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Saran wrap for Petri Dish / Agar work


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#1 raymycoto

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Posted 05 November 2018 - 06:13 PM

I started some agar work and found I had a bunch of topless plates. Guess I broke more tops than bottoms. But I decided to pour them and use saran wrap for cover.
 
I figure that saran is sterile on the inside fresh off the roll.
 
I poured 4 like this and here are a few. Blue tint is from UV light.
 
20181028_230318.jpg
 
The saran stuck nicely to the plates and is certainly cheaper than parafilm or even the grafting tape alternative.
 
20181028_235251.jpg
 
I had been at some point concerned about gas permeability but the metabolic level in on a plate is, so it seems, so low that it does not need any gas exchange, at least or a few weeks. And that is likely to be the time of use of the plate. 
 
Note on this plate, the small piece of micropore. I stuck a needle through the saran and sampled the gas and could find no detectable CO2 (or at least less than 1mm Hg, the threshold for my monitor)
 
20181105_063719.jpg
20181105_063736.jpg
 
The wrinkled up part is on the bottom of the plate. The top of the saran is pulled tight and is transparent.
 
So far, 4 out of 4 plates are not contaminated and growing well.
 
I’ll be trying this again, but so far:
 
Conclusion:
 
Saran wrap fresh off the roll is clean, clear and seals your petri dish quite well. 
Cost-wise it’s basically free since you probably have some already. 
It’s much easier to apply the saran than it is to wrap parafilm or equivalent carefully around the perimeter of your plates while making sure the parafilm doesn’t slip off yet covers the gap between the plates.
Unlike parafilm, it’s easily opened and reused / resealed in the hood or SAB.
It’s probably not permeable but I’m guessing that it won’t matter for at least a month, well into and possibly beyond your use of the plate.

Edited by raymycoto, 05 November 2018 - 06:17 PM.

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#2 Billcoz

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Posted 05 November 2018 - 09:17 PM

That's what I'm thinking as well. Ive been reading upon agar(never done it, seems easy enough) and I

 

have no parafilm and wanted to do some for more short-term and some for long-term, and Iv'e read that

 

it needs to breathe a lil bit if stored for a long time, petri dishes have loose lids and pfilm breathes some,

 

and they said suran was alright for a couple weeks. Someone experienced should verify. Id like know,

 

can double or triple layered micropore tape be used like parafilm? 


Edited by Billcoz, 05 November 2018 - 09:18 PM.


#3 raymycoto

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Posted 06 November 2018 - 11:01 PM

I have noted that, long term, with parafilm under room temps, your plates will dry out (2+ mo) due to air exchange. Of course, your specimens will be well past their prime at that point.  I think you could certainly use micropore. I have also heard of using masking tape or use nothing and just throw the plate in a zip lock.
 
You do want to keep out airborne contaminants so all of the above fulfill this requirement.
 
I'm not sure what you mean by "long term". Storage of myc long term in the presence of nutrients and oxygen is not the best way to go even if refrigerated, or so I have read. I have refrigerated plates just for a week or so then brought them back out and they seem to lose some vigor.
 
I'm not well versed on long term storage but there are those who are (on this forum).
 
Here is a discussion of storage with a nice reply by @CatsAndBats
 
 
I would be inclined to take the scraping of a vigorous, youthful, clean specimen on agar and place in sterile water without oxygen. You could use a large red top vacutainer as those are sterile inside and evacuated. Use a large bore (16 or so) needle to aspirate the specimen with the desired amount of water then insert into the vacutainer and the vacuum will suck the fluid into the tube. The vacutainer tops seal well after you remove the needle.
 
I have not done this approach so I can’t tell you exactly if that is the approach to use. I do want to try it. 
 
I am wondering what is the best way to collect as little agar as possible when collecting the mycelium as the agar would be an unwanted nutrient.

Edited by raymycoto, 06 November 2018 - 11:02 PM.

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#4 Billcoz

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Posted 07 November 2018 - 06:32 PM

I just meant a couple months(long term) vs. a couple weeks(short term).



