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OfflineMachiavelliavore
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How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP
    #22535684 - 11/17/15 01:07 AM (8 years, 4 months ago)

Hey,
      I'm working on a microwave only pour agar tek, and I'm wondering just how long I need to wait to see bacterial bloom.
Now since these bacteria would be in the agar not on the agar, I'm guessing it would be longer than normal.

My first test batch was microwaved under mild pressure for 45 minutes.  Given the conductivity of liquid, perhaps that's enough, perhaps not.  Any input on sterilization times for liquid media at atmospheric pressure would also be appreciated.

The blank plates haven't shown any contamination after a week so far.  I'm thinking I'll wait an entire month the open up the plates and chop'm up to inspect for any contaminants within.


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


I spawned some popcorn casings and had double-overlay cause I didn't put enough hydrogen peroxide in my automated aquarium mister.  I only got one mushroom so I cut off the head part where the seeds fall from and put it in a jar of LC and sprayed it all over a tin of PF cakes I made with gravel, cardboard, and bisquick in my microwave.  I think it will be good cause B+ is so potent.
Triggered yet?

Only a square would say "a cube is a cube."


No, this does not look right...

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Invisiblethe_r3dz
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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Machiavelliavore]
    #22535817 - 11/17/15 02:07 AM (8 years, 4 months ago)

I don't have anything useful to contribute, I'm just curious what it was like to run your microwave for 45 minutes. Did you put it outside?
I sort of think microwaves release at least a small amount of that shit when you use it, I'd be incredibly weary to have to run it for so long and if I couldn't put it outside I definitely wouldn't go into the room it was cooking in.

But I might be crazy or something. I'm also curious to see if this works for you, not going to bother with the obvious telling you to buy a PC but I feel like the cost of electricity should be a deciding factor? Can't be cheap to run that machine like that, but I have no idea how expensive microwaves actually are as far as energy goes.

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OfflineMachiavelliavore
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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: the_r3dz]
    #22535825 - 11/17/15 02:12 AM (8 years, 4 months ago)

I already have a PC, but I'm developing this for people who don't.  Microwaves aren't dangerous, the waves can't escape through a hole under some absurd size, like a golf ball or some shit.

Not sure about the energy cost, I only ran the microwave at 30%, which basically means it's only running 30% of the time, so you can say that was the equivalent of running it 13.5 minutes normally.

I don't think anyone flips a bitch over nuking a frozen lasagna :smile:

Edit Re: Power cost
MW uses about 1.2kW when active.  The usage time as stated is 13.5 minutes effective, or .225 hours, giving a power usage of .27 kW*hours, costing about $.05 here.


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


I spawned some popcorn casings and had double-overlay cause I didn't put enough hydrogen peroxide in my automated aquarium mister.  I only got one mushroom so I cut off the head part where the seeds fall from and put it in a jar of LC and sprayed it all over a tin of PF cakes I made with gravel, cardboard, and bisquick in my microwave.  I think it will be good cause B+ is so potent.
Triggered yet?

Only a square would say "a cube is a cube."


No, this does not look right...

Edited by Machiavelliavore (11/17/15 02:45 AM)

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Invisibleblindingleaf
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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Machiavelliavore]
    #22535889 - 11/17/15 03:03 AM (8 years, 4 months ago)

im not sure how long it might take.
only time i saw bacteria on uninnoculated plates was when i used ground WBS in the recipe.  about a month later, i was getting bacteria on plates with cultures, but in weird spots, not transfer sites.  looked at remaining 3 plates, and the bacteria was forming on the bottom, where the larger pieces of crushed WBS ended up.  I'm guessing because i put them in dry, and only cooked 40 minutes, it only killed x amount of endospores.

so i would guess about a month to see bacteria??
u can keep it in warm temps to make it go faster just to see if it shows.


--------------------
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MICROBIAL HUSBANDRY!!!!

The whole is greater than the sum of its parts

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OfflineMachiavelliavore
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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: blindingleaf]
    #22535950 - 11/17/15 04:06 AM (8 years, 4 months ago)

Warm temps, good idea!  Thanks BL.  I made it with potato flakes like pasty plates.  Not sure what the endospore load there is.  Low I'd guess.

If I have problems that can't be solved by microwaving longer, maybe I'll try like SITR + Cornstarch or something.  I don't think I will though.


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


I spawned some popcorn casings and had double-overlay cause I didn't put enough hydrogen peroxide in my automated aquarium mister.  I only got one mushroom so I cut off the head part where the seeds fall from and put it in a jar of LC and sprayed it all over a tin of PF cakes I made with gravel, cardboard, and bisquick in my microwave.  I think it will be good cause B+ is so potent.
Triggered yet?

Only a square would say "a cube is a cube."


No, this does not look right...

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Invisiblethe_r3dz
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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Machiavelliavore]
    #22535995 - 11/17/15 04:42 AM (8 years, 4 months ago)

Well, that was unexpected. Thanks for the comprehensive reply ha I feel like I've learned something.

I still fear the microwave. Toaster ovens all the way!

I'll stop derailing your thread now, right on with trying new things :thumbup:

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OfflineMachiavelliavore
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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: the_r3dz]
    #22536051 - 11/17/15 05:17 AM (8 years, 4 months ago)

Nah you're just bumping it :smile:  Dunno why you fear the microwave.  My method should work with a boiling water bath as well, but will just be a bit more work and more energy cost.


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


I spawned some popcorn casings and had double-overlay cause I didn't put enough hydrogen peroxide in my automated aquarium mister.  I only got one mushroom so I cut off the head part where the seeds fall from and put it in a jar of LC and sprayed it all over a tin of PF cakes I made with gravel, cardboard, and bisquick in my microwave.  I think it will be good cause B+ is so potent.
Triggered yet?

Only a square would say "a cube is a cube."


No, this does not look right...

