 years, 3 months ago)

Oh, it is a SAB, though since we always wear gloves in a SAB I really don't see the difference. Wouldn't everything inside the PC be sterilized including the air and the PC itself? How could outside contaminants be introduced while cooling? Until the pressure cooker reaches near room temperature, and especially until the pressure has equalized, things will only leave the PC, not go into it.

How would I use a SAB without a rack of somesort? Unless I hold everything in my hands where am I supposed to set things down besides in the contaminated soapy water?


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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: krypto2000]
    #18953878 - 10/09/13 11:30 AM (10 years, 3 months ago)

Hot air expands as it cools it draws air into the PC. No contamination gets through the foil cover for me.
If you wipe the table with alcohol or 10% bleach solution pryor to placing the 3 necessary items(dishes, agar & wrapping) on it you'll be fine. Lightly mist the walls of the tub, not dripping, this catches any particles that touch it.


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Offlinekrypto2000
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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: skippydude]
    #18953981 - 10/09/13 11:53 AM (10 years, 3 months ago)

So wipe it with bleach and I can just set my items directly into the bleach solution?


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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: krypto2000]
    #18954048 - 10/09/13 12:08 PM (10 years, 3 months ago)

A light wipe down to sanitize, I'm  not soaking it.
A light mist on the inside walls of the SAB too, not dripping wet
Wait at least 20 minutes before starting work to let the air inside the box settle
Don't hurry your movement when entering and exiting the box, be slow and sure
With time and practice pouring gets easier and cleaner


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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: skippydude]
    #18954086 - 10/09/13 12:18 PM (10 years, 3 months ago)

My SAB is upside-down on a damp towel. The towel serves as a gasket and a trap. It almost doesn't matter what wetting solution you use because the point is to have still air and to have something wet for potential contams to stick too. If you think you are killing anything to the degree of usefulness, you are only kidding yourself. I use Dr. Bronner's Magic Soap diluted with water.

I think you will find it difficult to suck up and squirt out agar. I've never tried it, but my guess is it will be too thick and cool too fast and clog your syringe.

As suggested, use a bottle with a filtered lid and pour your agar. As also mentioned, your past issue may be the unsealed glass petri dishes. I would think it would get contaminated sometime between cooling and transferring to your SAB.

If you are using pre-sterilized petris, you can just pour a few if you want. I put the left-overs in a new 1qt ziplock. 4 fit easily. Then 5-6 of those baggies will fit in a gallon size ziplock.

You can also pour the dishes and store them this way as well. But you don't need to parafilm or wrap unused dishes.

I use Snapple bottles for small pours.

You don't need to tape your gloves to your sleeves.

Tips:

Don't allow hands or tools to hover over your open containers. I lift the lid and pour right at the edge to help prevent something from falling in.

Work smoothly, but diligently. Try not to make too many turbulent movements, but don't move at a snails pace either.

IMHO, I think you will have better success with pouring than you did with sterilizing the agar in the dish. Good luck and keep us posted on your progress.


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Edited by SpitballJedi (10/09/13 12:20 PM)


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Offlineskippydude
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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: SpitballJedi]
    #18954158 - 10/09/13 12:36 PM (10 years, 3 months ago)

I didn't catch that you are using glass dishes. When I use my glass it gets wrapped in foil and PCed with the agar. When I first started I thought glass was the way to go, save the planet and all that jazz. I now have about 50 glass dishes that rarely get used. Now I buy a case of 500 plastic dishes in sleeves of 20. Plus you never know when you may want to trade cultures and you won't want to give up your glass


Edited by skippydude (10/09/13 12:37 PM)


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Offlinekrypto2000
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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: skippydude]
    #18954253 - 10/09/13 12:55 PM (10 years, 3 months ago)

I'm not using glass, I have some plastic dispoasables. So skip the syringe, wait for the agar to cool before pouring, skip the tape. Otherwise everything look good? So should I just take the whole plastic sleeve into the box and just set it down in the soap cleaning it off afterwards? I assume I just just seal that bag up and the remaining petris will be safe inside of it?


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Offlinekrypto2000
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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: krypto2000]
    #18954652 - 10/09/13 02:27 PM (10 years, 3 months ago)

So the pcer just released pressure. How long do you guys let it cool before pouring?


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OfflineOregonMushys
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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: krypto2000]
    #18954717 - 10/09/13 02:39 PM (10 years, 3 months ago)

The bottom of your glove box shouldnt necessarily be soaked in soap water just wiped down with it prior, and maybe misted a tiny bit. For the petris, i would find some sort of small rack like in RR video and wipe the rack down with iso prior setting in SAB. Before pouring the dishes, i would just set the petri sleeve on a folded up paper towel lightly soaked in iso :shrug:

I actually havn't done agar yet, i need some type of rack to set my petris on. Could i just set the petris on a iso soaked paper towel? Oh, and put the agar bottle in the SAB and get to work as soon as its cool enough to handle but not solidified.


