94 replies to this topic

[ipc]

that is some thriving myc

[CatsAndBats]

nice thread cat!! i'm not sure how i missed this.

agreed with peace, once you learn to use agar you'll never go back. i have no idea how i managed without it.

pouring agar into petri dishes is simple. lucky for me i get my petri's for free these days.

 

 

Thanks man, your input on any of my threads is welcome.

 

whitethumb! Good to see you on here, bro.

 

Another key member that I learned the way of the agar from.

[MsBehavin420]

Um. Can I pour plates before I Pc them? Am I in the wrong a here? I poured my jars before I PC'd them. However that was glass.

Edited by MsBehavin420, 09 April 2018 - 09:31 AM.

[CatsAndBats]

Um. Can I pour plates before I Pc them? Am I in the wrong a here? I poured my jars before I PC'd them. However that was glass.

 

 

No. Petris are poured after PC'ing. Maybe if they're glass dishes but I have no experience with that.

Edited by CatsAndBats, 09 April 2018 - 10:13 AM.

[MsBehavin420]

Sigh

[raymycoto]

 

Um. Can I pour plates before I Pc them? Am I in the wrong a here? I poured my jars before I PC'd them. However that was glass.

 

I started that way, pouring first. Freaking headache - I will never do again. My idea was that they would be totally sterile. Maybe in some sort of production lab it might work but here are the problems I had:

• agar sloshes around, gets up onto the edge of the lower plate an even the upper leading to contam later on
• massive condensation problem on the cover - once again leads to contam later since it provides a fluid path to the outside as it drips around the edge
• If you do let it cool in the autoclave they might be tilted and the agar will be uneven. Not a big deal unless it's way off.

Yes, sterilize your plates first then fill in a laminar flow air stream or well cleaned still air box. Leave the lids half off until the agar cools. Then leave open longer until condensation on the lids goes away. Use as soon as possible or seal them in zip locs or put 2 wraps of parafilm or equivalent around the edges ASAP or both to prevent contam.

 

I recently tried a different sterilization tech and it worked so far on about ten plates with no contam.

• Clean your workspace well - alcohol, bleach, etc. Clean hands well and / or wear gloves.
• Clean the petris in the dishwasher or with a bleached alcohol towel.
• Put a couple of cc of 3% H2O2 peroxide in each one and close the lids.
• Put them on a clean tray or plate in the microwave - OK to stack them.
• Cover with a clean towel or PP breathable drape.
• Microwave on high until peroxide is about gone. Everything will be really hot.
• Move them to your pouring area and pour them
• There are peroxide sterilizers that use H2O2 in some way to sterilize and I think that's what you are simulating.
• Works with polypropylene and HDPE plates
• PP plates will autoclave but HDPE may or may not survive PC or autoclave
• PS (polystyrene) will definitely self destruct in the PC but I have not tried them in the microwave.

[MsBehavin420]

I don't microwave(and neither should anyone ever). Though I totally appreciate the tips otherwise.
I did my jars in the Pc, however they were glass and taller. I was planning on shelfing them inside my Pc with some baskets(metal) I have that fit and stack well.
I don't have a hood but I have an air scrubber that runs when I'm home.

That condensation though.. That is a bummer

[Sidestreet]

Hey Cats, what kind of lids are you using on those no-pours?

 

I ordered a sleeve of petris to get started but, you know, I have a bunch of half-pints sitting around.  Why bother with petris at all, especially if they aren't reusable?

 

I can still see why I'd use test tubes for slants...

[raymycoto]

 

I ordered a sleeve of petris to get started but, you know, I have a bunch of half-pints sitting around.  Why bother with petris at all, especially if they aren't reusable?

 

Consider the microwave peroxide sterilization for your plastic petris. That's the whole point of doing that - reuse your non-PP It's quick, don't need PC or autoclave and you can do plastic, but I wouldn't guarantee that PS won't melt but I bet even that will hold up in the microwave. For sure works with PE and PP.

 

Worth a try anyway. Don't throw them out but clean them, and give a try. I would want to autoclave for guaranteed effect but I did another batch of peroxide plates 3 days ago and so far so good.

 

No pour half pints will give a super sterile prep. I have found, sampling CO2 on no pour agar, totally sealed with standard ring / band  - There is so much air and the with the small amount of myc that you can probably get by with just leaving it sealed for quite some time, like a couple weeks at least. Then, perhaps open in SAB if you like to vent it and reseal if you are still using it.

 

I'm really liking PP agar plates I recently got. Reusable and totally autoclave safe even up to 30 PSI. Light weight, more compact than glass, better lid fit, really cheap and they come sterile when you get them. Amazon.

 

 

[Microbe]

Hey Cats, what kind of lids are you using on those no-pours?

I ordered a sleeve of petris to get started but, you know, I have a bunch of half-pints sitting around. Why bother with petris at all, especially if they aren't reusable?

I can still see why I'd use test tubes for slants...

Plates allow you to get a clear view of whats going on. You are getting angled looks at the agar surface in jars. Not saying you cant get a clear view if whats going and it requires different mechanics to work with them I.e. or make transfers from and or inoculate.

Glass plates are reusable and i used them for years and still do on occasion if im cleaning up a culture or the occasional spore germination of and again, a dirty syringe. I have gone through 50 plates cleaning shit up which consumes considerable amount of resources and why i use 60 mm glass plates for that purpose......less agar and reusable plates. Sucks germinating a spore and it has only grown across a fractional amount of the agar surface in a 100 mm plate before having to tramsfer it. Getting a little off track here.

Not to sound like a dick, but there is a reason why you dont see cases of squats in laboratories. Before anyone flips their shit and starts throwing it at me, I think its ideal in certain situations and people have been using them for decades so they certainly have their place.

