Microwave Sterilisation
Cathal Garvey
So, I'm researching microwave sterilisation. So far, it's looking really
good for small volumes of media, say 5mls in a glass test tube. I think
one reason microwaves seem to work so well for this is that they
continue to heat the vapourised water, whereas in a conventional
heat-under-the-water scenario the steam leaves the surface at 100C and
doesn't gain significantly more heat.
Anything that makes DIYbio cheaper to dive into is a good thing in my
view, and having to invest in/learn to use a pressure cooker is a pain.
Here's some refs of note:
http://aem.asm.org/content/15/6/1371.short
http://jcm.asm.org/content/6/4/340.abstract
And here are some refs I can't access thanks to dirty, SOPA-supporting
paywalls, but would love to see:
http://www.sciencedirect.com/science/article/pii/0022098188900639
http://www.sciencedirect.com/science/article/pii/019567019190241Y
I may try to test this concept soon, it'd be a really welcome shortcut
from the 1.0+ hour job of sterilising with the pressure cooker on a
1.5kw hotplate.
--
www.indiebiotech.com
twitter.com/onetruecathal
joindiaspora.com/u/cathalgarvey
PGP Public Key: http://bit.ly/CathalGKey
Mega
the pressure cooker is enough'. He said they have done research for a
company which produces them it actually makes 119°C (250F) and quite
no organism could survive that for longer than a few minutes.
On 16 Jan., 17:09, Cathal Garvey <cathalgar...@gmail.com> wrote:
> Hey all,
> So, I'm researching microwave sterilisation. So far, it's looking really
> good for small volumes of media, say 5mls in a glass test tube. I think
> one reason microwaves seem to work so well for this is that they
> continue to heat the vapourised water, whereas in a conventional
> heat-under-the-water scenario the steam leaves the surface at 100C and
> doesn't gain significantly more heat.
>
> Anything that makes DIYbio cheaper to dive into is a good thing in my
> view, and having to invest in/learn to use a pressure cooker is a pain.
>
> And here are some refs I can't access thanks to dirty, SOPA-supporting
Dakota Hamill
It'd be interesting to sterilize batches of media side by side, one in pressure sterilizer, one in microwave, one left unsterilized as a control, then leave them side by side and see if anything/when anything grows
Cathal Garvey
it up to the temperature, not keeping it there. On the cheap hot-plate I
use in my lab and with the high-capacity pressure cooker I have, it
takes a long time to reach 120C, maybe 30+ minutes on a bad day!
It also takes quite a while to cool down, maybe another 10 minutes.
Especially with agar, you can't force-cool or you risk the sample
frothing up and boiling over the plate/tube as the inner pressure of the
cooker drops suddenly. So, you let it cool on its own.
I should probably start using my smaller pressure cooker more often
again, or perhaps insulate the sides of my large cooker with Kapton Tape
or something to help it heat rapidly.
Cathal Garvey
I'll see if reading through the literature I have can result in a nice
protocol we can put together; I think this is a promising angle to
pursue for beginners and as a general time-saver!
:)
On 16/01/12 16:35, erik wrote:
> http://www.mediafire.com/?5dvufmkhwunrk6i
> http://www.mediafire.com/?lcjfks3beuv3ze7
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Cathal Garvey
with the anti-SOPA activism and your mention of DIYbio discussion there;
is there a dedicated sub-reddit for DIYbioism?
For 5mls or less, it appears 45s or 1m is all it takes, which is enough
for test tube slants or smallish petri dishes, although I'm not sure
were they done individually or together in the experiment. Perhaps the
additional papers will enlighten me here. Maybe we can bring this down
to a simplish equation between volume needing sterilisation and the
wattage of the microwave..
Nathan McCorkle
> Hey all,
> So, I'm researching microwave sterilisation. So far, it's looking really
> good for small volumes of media, say 5mls in a glass test tube. I think
> one reason microwaves seem to work so well for this is that they
> continue to heat the vapourised water, whereas in a conventional
> heat-under-the-water scenario the steam leaves the surface at 100C and
> doesn't gain significantly more heat.
>
> Anything that makes DIYbio cheaper to dive into is a good thing in my
> view, and having to invest in/learn to use a pressure cooker is a pain.
>
This is something home mycologists have used for a long time... sorry
I never realized it was uncommon!
What I've always wondered about this method though, is how do you
calibrate for different microwaves that have different power output,
and even failing power supplies (power output is less than what the
label reads)? A pressure cooker/autoclave has a pressure gauge (my big
cooker goes up to 25 or 30psi), or at least you know that its up to
pressure based on when the air hole weight begins spewing steam.
