5 replies to this topic

[Dott. Berrino]

Hi everybody,

I'm trying to set up some E. Coli culture in a microplate (96 wells), has someone experience in that? Do you have some hints?
I keep the microplate (200ul/well) in the oscillating incubator at 37°C but first I'm not sure if bacteria are enough aerated and second if I leave them o.n. they dry almost completely. I found out there are some "gas permeable microplate seals" have someone ever used them? Is it worthy to buy them?
I need to constantly monitor OD, so now I'm covering the microplate with a normal adhesive sealer every time I need to bring it to the microplate reader, really uncomfortable. I have no idea if these "gas permeable microplate seals" will also allow me to read the plate in the microplate reader without changing the seal every time, this will be really wonderful!  

Thanks

[bob1]

I have heard good things about the gas permeable seals, but have never used them myself.  I think they will probably be OK with your spec, so long as they don't absorb at your measurement wavelength.  You would also need to make sure that you don't get any bubbles trapped under the seal as these will interfere with readings.  I don't know how getting rid of bubbles would interfere with the aeration of the plate.

Have you tried putting a lid on and wrapping the edge in parafilm?  If you have to make frequent readings, removing the lid to do them should be enough to replenish the oxygen.

[perneseblue]

what is the purpose of these small cultures?

Give the small size of the culture, I would guess that these E coli are better aerated than the average large culture in a conical flask.

[pDNA]

This should be a useful publication for you:

Scale-up from microtiter plate to laboratory fermenter: evaluation by online monitoring techniques of growth and protein expression in Escherichia coli and Hansenula polymorpha fermentations

Regards,
p

Edited by pDNA, 18 May 2010 - 09:02 PM.

[pito]

Dott. Berrino, on May 19 2010, 12:15 AM, said:

Hi everybody,

I'm trying to set up some E. Coli culture in a microplate (96 wells), has someone experience in that? Do you have some hints?
I keep the microplate (200ul/well) in the oscillating incubator at 37°C but first I'm not sure if bacteria are enough aerated and second if I leave them o.n. they dry almost completely. I found out there are some "gas permeable microplate seals" have someone ever used them? Is it worthy to buy them?
I need to constantly monitor OD, so now I'm covering the microplate with a normal adhesive sealer every time I need to bring it to the microplate reader, really uncomfortable. I have no idea if these "gas permeable microplate seals" will also allow me to read the plate in the microplate reader without changing the seal every time, this will be really wonderful!  

Thanks

So if I understand it correctly: you grow your plates overnight without a plastic seal? And when you want to measure them you paste a seal on the plate in the laf and they you measure your plate and them put it back in the incubator after you removed the plastic?

And for what purpose are you doing this? whats your experiment?




anyway

I have never grown 96 well plates without using a plastic seal, so dont know if its normal they dry out when not using a seal.


I have stored masses in 96 well plates with only 100-150µl and cultured them overnight or even 2 nights and never had a dried out culture because I use plastic seals. (normal ones, not the special gas permeable microplate seals)


I use plastics to seal the wells , there is no problem with the aeriation.
If you do use plastics and remove the plastic to measure: you need to use a new plastic in order to prevent contamination.

So that brings us to the gas permeable microplate seals that you want to use to keep on the plates all time. Well I dont think you can use them. They arent really transparent and they will interfer with the reader I think.
You could look for some newer ones , but I doubt they are ok to use.


PS. I have also never seen someone reading his plates when there was still a seal on the plate
(when using a seal you will get some condens, fluid drops on the seal when incubated+ maybe some airbubbles.. those things arent good when reading a plate....  Its almost impossible to manually paste the seal 100% perfect on your plate...so I wonder how you do it)

[Dott. Berrino]

Thanks to all for your reply and sorry if I can answer only now.

bob1, on May 19 2010, 02:36 AM, said:

I think they will probably be OK with your spec, so long as they don't absorb at your measurement wavelength.  You would also need to make sure that you don't get any bubbles trapped under the seal as these will interfere with readings.  I don't know how getting rid of bubbles would interfere with the aeration of the plate.

Yes that was also my doubt, but the fact is that I have even never seen those seals, so I have no idea if they can be used for that!

bob1, on May 19 2010, 02:36 AM, said:

Have you tried putting a lid on and wrapping the edge in parafilm?  If you have to make frequent readings, removing the lid to do them should be enough to replenish the oxygen.

I'm growing bacteria in a colture microplate covered with the lid and everytime I have to read the plate I take off the cover and put a plastic seal on it, this is becuase if I read the plate directly in the plate reader will be better for my reading but not for the reader, I don't wont to contaminate the reader with bacteria I think other people in the department will kill me!  

pito, on May 19 2010, 02:40 PM, said:

So if I understand it correctly: you grow your plates overnight without a plastic seal? And when you want to measure them you paste a seal on the plate in the laf and they you measure your plate and them put it back in the incubator after you removed the plastic?

yes something like that.


The purpose of my experiment is just to find a faster way of screning several clones trated with different drugs that should alter bacterial growth. I need to do several readings and I have several clones, so doing it in a microplate instead of doing into tubes and then reading using a cuvette will be more handy and faster!... if it works...  

Anyway thank to all for your suggestions. Reading some litterature I also found out I should agitate faster than I was doing (225rpm) to provide enough areation.

Regarding putting the seal on the plate before reading I explained my motivations before , anyway I change it everytime to avoid condense and drops on the top and... yes they are not completely transparent so the also absorb, but I have some empty wells and some wells with just medium I can use as blanks.

Probably these gas permeable seal wont be good anyway for my purpose, but at least I could use them for overnight cultures... let's seen when I will have them...

Bye