IMO if one does not have a flow hood or lacks discipline to execute flawless technicue then squats are the way to go for sure. But if one has a flow hood and has some cash to spare, then glass plates are the way to go. I use plastic plates and probably have 1500 of them because i hate washing glass......call me lazy or whatever but i cant stand it and got burned out it ling time ago. I was washing 100 plates every few weeks........now i don't pour as many plates as i once did so the occasional plate washing is no biggy and who knows, once i use up all my plastic plates in the next decade, i might consider investing in some 100 mm glass plates.

Finally im not saying squat jars are inferior as each has its own pro's and cons. It comes down to your own situation such as available equipment, enviroment and or skill set.

Do you own a flow hood?

[CatsAndBats]

"catsandbats democratizes agar"

 

[Microbe]

"catsandbats democratizes agar"

Add the blue to the red and not the other way around or else you will end up purple....

You can add the red to the blue only if you add a green stabilizer to the red first and the most important thing is you need to use the green stabilizer at 68° F and 52.453 RH or a violent reaction will take place and flood your lab with pink foam......its all kinds of bad.....almost criminal like.

Edited by Microbe, 12 April 2018 - 05:15 PM.

[CatsAndBats]

^^^ this fucking guy. 

[Microbe]

^^^ this fucking guy.

[scott_1971_h]

pouring agar into petri dishes is simple. lucky for me i get my petri's for free these days.

Where do you get your dishes? lol

[Sidestreet]

 

Do you own a flow hood?

 

 

Not yet.  I've mostly lived in apartments without a good spot for one.  Soon we'll be in a house for good but in the meantime I'm still using the bathroom as a lab.  I should try to find a little one to take in and out of there.

 

I figure the glove box will work in the meantime if I'm very careful.  What do you think?

 

My very first plates, MEA, scalpel, lamp, and parafilm are coming today!  I'm going to get an oyster culture and go agar > grain > woodchips/soybean hulls (if I can find them). 

Edited by Sidestreet, 13 April 2018 - 04:16 AM.

[Microbe]

Do you own a flow hood?

Not yet. I've mostly lived in apartments without a good spot for one. Soon we'll be in a house for good but in the meantime I'm still using the bathroom as a lab. I should try to find a little one to take in and out of there.

I figure the glove box will work in the meantime if I'm very careful. What do you think?

My very first plates, MEA, scalpel, lamp, and parafilm are coming today! I'm going to get an oyster culture and go agar > grain > woodchips/soybean hulls (if I can find them).

I poured plates in a SAB using my no tilt jar lids. I would draw the agar into the syringe through the luer lock port then aspirate it into the plates. By the time I started actually pouring i had built a flow hood.

You can pour plates in a SAB with proper operation of one. I bought a alcohol lamp and about 5 minutes after lighting the wick i placed the cap back on it and donated it. I found a small butane torch to be better. Took what seemed like minutes to get 16 gauge sharps or larger glowing.

Your agar to grain to wood chips sounds like a plan to me.

[raymycoto]

 

I found a small butane torch to be better.

 

@microbe - absolutely. Small butane torch like for cooking is very maneuverable and awesome. I have a piezo propane torch head that pops onto a standard small canister. Turn it on and off in a second for a quick blast.

 

Have alcohol lamp but never use it now.

 

Uses I have found for the propane torch / head

 

• instantly sterilize your instruments - scalpel, needle, wire loop, pickups
• flame your flask or bottle top before (and after if planning to store it) pouring agar
• gently hit the top of a cracked-open freshly poured petri to evaporate condensation
• salvage and sterilize the wound edge of a "surgerized" petri where I cut out some rapidly growing mold on the edge of the agar - worked several times! Even though the mold has sometimes turned black with spores I seemed to have lucked out and it doesn't spread.
• gently reheat a flask of agar that you are pouring if its getting a bit too cool and begins to gel

Edited by raymycoto, 13 April 2018 - 09:08 AM.

[Microbe]

I found a small butane torch to be better.

@microbe - absolutely. Small butane torch like for cooking is very maneuverable and awesome. I have a piezo propane torch head that pops onto a standard small canister. Turn it on and off in a second for a quick blast.

Have alcohol lamp but never use it now.

Uses I have found for the propane torch / head

• instantly sterilize your instruments - scalpel, needle, wire loop, pickups
• flame your flask or bottle top before (and after if planning to store it) pouring agar
• gently hit the top of a cracked-open freshly poured petri to evaporate condensation
• salvage and sterilize the wound edge of a "surgerized" petri where I cut out some rapidly growing mold on the edge of the agar - worked several times! Even though the mold has sometimes turned black with spores I seemed to have lucked out and it doesn't spread.
• gently reheat a flask of agar that you are pouring if its getting a bit too cool and begins to gel

Heating up a flask of agar! That is brilliant and wish I would have thought of that before also. I have a stir plate with heating element now but i cant tell you how many times I was pouring agar clots into plates.

[raymycoto]

 

Heating up a flask of agar!

Yeah, have to do it gently. That torch is hot and could crack the glass if just a jar or maybe burn or cause a sudden 'burp' of the agar it if not careful. A flow hood or lami flow device seems to cool the agar container too fast sometimes. If you are pouring and anticipate it being a length process or just fiddling with your plates for a while then you may start hitting it early to keep it in the zone.

 

I'm playing a bit with peroxidated agar. One must add the peroxide at a temp of not much above 50c so as to preserve the H2O2. But this is getting a bit cool to allow much time.

 

I like your idea as well of a hot plate (or water bath maybe to keep it at 50C).

 

I'll report sometime when I have enough experience with the perox plates to have an opinion. They do have their uses, I believe for mold spore resistance and for 'cleaning' up spore germinations (grow thru agar is cool - I'll report soon).