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OfflineDrCrumbs
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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Machiavelliavore]
    #22537174 - 11/17/15 11:51 AM (8 years, 4 months ago)

I had a chem prof that would tell stories of sailors warming themselves from the ships microwaves during WW2 without ill effects.

I have two pasty plates that were PC'ed at 15PSI for 20 min. that are about 6 weeks old, never opened without showing contam's. I dunno if that helps anything as it kind of doesnt apply, just thought I'd throw that out there.

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OfflineMachiavelliavore
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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: DrCrumbs]
    #22537286 - 11/17/15 12:16 PM (8 years, 4 months ago)

Perhaps just warming themselves from radiant heat.  No ill effects reported before being murdered by German MG-42's on Omaha.

Well, I did some research:

Blackout: shorter microwave times than I used for microwave agar
Violet: way shorter times, but multi-treatment quasi-tyndalization
_OttO_: Liquid culture with no potato flakes, but 30 minutes at atmospheric boiling seemed to work for him.  Then again, a lot of shit seems to work for him :smile:

I think I'm good but I'll wait the month before releasing the tek and make sure the plates are clean.  Also need to see if PP5 containers can be effectively sterilized in the microwave by adding water and nuking them to steam the inside.  That would be way more convenient than steaming in a pot, though I'm concerned about the lip harboring contaminants.  I will probably try putting water in the lid lip before closing it to attempt to boil water there.


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


I spawned some popcorn casings and had double-overlay cause I didn't put enough hydrogen peroxide in my automated aquarium mister.  I only got one mushroom so I cut off the head part where the seeds fall from and put it in a jar of LC and sprayed it all over a tin of PF cakes I made with gravel, cardboard, and bisquick in my microwave.  I think it will be good cause B+ is so potent.
Triggered yet?

Only a square would say "a cube is a cube."


No, this does not look right...

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Invisiblemorrow
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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Machiavelliavore]
    #22537458 - 11/17/15 12:51 PM (8 years, 4 months ago)

Hei.

I recently played around with agar and microwaving.

Started off boiling a batch of LME agar on a gas stove with no lid. Poured into shallow PP5 food containers in open air. Laugh all you want: this actually was the point, to see what species of nasties I have floating in my home and how fast they grow, what color they are etc. Anyway, left them alone in a drawer for three days. During that time visible colonies of up to 2mm diameter had formed on the surface. Colonies had also formed suspended in agar. But they were much, much smaller.

I then decided to experiment with microwaving them over a course of three consecutive days. I only microwaved them until I detected a boil. Which in my case was exactly 40 seconds per container. The result so far is: all of the dead colonies have sunk to the bottom and have not revived. There are no new colonies on the surface, neither suspended in agar. They have been sitting around in the dark for 4/5 days now.

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Offlineblackout
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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Machiavelliavore]
    #22538047 - 11/17/15 03:08 PM (8 years, 4 months ago)

Quote:

Machiavelliavore said:
My first test batch was microwaved under mild pressure for 45 minutes.



How did you create pressure? if you have a filter on a jar it will usually be under some pressure. If you have plastic containers the lids can blow off, so you can either lower the power or increase the filter size or filter type. Glass jars risk exploding.

Most microwaves do not regulate the power, but rather regulate the time on and off, so you can get surges of boiling. Agar can be tricky as the bubbles if forms can be huge and you really need a large jar just partially full, LCs are easier.

I use shorter times as there is usually less heat up time in a microwave, most PC times are taking into account the long time to reach temp throughout. Once boiling in the microwave it stirs itself up and power can be lower.

I have microwave agar a few months old which is still OK. I would not add grains or grain flours, I believe these do need PCing, but repeated treatments may work.

You must also account for moisture loss, adding more water at the start. I mark jars with a permanent marker so I know when enough moisture has been driven off.



Quote:

Machiavelliavore said:
Also need to see if PP5 containers can be effectively sterilized in the microwave by adding water and nuking them to steam the inside.


Yes, I have done this many times. I usually use containers within filtered larger containers. I also put my LC jars or agar jars in plastic bags with a little water in them.

Putting them inside a larger fitlered container means when cooling down they are drawing in filtered air, this means my small dishes do not need filters. I have little dip trays I use for agar, I can fit loads into my 4.5L PP5 filtered container.

Not only is the air filtered but if you add water to the large container then the small containers will be properly steamed on the outside too. I would put water in the small tubs too, any left over water can be poured out in a SAB. You can boil them dry but it is risky, you should always have water in the microwave, even if its in a glass, otherwise you risk the plastic melting. I stupidly broke a few pyrex petris by boiling dry in a microwave with no additional water in it.

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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: blackout]
    #22538300 - 11/17/15 04:40 PM (8 years, 4 months ago)

Lots of people sell their old or never used PCs for cheap or even free. All you'd need is a new seal which are only like $10.

There's a bidding page on Facebook wear I live where I've seen a few people giving away pressure cookers.


--------------------
http://www.shroomery.org/forums/showflat.php/Number/22409475
“Psychedelics are illegal not because a loving government is concerned that you may jump out of a third story window. Psychedelics are illegal because they dissolve opinion structures and culturally laid down models of behaviour and information processing. They open you up to the possibility that everything you know is wrong.”
― Terence McKenna

Edited by Arush (11/17/15 04:45 PM)

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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Arush]
    #22540807 - 11/18/15 08:16 AM (8 years, 4 months ago)

I've always seen bacteria within a few days of an exposure in the dish. I can't even be sure I've had it take 4.  The one contam of most recent project with plants, gloves, and no clean air, had bacteria on a very light recipe of agar and sugar, and it had its blotches by the end of 2 days.
Buuuuut its buddy has a nice vigorous specimen growing in it with no contamination, 4 days later.  So especially since they both have a little pool of water in them, and bacteria would have shown by now.
Yeah they have the strength of an agar recipe made with 2.5g per 500mL and it is regular sugar, with ferts and for plants.  But bacteria showed up in no time on agar with the same typical speed, and a clean plates showed nothing past the clear time for bacteria.
By the way, the clean one was microwaved on 40-50% for like 12 minutes. This was only one time. I use a weak recipe of agar also, because I want the softest yet immobile gel, and a noteworthy amount of water is lost to steam, maybe 15-30mL per container at a guess.