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Offlinekrypto2000
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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: krypto2000]
    #18954783 - 10/09/13 02:51 PM (10 years, 3 months ago)

Ok, well I ended up with three dishes, we'll see if they contam. The procedure I outlined was slightly different in practice. It was more like get drunk, stumble into GB, pour shit in dishes, cuss at process, put dishes in ziplock. Let's hope they're ok. I dont think they'd hire me in a lab.


Edited by krypto2000 (10/09/13 02:52 PM)


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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: krypto2000]
    #18954945 - 10/09/13 03:32 PM (10 years, 3 months ago)

Quote:

krypto2000 said:
I'm not using glass, I have some plastic dispoasables.




My bad. I misread.

Quote:

krypto2000 said:
I'll be pressure cooking the agar in a closed jar (in the past I've used a pyrex measuring dish with foil on top). Ok, so now I'm clean, the agar is cooling.




That's why you've been having cantam issues. You have to use a filtered lid. Foil alone will not suffice.

I let the agar cool enough to handle with my nitrile gloves on. I usually have to make more than I think I need because some of it will cool and congeal inside the bottle.

I have a little wire rack shelf in my SAB for when I pour agar. I don't like sitting my petris on the bottom.

Some people do put the dishes back in the original bag. I find it a PITA. That's why I use ziplocks.

Put the whole sleeve in the SAB before opening. If you expose the petris to outside air, they will no longer be sterile.

Sorry I'm late to the party, I had to go back to work.

Keep us posted.


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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: krypto2000]
    #18954958 - 10/09/13 03:34 PM (10 years, 3 months ago)

I wait till the PC is cool enough to pick up with my wrists. If the PC is warm, but doesn't burn me, it's the perfect temperature to pour.
Agar starts gelling at about 88F so don't wait too long.


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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: skippydude]
    #18956681 - 10/09/13 09:33 PM (10 years, 3 months ago)

Maybe try a no pour tek? FooMan, DocT, Professor Pinhead and others have posted teks. Seems to work pretty good.


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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: Pitbullgrin]
    #18958660 - 10/10/13 10:39 AM (10 years, 3 months ago)

No pour jars can be a PITA to work with. Some don't mind, but I'm glad I switched to pouring petris.


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Offlinekrypto2000
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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: SpitballJedi]
    #18958864 - 10/10/13 11:22 AM (10 years, 3 months ago)

By no pour jars you guys are referring to pcing them in half pints and using those instead of proper petris? I've thought about doing that, but it seems like a half measure. The only real benefit I see with it is ease of doing MS or liquid culture since you can inoculate them without opening them, but if you ever want to do isolation work I don't see the point, and if all you're doing is nocing up a petri to transfer to grain you might as well skip it and just do a g2g.


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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: krypto2000]
    #18959004 - 10/10/13 11:50 AM (10 years, 3 months ago)

A Jedi you will become, Pattawan.


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Offlinekrypto2000
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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: SpitballJedi]
    #18959056 - 10/10/13 12:04 PM (10 years, 3 months ago)

I hope so :mushroom2:


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InvisiblePitbullgrin
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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: krypto2000]
    #18961803 - 10/10/13 10:24 PM (10 years, 3 months ago)

10g agar 10g LME 500ml/2cups h2o.1/4 pint jars with plastic unfiltered lids PCd for 45 min. Used for print to tissues. Use what works for you. Agar is fun!! :headbang3:


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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: Pitbullgrin]
    #18962802 - 10/11/13 07:09 AM (10 years, 3 months ago)

Quote:

Pitbullgrin said:
10g agar 10g LME 500ml/2cups h2o.1/4 pint jars with plastic unfiltered lids PCd for 45 min. Used for print to tissues. Use what works for you. Agar is fun!! :headbang3:




I always got mad condensation using those with metal filtered lids. Unless your talking the whole jar being plastic like the 1/4pt zip locks those implode without a gas exchange.


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Offlinekrypto2000
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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: bodhisatta]
    #18963003 - 10/11/13 08:47 AM (10 years, 3 months ago)

Is condensation bad other than it obscuring your view?


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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: krypto2000]
    #18963020 - 10/11/13 08:57 AM (10 years, 3 months ago)

Yes it aids in bacterial infections. It makes myc growth happen everywhere if you use spores, which isn't good for sectoring and isolation. It makes isolation hard to do since the water carries everything across the agar's surface.

I had these problems on every no pour jar I ever made using 10g agar 10g ELME/LME and 500ml h2o


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Re: Check my SAB/Agar procedure and a couple questions on handling petris. [Re: bodhisatta]
    #18963102 - 10/11/13 09:25 AM (10 years, 3 months ago)

.5ml Gentamicin per 500ml agar would eliminate most of your bacteria problems, but condensation is still bad. Store your dishes where the temperature is stable to avoid it. Storing dishes upside down helps too. In my culture fridge the slants are kept in a thick Styrofoam box as a buffer against temperature change from opening the door.


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