#5 onediadem

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Posted 07 November 2018 - 10:36 PM

I have used saran wrap cut into 1-inch rolls for over 15 years. Plates last a good long time, (over a year). I also pour a lot of plates at one time for use later so when I go to spores, I already have h202 plates standing by for transfers. I just poured 40 plates myself.



#6 raymycoto

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Posted 08 November 2018 - 12:10 AM

I have used saran wrap cut into 1-inch rolls for over 15 years. Plates last a good long time, (over a year). I also pour a lot of plates at one time for use later so when I go to spores, I already have h202 plates standing by for transfers. I just poured 40 plates myself.

 

OK, cool. You are using it around the edge like parafilm or other tape.

 

BTW, What is your ratio of H2O2 to agar? I have tried this and I think I did 2cc of 3% H2O2 per 250cc agar. And wait until the agar is around 130 deg F so it does not decompose.

 

I did a peroxide agar 'sandwich' of the new transfer to clean it before going to LC. Grows through pretty quickly.



#7 onediadem

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Posted 08 November 2018 - 01:20 AM

I use 1cc per 100 ml.


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#8 tricktek

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Posted 08 November 2018 - 07:28 AM

your saran covers looking good, ray, very creative.  as far as parafilm goes, I've been using electrical tape for years. The kind with a duck logo works great. A friend of mine uses the saran wrap cut into small rolls as mentioned by onediadem.  my bud cuts the rolls with an electric knife.


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#9 raymycoto

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Posted 08 November 2018 - 11:55 PM

Update, since we have talked about the air exchange and because I can't resist showing some mycelium on agar porn.

 

Crazy but the saran plates are doing better than my double glass with the parafilm substitute.

 

These plate are now 2 wks post transfer. I'm wondering if they are a single clone. Hard to say whether I'm looking at different sectors or just some rhizomorphism. I'm going to guess I have a single clone on these plates because I want to proceed to the next step. Should have transferred earlier if I was going to divide them another time, anyway.

 

20181108_222344.jpg    20181108_222325.jpg

 

20181108_222307.jpg    20181108_222404.jpg

 

The one labelled 'C' had no detectable CO2 about a week ago. Today, I sampled thru the saran with a 20ga needle again then covered with another layer of micropore tape. I got a CO2 level of 9 mm Hg

 

On the myco level, that's really low. I've found that mature grain jars doing really well with vented tops and micropore have a CO2 level of 150 to 175 and an O2 level of 13% or so. The O2 level was 19% in this plate.

 

20181108_222841.jpg

20181108_222855.jpg

20181108_222905.jpg


Edited by raymycoto, 08 November 2018 - 11:58 PM.

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#10 Billcoz

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Posted 09 November 2018 - 01:38 AM

Well the grains have more air throughout the jar than a tiny dish, so the myc is growing bigger and breathing more o2 up and producing more co2. If you think about it the nutrition in a grain jar would probably not last as long as the nutes in a tiny pitri dish either, or we could keep grains longer, & i guess they might rot or something after a while. That's cool your measuring co2.



#11 raymycoto

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Posted 09 November 2018 - 02:32 PM

I refurbish and resell medical equipment and usually have one of those machines around so it's handy to use to sample air in jars and bins. Interestingly, as I mentioned above, myc really does well in a high CO2 atmosphere with low O2 from its own metabolic processes.

 

I have observed that too much FAE (even with a good submicron filter) in a jar leads to less success. It may be that the sweet spot of CO2 level around 150-175 torr inhibits some of the contaminants but is not too high for the myc to survive.

 

CO2 level is also good to sense activity in a LC or in a jar with no visible activity. Rising CO2 in an apparently inactive jar will show up as bacterial contamination eventually.

 

I have never tried to control the CO2 level. I don't have that equipment but I know that it's possible to regulate CO2 with sensors (and gas electromechanical valves, I guess). I suppose one could supplement with extra O2 in a jar. I do have O2 cylinders and could try that. 

 

Any opinions on whether hyper-oxygenated environment could be beneficial? Or supplemental CO2?

I realize I'm making it way too complex by considering this but could be interesting. Maybe I'll noc up a couple of sealed jars - one with air and one with 80% O2, 20% CO2 (150 mm Hg).