My recipe:
Firm agar for mushroom transfers should be made with just a slight reduction of agar-agar powder from the normal recipe.
17-18 grams per 1000mL of solution before division.  Expect water loss during heat treatment.  Final recipe (essentially agar-agar percentage near 2%) can be determined by measuring by the cc on the side of the container before and after the treatment, with tape marked with the measurements in advance. Personally I don't bother, I just wing it all.

The heavier your agar of mixture is, the more that pressure cookers and microwaves can create volatile spill-overs of boiling agar foam.  With a firm mix for mushrooms, you will need to familiarize yourself with microwave power selections.  This is done by inputting your amount of time to microwave, then press power level (etc.), then press 1, 2, or 3 for 10%, 20%, or 30% of power level. Each have different intervals of microwave blasts that give your agar enough time to settle the boil back down before stoking it up again.
The goal is to run it until your agar boils, then inputting the setting of 10-15 minutes with the power level for Your microwave low enough to cause the boil to rise only high enough to not risk reaching the container's lid.  It isn't a loss if agar reaches the seal, but it's nicer not to.

.5 or .6 grams powder per 40mL no-pour agar dish should firm up to 25-30 mL.  The smaller amount boils sooner, and then is more easily stoked back to a boil.  This will involve low power levels, likely even just 10% depending on the strength of your microwave.
The low amount of moisture in the small and shallow agar dishes means that an otherwise small loss is a significant portion from a single dish for 2D culturing.

To lock in best odds, microwave treat the agar 2 times.  The ideal timeframe may be 14-18 hours after the first time.
With a fert-ferment sub such as the pom tek recipe, wait 24hrs and microwave extra thoroughly the last time.

My microwave agars have shown no sign of contamination if unopened. None at all.  Some of my first time containers didn't get contamination at all by the time I repurposed them for something else, going like 10 or 12 days and likely longer.



WAY LATER EDIT / NOTE TO ANY READERS:  I have totally trashed the recipes here.  Where I was indeed getting success, I was still tuning, and don't want you to go by any of this!  Check my threads for final recipes!


--------------------
Intentionally or not, here in mushcult we are purveyors of love culture and enlightenment movement. Let's try to act like it!

PODS TEK - Growing Invitro with BRF/verm or Grass Seed containers
The simplest, quickest, safest tek!  For beginners, culturers, lazy people, stealth lovers, contam haters, and alternative seekers!
Violet's Teks and Posts

Edited by Violet (12/29/15 11:22 PM)

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OfflineMachiavelliavore
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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Violet]
    #22540976 - 11/18/15 09:09 AM (8 years, 4 months ago)

What are you growing Violet, psychedelic lichen?

I have apparently solved most of this on my own similarly to you and blackout.  Here is the tek (still rough:)
http://www.shroomery.org/forums/showflat.php?Cat=0&Number=22526843&page=0&vc=1#22526843

Just passed a week with no contams on the plates today.

Basically it uses a plastic jar lid on a mason jar with a small hole covered in tyvek to control the pressure release.  This keeps the boil down quite effectively, though cracking the jar would be rather surprising unless it was microwaved SUPER hard.  It should work in a boiling water bath as well, though I don't see the appeal of doing it that way.

According to blackout, the containers for pour plates work fine when microwave sterilized with water, which is great.  For poptops, I need to check if they sterilize under the lip.  Planning to swab 360deg around the lip with some sterilized que tips and swipe them on an agar plate.

Anyway, trying to bring culturing/cleaning for PF tek users, especially those that start with spore ring prints or marketplace prints.


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


I spawned some popcorn casings and had double-overlay cause I didn't put enough hydrogen peroxide in my automated aquarium mister.  I only got one mushroom so I cut off the head part where the seeds fall from and put it in a jar of LC and sprayed it all over a tin of PF cakes I made with gravel, cardboard, and bisquick in my microwave.  I think it will be good cause B+ is so potent.
Triggered yet?

Only a square would say "a cube is a cube."


No, this does not look right...

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InvisibleViolet
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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Machiavelliavore]
    #22541345 - 11/18/15 10:48 AM (8 years, 4 months ago)

That's exactly what he and I were doing it it mind for, more than anything else at least.  He did it well before I did, although I didn't know about it and started doing so on my own out of curiosity and positive suspicion.

But you'll probably want to take note of the treatment times I gave above.  I haven't microwaved these things for more than 15 for sure! And I only say it that way because I can't remember what the most I have nuked them for was, maybe only 12.  Poms1 sub using just 1/8 of the normal agar-agar thanks to the right thickness of rice starch, is microwaved on 50-60% power thanks to a lower boil intensity, but you want to totally avoid messes around the seal if you can.

A larger amount of liquid in the container will require lower powers and increased times.  Small containers CAN have their agar sterilized quickly but the concern is also steam sterilizing the inside of the container as a whole.  Waiting some time for a second solid treatment reduces the total time necessary by destroying the germinating wave of any surviving endoscopes and germinating mold, as well as further decreasing the odds of endoscope straggler survivors.

I should add to my recipe, all times that I generally give are AFTER running the microwave regularly until I see the agar first begin to boil.

In my crappy microwave, I have treated a recipe of firm brown rice agar at 20 or 30 percent power 2 times for 10-12 minutes and had no contams.
Maybe even shorter times can be pulled off for one or both treatments.