Are the cheap IR thermometers actually reliable? Would they work
through the wire mesh in a microwave? Would simply having a beaker of
water in the microwave with a glass thermometer in it, so you could
read through the window suffice for calibration? Would you want the
microwave carousel to turn, or be stationary? Would you mark a
footprint in the microwave and always place you media container there?
--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics
Nathan McCorkle
the mycology techniques, because I've always been scared I would fail
and waste my media to contamination!
I was also concerned with water loss... I'll have to read the articles
that were linked to earlier, maybe they talk about this.
I found a table showing mass lost after microwaving (said 800 watts)...
start mass 121g (inlcuded 53g water to begin, along with solid
nutrients and inert filler)
after 1 min 118g
after 2 min 103g
after 3 min 89g
Patrik
microwave. Some microwave ovens do a *really* poor job at this, with
very hot hotspots, and very cold coldspots. Here's an amusing approach
to DIY microwave diagnostics:
http://www.evilmadscientist.com/article.php/microappalam
Most microwaves these days come with a rotating platform to even out
the hotspots to some degree. I've also seen plastic windup carrousels
to achieve the same effect. For more reliable sterilisation it may be
worth putting one of those off-center on top of the rotating platform
to get an even more even distribution.
On Jan 16, 9:56 am, Nathan McCorkle <nmz...@gmail.com> wrote:
> By the way, I've never actually tried using the microwave according to
> the mycology techniques, because I've always been scared I would fail
> and waste my media to contamination!
>
> I was also concerned with water loss... I'll have to read the articles
> that were linked to earlier, maybe they talk about this.
> I found a table showing mass lost after microwaving (said 800 watts)...
> start mass 121g (inlcuded 53g water to begin, along with solid
> nutrients and inert filler)
> after 1 min 118g
> after 2 min 103g
> after 3 min 89g
>
>
>
>
>
>
>
>
>
> On Mon, Jan 16, 2012 at 12:39 PM, Nathan McCorkle <nmz...@gmail.com> wrote:
Cathal Garvey
maths there to calculate the coverage of different areas..
It's true that hotspots and coldspots would prove problematic.
Particularly as most of the tested protocols involve small volumes, such
as 5mls in a test tube, rather than larger single-item volumes. Single
items full of liquid would be more likely to achieve homogeneity from a
microwave even accounting for awful hotspots, whereas a rack full of
test tubes might instead result in some cold tubes and some evaporated
tubes.
One of the papers that Erik was kind enough to source actually discussed
this briefly, I think, and most of the papers have some mention of
methods to account for hot/cold spots, though they weren't much more
complex than "remove and rotate 180 halfway through".
There's also disgreement between those who feel that microwaves only
work due to thermal energy and those who think there's something special
going on; any opinions out there to share?
Personally, I'd be surprised if it was a thermal-only effect, as you
typically need to achieve 120C for 15 minutes to sterilise by wet heat
alone, whereas these microwave protocols can work in less than a minute
for small volumes.
I personally, and without evidence to back me up, suspect that the
complex shells of hydration that support protein structures in the
target cells are getting shaken apart, leading to low-temperature
protein denaturation. Like a "virtual solvation" effect, where parts of
the protein that normally remain on the outside due to water binding
instead snap inwards as water shells momentarily "vanish" under
microwave excitation.
Thoughts?
Ethan
cooker has to do with the fact that heat transfer from a gas phase
(superheated steam) to a solid/liquid phase is somewhat slow
especially without convection and stirring. It relies completely on
conduction of the heat through the media. In something like a 500ml
media bottle, there is a lot of mass that needs to be heated. In the
microwave, the energy penetrates the media and heats up all parts
simultaneously (ideally is it even heating, but that is not so). I do
imagine that there is some added effect from direct interference with
macromolecules in the microorganisms.
One thing I would be worried about with sterilizing small amount of
media or empty glassware is that there might not be enough mass to
absorb the microwaves. It is recommended that microwaves are not run
empty because it can lead to feedback. It might be something to look
into.
Nathan McCorkle
Sent from my mobile Android device, please excuse any typographical errors.
On Jan 17, 2012 5:45 PM, "Ethan" <argen...@gmail.com> wrote:
>
> I think that a large part of the long times for autoclave/pressure
> cooker has to do with the fact that heat transfer from a gas phase
> (superheated steam) to a solid/liquid phase is somewhat slow
> especially without convection and stirring. It relies completely on
> conduction of the heat through the media. In something like a 500ml
> media bottle, there is a lot of mass that needs to be heated. In the
> microwave, the energy penetrates the media and heats up all parts
> simultaneously (ideally is it even heating, but that is not so). I do
I agree, microwaves cause the water molecules to immediately start vibrating, rather than first vibrating the pressure vessel and glassware/plasticware
I remember reading about a nurse who caused a death by microwaving blood for a transfusion rather than warming via water bath or dry heat... I'll try to find a reference ... I remember also reading a lot of nutrition articles( maybe not scientific) regarding degradation due to microwave vs conductive cooking
Pat
gotten contamination due to lack of sterile media. On occasion I do
use an autoclave but using the microwave at home is so much easier and
faster.