Edited by raymycoto, 09 November 2018 - 03:44 PM.

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#12 Billcoz

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Posted 09 November 2018 - 09:06 PM

Ha, that's fkin cool. Wouldn't the myc try to pin with oxegen being fed into it? seems like it might fruit

 

in-vitro, but IDK it's a good mad scientist experiment. For me, just growing out a few brf jars & a

 

couple FCs is a lot to keep track of, I took prints & I'm getting ready to do some agar and that'll be a

 

bitch for me, but some people on here do these elaborate experimental teks and write-ups that are so

 

damn organized, I'm jealous of the patients and creativity y'all have lol. I actually got nervous the first

 

couple times when I'd prepare to knock up some jars or do sterile work cuz it seemed tedious and I

 

wouldn't be able to stop once I started,but it got easier to do. I'd like to  test my spore prints, now I feel

 

like they might notta been dry enough when I bagged em so keeping track of agar plates(jars) is i

 

intimidating. I don't have my stuff yet, couldn't find agar or malt extract at wally mart's, I'll check the

 

health store or order. This has prolly been asked too much, but does anyone know how good instant

 

taters work? And can they be the powdered or only the flakes?


Edited by Billcoz, 09 November 2018 - 09:11 PM.


#13 raymycoto

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Posted 09 November 2018 - 10:02 PM

I have a couple of thoughts on agar plates. There are various formulas and nutrients. This is off the top of my head, so go ahead and research it to get the correct info.

A few types you can make or buy. . . 

PDA - potato dextrose

PDMA - potato dextrose malt

PDMYA - add yeast to the above

Other - add some of your own nutrients - perhaps some water from steeping your grains. Be careful - sometimes adding these things can inactivate the gelling properties of your agar and you will have a mush that won't gel or will gel, then later liquify.

I got malt from a beer store or order on Amazon. Caution: Malt powder absorbs moisture and you had better seal it up well immediately after opening or it turns into a rock.

Cheapest option - buy Chinese ebay agar powder then find a recipe for how to add the potatoes. Sometimes it's good to have some plain agar anyway to add to things.

 

Best option for beginner - keep it simple - Buy the premixed Amazon PDA and just follow the recipe - powder plus water. More expensive but you will have enough for gallons of agar.

I make one liter at a time and divide it into 4 x 16 oz  mason jars (each half full so they don't boil over when you PC them). Let your jars cool in the PC with the tops on as if you are canning something so the result is four vacuum sealed jars with 8 oz agar in each. Then use each one when you need it. Warm each jar as needed in a pot on the stove with about 1" of water, light boil an cover the pot to keep the steam in. Loosen the band top of the jar when heating. Wait for it to liquify. Prep your plates. Pour as much as you need from the jar. I have successfully returned the unused portion in the jar to the double boiler for a while and resealed it with the band after I used what I needed from the jar and the jar remained uncontaminated.

 

Or you can PC about 1/4" of agar in the 'squatty' 4 oz, jars if you don't have petri dishes. The bottoms of these jars are raised in the middle so you have to pour enough to cover the center and thus it's extra thick on the edges. Not a prob but you do use about twice the agar for the same 'plate' size. BTW, speaking of air, you will have all the air you need in these jars! And it is somewhat convenient to have a screw lid. OTOH the high sides of even the squatty jars do get in the way when instrumenting the agar surface.


Edited by raymycoto, 09 November 2018 - 10:05 PM.

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#14 Billcoz

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Posted 09 November 2018 - 11:26 PM

I've been reading about this exact subject for a couple days lol! I was messin around with brf/water

 

like 1/4" deep in 1/2 pints onlyto play around until I get agar but that was 2 days ago and I don't see

 

anything yet and it's really opaque like vanilla ice cream so its hard to see thru even with light shining

 

and the lid makes it hard to see. I probly squirted too much solution, it popped out. Only had less than

 

a 1/2 a cc of solution from when I did my last brf jar so I decided to play around with that but I'm goin to

 

the health store tomorrow to check fragar. which'll be fuckin expensive, probly go online I don't know 

 

where there's any malt extract so I was gonna use instant potatoes, they only have powdered tho and i'm

 

not sure cuz I've read a few things sayin flakes, so IDK, is the powder any good? Are the 1/2 pints too 

 

big r too much air? Thx man.