45 minutes just seems like a really long time to microwave.
I have recently microwave prepped low-sugar recipes of soft agar. A single 14ish minute period of microwaving at like 40% power apparently sterilized 2 containers simultaneously in the mic together, each with 1cup in them. Looks like they lost about 1/8 of their moisture, like 30mL.

If the container with water, light agar, and some sugar to show contams are sterilized with a single session of like 10 minutes of half power, it tells me that anything beyond that would only be necessary as the contents specifically call for.
For instance, with fert-ferment nutrient-dense grain agar I wouldn't double up containers together and would likely want to do a second treatment 18-24 hours later.

Try a few simple containers with shorter times!


What I've been growing is plants!


--------------------
Intentionally or not, here in mushcult we are purveyors of love culture and enlightenment movement. Let's try to act like it!

PODS TEK - Growing Invitro with BRF/verm or Grass Seed containers
The simplest, quickest, safest tek!  For beginners, culturers, lazy people, stealth lovers, contam haters, and alternative seekers!
Violet's Teks and Posts

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OfflineMachiavelliavore
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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Violet]
    #22541396 - 11/18/15 11:05 AM (8 years, 4 months ago)

I think my next experiment will be 2 cups of agar, 30 minute sterilization, 30% power.  I'll also be microwave sterilizing pp5 containers with boiling water, rather than using pressure sterilized containers.

I'll try some no pours in a space efficient container such as a miniround or hummus half pint pp5 and see if the boil is controlable at low power.  It would be easier for n00bs.

Pour Agar has some advantages related to microwaving especially:
Easier to track and regulate moisture content
Easier to effectively sterilize containers
Easier to correctly measure and distribute bar or pasta type agar if powder is unavailable, no grinding required
Less Condensation
Better sterilization of inside of container due to pressure buildup/increased microwave times

Downside: Pouring.


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


I spawned some popcorn casings and had double-overlay cause I didn't put enough hydrogen peroxide in my automated aquarium mister.  I only got one mushroom so I cut off the head part where the seeds fall from and put it in a jar of LC and sprayed it all over a tin of PF cakes I made with gravel, cardboard, and bisquick in my microwave.  I think it will be good cause B+ is so potent.
Triggered yet?

Only a square would say "a cube is a cube."


No, this does not look right...

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InvisibleViolet
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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Machiavelliavore]
    #22541427 - 11/18/15 11:18 AM (8 years, 4 months ago)

Oh I see, just now understood for sure that you're pouring.

2 cups of water is almost the regular 500mL recipe for agar.  If you use 500mL, the water loss will actually result with about 2 cups.  That's why I modify the recipe to 8.5-9 grams agar-agar per 500mL for firm agar, and down to just 1.2-1.5 grams for very soft gel.

The condensation improvement is likely with the poured dishes, but I haven't ever been really bothered by condensation unless there was bacterial contamination which is usually just tossed, and I almost never see it with my agar techniques anyway.

Why do you think it sterilizes more effectively in one large container with the other ones just getting water?
The way I see it, if you're going to microwave sterilize the receiving containers with just water beforehand anyway, might as well just have the final substrate in there, right? Entirely eliminates your extra long cook time on a large volume of liquid with a very small proportion of overhead boil space under the lid.

I can see where it would be easier to track the moisture loss, but once you do understand it you can simply adjust your recipe and use no-pour containers for ease and efficiency.

Also; don't you have Ziplocs?


--------------------
Intentionally or not, here in mushcult we are purveyors of love culture and enlightenment movement. Let's try to act like it!

PODS TEK - Growing Invitro with BRF/verm or Grass Seed containers
The simplest, quickest, safest tek!  For beginners, culturers, lazy people, stealth lovers, contam haters, and alternative seekers!
Violet's Teks and Posts

Edited by Violet (11/18/15 11:26 AM)

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OfflineMachiavelliavore
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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Violet]
    #22541672 - 11/18/15 12:32 PM (8 years, 4 months ago)

My container lid appears completely gets rid of boilover issues.  Water loss for 1 cup of agar over 45min @ 30% was only about 1/3 cup.

Sterilizing the agar containers with water instead of agar, they can be nuked longer and harder to better sterilize the entire interior.  The main pour container pressurizes mildly, which should help maintain the temps throughout.  I guess I'm also a little miffed that you can get away with so much lighter sterilization protocols :blast:, and I've really been enjoying pouring for some reason lately.  It's helping me eliminate variables in my own agar prep, like water loss in pc, uneven distribution, etc.

I'll definitely be trying out nopours and trying to evaluate what seems easier for a scrub.

I do have betty crocker twist tops, but I don't use them for agar, as they take up too much space.  Part of that is that I maintain huge redundancy to compensate for any screwups I may make, or late discoveries of great strains.  1:4 dilute grainwater this batch, with air filters, if this doesn't fix it, it must be either the quantity of agar or the cultures (5/5 slowmentose would be waaaay to weird though.)  Pasty plates ended up being about 5% slower than your undiluted grainwater agar.

Next up, ziploc steamer bag microwave sterilization of BRF + straw substrate and/or LC hydrated straw :smile:

Maybe this pour method will have more appeal to more experienced cultivators, though I kinda doubt anyone with a PC will opt to trust it.

Plants, huh?  Tell me more about these "plants."


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


I spawned some popcorn casings and had double-overlay cause I didn't put enough hydrogen peroxide in my automated aquarium mister.  I only got one mushroom so I cut off the head part where the seeds fall from and put it in a jar of LC and sprayed it all over a tin of PF cakes I made with gravel, cardboard, and bisquick in my microwave.  I think it will be good cause B+ is so potent.
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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: the_r3dz]
    #22541696 - 11/18/15 12:38 PM (8 years, 4 months ago)

Quote:

the_r3dz said:
I don't have anything useful to contribute, I'm just curious what it was like to run your microwave for 45 minutes. Did you put it outside?
I sort of think microwaves release at least a small amount of that shit when you use it, I'd be incredibly weary to have to run it for so long and if I couldn't put it outside I definitely wouldn't go into the room it was cooking in.