There is a chart in this pdf: http://www.phytotechlab.com/TechInfo/O788-Info.pdf
on page six, ill reproduce some of it below.
mL media Autoclave time Microwave Time
25 15-20 4-6
50 25 6-8
100 28 8-10
250 31 10-12
1000 40 NR
2000 48 NR
4000 63 NR
On Jan 17, 6:26 pm, Nathan McCorkle <nmz...@gmail.com> wrote:
> Sent from my mobile Android device, please excuse any typographical errors.
Cathal Garvey
Mind if I ask; does that tissue culture media contain a carbon source,
or are the cells grown under light conditions? I ask because the
sterility requirements in plant tissue culture are often lower when
there's no carbon/nitrogen source present in the agar. If you're using
stuff with carbon in it, it makes the microwave sterilisation look even
better.
It's great to know that this works well for you; I'm pretty convinced
that I should adopt this technique myself.
Off to grab a spare microwave, then! :)
-Cathal
Ethan
completely boiling over? I run into that problem even when I am just
melting agar for short times never mind longer sterilization.
Cathal Garvey
I suppose you could add silicone anti-foaming agents? Available either
from chemical suppliers like mistralni.co.uk or from pharmacies as
anti-colic agents for newborn babies. You'd have to get a brand that
doesn't have any additives or flavourings that might affect your
experiments. Also, some of these might be surfacants that would interact
with or damage cell membranes, so they might be bad enough on their own.
At least some anti-foaming agents are biocompatible, as they're
routinely added to bioreacters and fermenters to prevent foaming-over
during agitation and sparging (aeration).
Ethan
to splatter out) than the mixture foaming up, though that is probably
an issue also. From my experiences in cooking (something that comes in
handy more than you would think in amateur biology), emulsifiers have
a tendency to work as anti-foaming agents. For example, if you want to
whip egg whites, you can't let any egg yolk get into the mix, or it
will not hold air. I would try mixing some egg yolk into media and see
what that does. I don't know if the egg yolk also contains lysozyme,
but that should be denatured through heating. Another thing to look at
would be soy lecithin (sold pure as a food additive/supplement). I
would test some of this out, but I am sadly away from my lab at the
present.
Jeswin
> When you sterilize media in the microwave, how do you prevent it from
> completely boiling over? I run into that problem even when I am just
> melting agar for short times never mind longer sterilization.
>
Easy. Keep an eye for boil-over and turn off the microwave asap.
Nothing else can be done. In chemistry, you can put boiling stones but
you can't with agarose.
Nathan McCorkle
Why can't you use boiling stones/chips with agarose?
Dakota Hamill
Pat
have no nutrients of their own so you have to supplement everything.
@Ethan: The lid usually fit on snug so there is no boil over and if it
appears like there will be I usually stop the microwave for a few
seconds.
Cathal Garvey
expect to see contamination by late-germinating spores by a week or so.
I have to say, it's mystifying to me how this could work if it were just
the heat; you need 15-20 minutes at 120 to sterilise things by
autoclave, so achieving comparable results by microwave for 5-10 minutes
is.. confusing.
But, if it works I can hardly complain! I have a microwave in the lab
now, and I'll start using it for small loads and see how it works out.
Question: If you're making agar plates, do you microwave the agar media
in the dish, or do you pressure/oven sterilise empty dishes and add
microwave sterilised agar afterwards?
Dakota Hamill
Pat
media it into tuber-ware containers. The tuber-ware containers with
the media then get microwaved for the appropriate time.
Check this video out you can start at 5:00:
http://www.youtube.com/watch?v=qxKgOBPT494&feature=related
Let me know how it works for you.
@Dakota If you have the right media it all depends on your sterile
technique and your access to a laminar flow hood or for other sterile
environment. A fish tank turned on its side and wiped down with
bleach and sprayed with 70% Etoh would do the trick. Hhaha nice
depending on the orchid you can make a sizable buck.
Jeswin
> I heard a story from one of my professors about a kid she went to grad
> school with or something of the sorts who used to go to orchid shows and ask
> for any extra cuttings the people had right before they were putting them on
> show to be judged, sort of like a dog show I guess, last minute repairs.
>
> Anyway, he'd culture them and sell them as really rare orchid breads and
> that's how he payed for school
This sounds oddly familiar. Wasn't this what you did back in college,
Simon? I wonder why he didn't chime in, since I thought he was an
expert in orchid culture.
I'm talking of Simon Field; not sure if there are other Simons on the list.