Edited by Billcoz, 09 November 2018 - 11:28 PM.


#15 raymycoto

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Posted 11 November 2018 - 11:56 AM

BRF won't work in place of agar. Among others, reasons to use agar are to clearly see what you are growing, isolate sectors and have some satisfaction that you might not have contamination.

 

You could use any jar for agar work. Of course the form factor of whatever you use for your 'petri' may interfere with your plans for the specimen on agar. No problem with too much air if use 1/2 pints.


Edited by raymycoto, 11 November 2018 - 11:57 AM.


#16 Billcoz

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Posted 11 November 2018 - 12:58 PM

BRF won't work in place of agar. Among others, reasons to use agar are to clearly see what you are growing, isolate sectors and have some satisfaction that you might not have contamination.

 

You could use any jar for agar work. Of course the form factor of whatever you use for your 'petri' may interfere with your plans for the specimen on agar. No problem with too much air if use 1/2 pints.

My brf/water pucks did germinate after another day. I know that it's not the same as agar, I just wanted to play around until I got all the agar supplies. The BRF/H2O is too deep and opaque to see through like you could agar.

 

The myc is bright white on the surface tho, and I think I could get a sector, I can see rhiz arms starting and it's still tiny. If I had added food coloring I bet it would be no problem to isolate from.

 

I also have to borrow the PC I use and didn't feel like asking again so soon(three days) and BRF is fine to steam sterilize in a pot, I was bored, and had spores.

 

I have read of people isolating successfully this way, they had nice pics and everything, then after a few replies, people who ignored the pics, tek, and the guy explaining that it worked over & over for him, started saying there's no way it could work lol. I think it will, but have my agar supplies now so I'll be doin that. 

 

BTW, I'm gonna make up some plastic lids, some for grain, some for agar jars. I have RTV for the self-healing port, and tyvec and polyfil, Would you recommend leaving the ones for agar without a 1/4" gas exchange? THX.

 

EDIT- Oh yeah, I don't think you mentioned, What kind of mycelium is on the plates?


Edited by Billcoz, 11 November 2018 - 01:04 PM.


#17 raymycoto

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Posted 12 November 2018 - 11:04 AM

OK, Cool, you are making it work! Yes, that's what this hobby is about, trying new things when you don't have all the ingredients or parts.  Yes, you could certainly grow myc on BRF for inoculating and looks like you might be able to isolate as well as you are doing.

 

Here is something you might try instead of PC for sterilizing plates. Put 1cc of H2O2 per jar or petri dish and microwave on high until dry. I know it sounds like something that would destroy your microwave but mine did survive. Haven't done it in a while but the plates I did this way performed well without contamination. BTW, this is the way that the Steris autoclave system works. H2O2 is injected in to the autoclave chamber and then microwaves vaporize the H2O2. 

 

I also have sprayed plates with alcohol in the flow hood and waited until it evaporated. That leaves them pretty clean as well. Don't think that would work in a SAB because the alcohol would not evaporate without air flow. 


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#18 Billcoz

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Posted 12 November 2018 - 11:44 AM

Yeah I've read about H2O2 plates working well. I ordered an 8 quart PC last night, it should be here Tues. It came to $33 something with free 2 day shipping. I will be using the brf plates for knocking up grains and maybe try to isolate from one to experiment. I mostly wanna test my prints for contams and I'm not sure if they'd show on the brf like you said, I can't see anything but myc so far so they might be clean, but that was a vendor syringe, not my prints.



#19 onediadem

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Posted 12 November 2018 - 12:43 PM

I tried the brf pucks years ago. 
The problem I had was getting the 
myc to jump off of it.



#20 Billcoz

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Posted 12 November 2018 - 02:03 PM

I tried the brf pucks years ago. 
The problem I had was getting the 
myc to jump off of it.

So if I put a piece in a grain jar, would it just take longer, or not even grow into the grains or do you mean to agar? Would it make a good LC, assuming it doesn't contam?






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