But I might be crazy or something. I'm also curious to see if this works for you, not going to bother with the obvious telling you to buy a PC but I feel like the cost of electricity should be a deciding factor? Can't be cheap to run that machine like that, but I have no idea how expensive microwaves actually are as far as energy goes.




Nah microwaves shouldn't escape in this day and age. I did hear stories of when they first came out that a lotta housewifes were turning up at the hospital with weird burns on their stomachs. Not sure if its true or not i was still young or unborn when the first microwaves were sold. I don't think its that far fetched though. Back then they were missing a lot of good shit.


Anyways im :takingnotes: on this one mach :billymaythumbup:

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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Machiavelliavore]
    #22541861 - 11/18/15 01:33 PM (8 years, 4 months ago)

Whew, you say a cup only lost 1/3 of a cup but that's a third of its total water!  Like 80mL.  That sounds about similar to the rate of moisture loss per container that I have gotten, about 2mL per minute in the nuke at a mid/low-power level occasionally stoking up the boil.

I just think that small no-pour containers, pre-loaded with their agar recipe, sterilized one, two, or three at a time for 6, 9, or 12 minutes respectively, can be prepared more easily with a lower total run time than one large container.  Should also involve substantially less energy used.
One guy in this thread has apparently gotten the surface to stay clean after just a few minute-long treatments.  If such an extremely low treatment time is possible in stages, 2 or 3 times for a minute or one time for 4-8 minutes.

This isn't to say you should do it that way instead if you have particular preferences - in fact it's awesome and best that we can make it work with apparently any combination.  Just that I think individual containers this way are the true motivation and best ideal function for preparing agar both on the fly as well as for people without pressure cookers.
For someone wanting to pour one large batch into several sterilized containers, they probably are doing the type of growing where they need a cooker anyway and certainly would be better off using that instead.


That's true about water being able to sterilize most easily.  Without agar, the boil won't really foam.  You can keep it full power as long as you want.

It shouldn't take long to sterilize.  Think about how peeps without cookers used to sterilize syringes... Getting water to a good boil on the stove, then repetitively drawing the boiling water into the syringe, waiting a moment, and shooting it back. Many times or for a few minutes.  If we can trust the syringe to be effectively sterile doing this for 5 minutes, then 5 minutes of turmoltuous internal boiling should do it. 

Oh and with the crazy boil of agar, thinner no-pour preps might even have their substrate sterilized as quickly.


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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Violet]
    #22542629 - 11/18/15 04:13 PM (8 years, 4 months ago)

The plants so far are just a flowering vine, a particular plant that's special to me :>
I'm learning the dynamics of using this kind of method for propagating plants from seed, and hopefully cuttings/tissue culture.  So far playing around is doing well.  My intent is to figure it out with plants like strawberries, cherry tomatoes, roses, maybe violets, and more. Ive seen variations of it work with both Orchids and Venus flytraps which I've seen some cool photos of in tissue culture.  Apparently almost all Orchids are started in such a fashion.


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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Violet]
    #22542979 - 11/18/15 05:27 PM (8 years, 4 months ago)

Quote:

Violet said:

That's true about water being able to sterilize most easily.  Without agar, the boil won't really foam.  You can keep it full power as long as you want.

It shouldn't take long to sterilize.  Think about how peeps without cookers used to sterilize syringes... Getting water to a good boil on the stove, then repetitively drawing the boiling water into the syringe, waiting a moment, and shooting it back. Many times or for a few minutes.  If we can trust the syringe to be effectively sterile doing this for 5 minutes, then 5 minutes of turmoltuous internal boiling should do it. 





My concern was with the low microwave times combined with the low conductivity of plastic (afaik.)  It might not hit or hold temperature with just the steam from a bit of agar done for a few minutes at very low power.  It definitely won't be holding temp long once the microwave shuts off.  Maybe it's fine, but that's what concerned me about it when I was considering it.  The syringes hit near boiling temperatures on all internal surfaces instantly and hold it pretty well.

Using water, the entire interior can be vigorously steam sterilized, and the plastic should hit and hold temperature for a good long while.

As for the cost, I think I calculated 45mins@30% of nuking to be about 5cents, so no worries there.  Just want the best efficacy so nobody ends up wasting their time.

This method should also should work fine in a boiling water bath, which is a nice bonus.  It can also be done easily regardless of the specifics of plate numbers and microwave powers to unique people, since they will just boil on past the prescribed time until the agar returns to the desired volume, which is easily visible.

Anyway, like I said I'll be trying out nopours.  I just didn't think it could be that easy :smile:



Interesting on the plant tissue.  I guess if you put any clean cell culture in an appropriate food bath, it will typically get rolling.


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


I spawned some popcorn casings and had double-overlay cause I didn't put enough hydrogen peroxide in my automated aquarium mister.  I only got one mushroom so I cut off the head part where the seeds fall from and put it in a jar of LC and sprayed it all over a tin of PF cakes I made with gravel, cardboard, and bisquick in my microwave.  I think it will be good cause B+ is so potent.
Triggered yet?

Only a square would say "a cube is a cube."


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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Machiavelliavore]
    #22542998 - 11/18/15 05:31 PM (8 years, 4 months ago)

Could you do this to sterilize used plates? Put them in a bag with a little water in each one, let it steam out? Or would they melt

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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: the_r3dz]
    #22543050 - 11/18/15 05:46 PM (8 years, 4 months ago)

Probably not since the working principle of the microwave only lets you achieve 100C at atmospheric pressure. Technically, a little higher, but as far as sterilization goes: NOT HIGH ENOUGH.

Your plastic container can not melt, since the temperature stays around 100C and most commonly used plastic polymers have a melting point higher than that.

Same goes for bacteria: the organic molecules that make up the little bastards will not be destroyed by such a low temperature in such a short time.

You either have to increase the temperature to ~120C by increasing pressure (the trusty PC) or increase time spent on sterilizing by keeping the temperature at 100C and doing multiple runs over a period of days. (what was in an earlier post referred to as: multi-treatment quasi-tyndalization)

Edited by morrow (11/18/15 05:48 PM)

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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: morrow]
    #22543780 - 11/18/15 08:04 PM (8 years, 4 months ago)

Idunno about that.  I think the microwave, perhaps the actual microwaves themselves, make things different.
I don't feel like I'm taking sterilization by the normal approach, much less fractionally.

Waiting for just the heat to conduct and reach sterilization temps in a cooker does take a long time, and is faster than steaming them.  But the microwave creates heat with trillions of heinous collisions in the materials themselves, especially water despite its highest specific heat.
So it seems as if I have great success steam sterilizing these containers with mere minutes in the nuke, and the way that microwaves specifically function is probably to blame.  The vicious boiling of agar mixtures may be related too, but I think that the combination of that, the boiling of the sub, and the near-constant internal steam positive pressure forcing out the threads and seal are all just backup to the destructive power of the waves themselves.  The microwave may very well destroy life forms, especially when any water at all is in them.  Hence why I have seen what appears to be complete sterilization from times at boiling-temp heat that otherwise wouldn't even guarantee molds to be dead.


Anyways, Machi.
That might tell you a little bit about how I think microwaves work so well, and why the aims of steam sterilization in microwave treatment are only to saturate all content and surfaces with moisture to be attacked by volley after volley of radiation.

Think: traditional treatments killing only via saturation of heat held for time, opposed to the quick infiltration of contaminants with water then breaking the surroundings of that water with The Force.

So I think a boiling water bath without microwaves should take longer than in a pressure cooker, if it works at all.


How are you measuring the energy use of microwave ovens?  This is relevant to my interests!


Yeah! The Morning Glories germinated and began expanding as seedlings on a soft water-only agar gel.  I have a couple plants now rooted in 1 cup of microwaved soft agar with ferts and 1g of sugar for each, in quart containers, and they're doing the best by far compared to the 4 that are planted into bottles of microwaved fert soil, lids on.  Very promising stuff.  Perhaps with variations of the recipe when necessary, any rooting hormones and all that, I can get all kinds of vegetables, fruits, flowers, succulents, and leafy greens to propagate quickly.


Ooh I got a better one!  Sneaking a ball of metal (water) into a guarded place (spore or endospore), so that Magneto could use his powers of magnetism (microwave radiation) to use the metal as a weapon from the inside out.


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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Violet]
    #22545360 - 11/19/15 06:33 AM (8 years, 4 months ago)

Microwaves heat polar molecules, mostly water.  They don't care about plastic, which is why I was saying the poptop gasket or the threads would need to be steamed.  There has to be water there to here them up.  Trying to get water into the threads or poptops is something I've considered and tried, but only a trivial ammount seems willing to stay in there, maybe enough to help though.  Certainly boiling water in a twisttop thats patially open will steam sterilize the threads much better, but the steam elevating the temperature all over in a poptop may be enough to get the lid gasket hot.

There should most definitely be water in contaminants though.  I just assumed if that was an effective strategy, people would be microwave sterilizing grains and stuff all the time.  Perhaps given their low water mass, heat bleeds off them too fast into an environment very low in temperature, preventing them from staying at temps that would kill them for too long.  Fuck, life is so unfair.  I wish I could get microwaved for 10 minutes and live.

I'm not sure how this explains blackout's experience of having plastics melt when they ran out of water.  Perhaps salts, polar organics, or trapped water is present receiving all the energy output of the microwave in an extremely condensed area.

Microwave power consumption is easy.  Most are rated at 1100 or 1200 Watts, which is a energy/time unit.  Power is sold in KiloWatt*Hours (which oddy is an energy unit because (Energy/Time)*Time gives units of Energy).  Anyway, suffice it to say you multipy the power usage of your microwave in KiloWatts (say, 1200W = 1.2KW) by the actual usage time in hours, say 45mins@33%=15mins=.25 hours

(.25 Hours)(1.2KW) = .3 KiloWatt*Hours  Round here it's about $.20/KW*H, so (.3KW*H)(.20$/KW*H)=$.06, most places it's more like $.10/KW*H
I honestly didn't think nuking would be quite that cheap.  It only costs $1 to leave a 100W lightbulb on for 50 hours.  $58 to leave a desktop on for a month.  $173 to run your microwave 24/7 full power for a month.  If you pay Kali4nia rates.  And if anyone gives shit about being green, steal their prius battery to power your microwave, after you electricute them with it a bit for good measure.

Might even be worth considering nuking all water for grain and/or bulk substrate prep.

I think I'll be trying all these methods out and swabbing from the lid gasket/threads with sterile q-tips onto agar plates.  Should be fun.  Hopefully I can get out a decent tek within a couple months though.


That plant stuff sounds pretty cool, and more doable with what I know than other plant cloning.  I wonder if it will work with a maple tree.  I might have to try it.  Does it extend the seedling growth mode?  It seems like if you put a seed in nute agar, it would give it like 100 times the nutrition to work with a normal seed would, maybe yielding a larger plant faster.


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


I spawned some popcorn casings and had double-overlay cause I didn't put enough hydrogen peroxide in my automated aquarium mister.  I only got one mushroom so I cut off the head part where the seeds fall from and put it in a jar of LC and sprayed it all over a tin of PF cakes I made with gravel, cardboard, and bisquick in my microwave.  I think it will be good cause B+ is so potent.
Triggered yet?

Only a square would say "a cube is a cube."


No, this does not look right...

Edited by Machiavelliavore (11/19/15 06:35 AM)

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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Machiavelliavore]
    #22545723 - 11/19/15 08:58 AM (8 years, 4 months ago)

Well microwaves do heat all kinds of things.  They don't entirely "ignore" plastic, as you'd find if you just put a container in the microwave for a while, but when microwaves melt plastic it's almost always the fault of the contents.

This is for the same reason that pressure cookers work on water, but the containers can and will warp and melt when the water runs out.

Water acts as a magnificent heat buffer and threshold setter.  Whenever something in the immediate presence of water tries to go above the water's boiling temperature, 212°F at common atmospheric pressure or ~250°F at 15PSI, the water essentially steals that energy and becomes steam, moving away with that excess energy.  So water's container cannot go above the temperature of that water's boiling point except where the passing steam has maybe a couple extra degrees.

However, other materials don't have such a boil point.  Lots of things will go above 212°F in a microwave, and it can get crazy.

Once I tried microwaving popcorn straight out of a bag of bulk popcorn, in a few ways to see which ways would result in easy and cheap popcorn.
I found that without something like butter or oil I was just making the kernels and the glass extremely hot!
But when I put oil on them in a container, before many of the grains popped the oil itself apparently got above the melting temperature of the tupperware!!!  AH!  It melted a bunch of holes and little bubbles into the plastic.  Obviously the same thing doesn't happen with the same grain kernels but in water.



The reason that microwaves are going to "work better" for these applications is because the energy goes straight to the moisture in the substrate... and the contaminants. 95% of the time waiting for heat convection in other treatment methods is skipped outright, and the continual application of new energy into heat is just as rapid and intense.  That intensity, if not the radiation itself, is what I believe is responsible for the much more rapid destruction of contaminants.  Those 2 factors are the only ways I can rightly explain my successes with such short treatment times in the nuke.

Only swabbing as you intend to will make more clear than it already is how well the thread area is sterilized by this process.  But keep these things in mind:  Even the steam attempting to escape is still getting hit by the microwaves, so we're not limited precisely to boiling temps in the steam like with methods of steam sterilization;  And any spores or endospores within the containers or thread area will most likely be hydrated enough for the combination of ridiculous volatile internal heating and radiation to decimate them in just a couple minutes.  So I don't suspect that watering around the threads and seal will help much but they sure can't hurt!



Well, it is POSSIBLE to prepare grains in the microwave.  The problems lie with immense water loss, bursting, and the difficulty of getting the saturating heat to the core and getting radiation on and into every last bit of them.  Obviously this is not only a problem with water, but is practically instant.
Blackout has prepared grains in the microwave several times, and I have *kindof* done it with grass seed a time or so.
He's gotten good at tracking the water loss and adding more to compensate in order to leave it at the right level.  I think I remember that the actual treatment is part of his grain prep for this reason as it reduces burst grains, but you'd have to ask him for sure.

But in short, the reason it isn't an effective strategy is because it is a complicated and problematic one, and an unsure one until it is dialed in and nailed by individuals themselves.


What stands to be effectively educated to people is the use of microwaves for water, agar, tools, casing layer, and maybe even batches of substrate just like casing.


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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Machiavelliavore]
    #22545786 - 11/19/15 09:15 AM (8 years, 4 months ago)

Quote:

Machiavelliavore said:
That plant stuff sounds pretty cool, and more doable with what I know than other plant cloning.  I wonder if it will work with a maple tree.  I might have to try it.  Does it extend the seedling growth mode?  It seems like if you put a seed in nute agar, it would give it like 100 times the nutrition to work with a normal seed would, maybe yielding a larger plant faster.



That's pretty much exactly what's happening - the sprouts planted in a nutrient agar are growing ABOVE the agar incredibly rapidly, MUCH faster than their soil in counterparts.  The difference is that the ones in agar have de-prioritized root development; more than 80% of new growth is the stalk etc.  It's the exact opposite to the ones that have gone to soil.
My next concern and area for observation is if cleaning and transferring the slender plants from agar, with smaller roots, will break them in the process or leave them "top-heavy" biologically.  Like will they dry out when put into soil?
It's different with the bulk propagation from seed or exoplant clone.  You get tons of them going, and "harden" them together.  I think my problem with this plant, Morning Glory, is specifically with the fact that it is a vine.  I suspect that, if it works at all, many of my other target specimens will do better in that regard, whereas this one excelled at vigorous germination and establishing itself as a seedling... because it is as much as a weed vine as a flower vine.
Maple might need to be germinated from seed, or cuttings/exoplants from a sapling etc..  Since it's a tree, the mother plant selection method would take a long time, and might be excessive/pointless.  Getcha some seeds, soak them in H2O2 with a touch of bleach for a while, dip them in sterilized water on the way to a dish!  (I have used water-only agar for germination, but because I've just done it open air)


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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Violet]
    #22545807 - 11/19/15 09:18 AM (8 years, 4 months ago)

Yeah, trapped water or other polar molecules can heat things up for sure.  And the steam will continue to be heated.

But what I'm saying is that if dry nuking contams in plastics worked, I would have expect it to be a popular method for sterilizing all kinds of stuff.  Otherwise the sterilization is most likely do steaming just like in a pot, though the steam temperature is probably slightly higher.


I wonder if grains can be effectively sterilized before hydration.  I believe one of your posts on grainwater nutes estimated 3% water in dry rice grain.  Maybe it can by tantalized without hydration in a sealed bowl, then have sterile-ish water added and have the hydration process completed in the microwave.  I think any contams coming in with the water would get totally wrecked immediately.

And I mosdef agree, skipping conduction heating with gas and hitting temperature immediately in a microwave will reduce times a lot!


Figured out my own casing prep with microwave treatment with materials I purchased when I started out.  pH balanced peat mixed with coir 70/30 fixes all the textural problems straight peat has, nukes better than verm, and comes away with a textural improvement.  Microwaves definitely have a lot of potential that is untapped.

I'm looking forward to trying some fractional sterilization of the pasty BRF+straw sub.


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


I spawned some popcorn casings and had double-overlay cause I didn't put enough hydrogen peroxide in my automated aquarium mister.  I only got one mushroom so I cut off the head part where the seeds fall from and put it in a jar of LC and sprayed it all over a tin of PF cakes I made with gravel, cardboard, and bisquick in my microwave.  I think it will be good cause B+ is so potent.
Triggered yet?

Only a square would say "a cube is a cube."


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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Machiavelliavore]
    #22545853 - 11/19/15 09:27 AM (8 years, 4 months ago)

Dry microwaving shouldn't work because there's no water.  Spores and endospores are usually just waiting on a hydrated area to germinate and check for food, so according to why I think microwaves can work well for mycology you can't count on those contaminants being destroyed without plenty of water or steam around them, thus in them.


A good thought about the grains...  but that post where I was figuring out the water content of grains, and the grain content of water post-prep,  involved nuking the SHIIIIIIT out of the rice.
Remember, for the same reason that water acts as a temperature buffer in PCs and microwaves is the same reason that it stops food from burning.  The rice kernels definitely burnt.  I'm not sure I'd want to see what they'd be like after an attempted preparation, but maybe it wouldn't be too bad.  But mainly I don't think that little water content will really help.  I'd more likely put my money on preparing more hardy grains in the nuke, from the top, in boiling water... which would then cause excess starch problems and stickiness, another compounding issue of not preparing brown rice, seed, and other starchy grains without excess water.


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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Violet]
    #22545882 - 11/19/15 09:34 AM (8 years, 4 months ago)

Sounds like oats might be a good candidate for getting boiled for a long time.

On thing I was considering for several uses way a layer of polyfill at the bottom of the container of whatever is being sterilized, to suspend the grains over a resevoir of steaming water.  Perhaps that would be an effective final stage of sterilization to prevent drying out while the final hydration level is reached.

Might try drynuking some rice and get a tempcheck at least.  EDIT:  Did it.  -1 PP5 pint.  There's definitely some water in there and it gets hot.  Started to burn around 6-8 minutes at 10%.  There might be some potential to very slow multiple heat applications.



With your plants, I wonder if you could do some kind of radiating mixture of agar and soil.  Agar in the center, then like agar and peat/soil mixed further out to ease it into the soil, if any root structure is developing at all.


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


I spawned some popcorn casings and had double-overlay cause I didn't put enough hydrogen peroxide in my automated aquarium mister.  I only got one mushroom so I cut off the head part where the seeds fall from and put it in a jar of LC and sprayed it all over a tin of PF cakes I made with gravel, cardboard, and bisquick in my microwave.  I think it will be good cause B+ is so potent.
Triggered yet?

Only a square would say "a cube is a cube."


No, this does not look right...

Edited by Machiavelliavore (11/19/15 10:22 AM)

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Re: How long for blank agar plates to show contams? Sterilization times of liquid media at atmosphericP [Re: Machiavelliavore]
    #22547393 - 11/19/15 03:43 PM (8 years, 4 months ago)

The best way to monitor moisture loss is to simply weight it. I have weights written on most of my jars and you can accurately measure it. I have spoke of making "agar trays" before. These were PP5 chinese takeaway containers. As the layer of agar is thin it is harder to monitor moisture loss if you are simply drawing lines to gauge.

For beginners working with questionable prints I have wondered about the hot agar pour technique. Where hot agar is poured ontop of growing myc with bacteria in it and the myc grows upwards through it while the bacteria remains behind, then you transfer it. But I wanted a simple way to do this. One idea was to get a filtered jar and microwave agar in it. While it is cooling you tilt it around so agar coats the sides of the jar. This can be run under a cool tap to make the agar set. Now you put a drop of spore solution in the bottom and when it grows you heat the side of the jar with a hair dryer or heat gun and the agar liquifies and pours over the bottom. No SAB needed, no new batch of agar etc.

I believe grains may be possibly be heat treated enough for our needs by microwaving while dry or only partially soaked (note I am careful not to say sterilized!). This is one old test worth a read

2 Step Microwave preparation experiment. 24hr interval. 100g of wheat.


The microwave can also be combined with a PC. I currently have a 3.5 or 4.5L container about 80% colonised maize. This is from another thread about it

Quote:

blackout said:
I am running my own tests on being more efficient with my workload trying to combine a PC and microwave to suit me best.

these are my notes on my recent one, I did not vent as the grains are submerged and not been heated by steam. This allows me to get to a high pressure/temperature much quicker, if it takes too long the grains will overcook, overhydrate and explode. I am trying to hydrate and sterilize at the same time using a small PC. I then put into a large container which would not fit in my PC. I preheat the grains in a microwave so they are past 100C, when I pour them in the boiling water it actually causes surges as it is hotter than the water.

975g unsoaked maize
heated in microwave until I hear 2 pops
add to approx 3L rolling boiling water in PC, seal, no venting
3mins 5psi
6mins 10psi
8mins 15psi
15mins 20psi
25mins turn off still 20psi
45mins vent at 3psi

1732g afterwards.
Put into 3.5L filtered tupperware container, put inside loosely sealed autoclave bag

microwave 4mins full untill puffed out as it is boiling and steaming,

30mins on defrost/low to continue boiling without excessive moisture loss.

The idea is the PC sterilizes, and only airbourne contams get into the container which is microwaved to kill off. Maize or popcorn is difficult to hydrate and so can be PCd at high pressure without exploding.




The grains are their own pressure cooker. I have got grains well past 150C, my thermometer maxes out at that. Popcorn will get to 135psi & 180C before popping. This thread may be of interest

http://www.shroomery.org/forums/showflat.php/Number/20938439

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