Tiny black particles vibrating in my cell culture...Contamination?
Sze Wah Agnes CHAN
Have any of you seen these before? If I pay very close attention, with only
200X, I saw tiny black particles, some spindle shapes, some more round,
vibrating in my cell culture flasks. Are they mere particles in Brownian
motion? Or some kind of "living" contamination?? Culture medium appears
clear and cells seem to be growing OK. Thanks in advance for sharing your
experience.
Agnes
---
redeamer
Hi, Agnes... you ve mentioned the Brownian motion... but in brownian
motion there is motion... so do you have a motion on your plate when
it is immobilized under microscope.. if yes with a great probability
you have bacteria proliferating there... if that stuff is growing in
quantity that is for sure prokaryotes...
At the other hand there are cells that really want to live in
different sort of garbage stuff, or that is the way we think, cause we
dont know for sure:) we just dont like it under microscope:) I had an
experiance of working with LNCaP human prostate adenocarcinoma cell
line... that was an interesting one... a lot of debris were floating
around, and the cells were VERY SATISFIED with that, and when i tried
to wash the plate ---> their cell cycle slowed down...
So just stay calm and wathc your cells..
Cheers -->
Drew
Wolfgang Schechinger
please give us some more details (cells, media, antibiotics, general
procedure)!
Hm *vibrating*.... If you relly see *vibrating* stuff, then I guess you
have living contaminants. Or a vibrating microscope.
Normally, debris has a different motion pattern than many contaminating
bacteria who a moving by active motion (using flagellae motors). If these
'bugs' move in an out of focus, or if you obeserve turbid precipitates even
without a microscope, that makes your cell culture look like yeast or E.
coli culture, then you should be worried.
Best, you find somone who is really experienced with TC and is able to tell
you just by looking through the microscope. This is much better than
'intelligent guessing'.
Make sure your supplies are not contaminated. e.g. by storing aliqots of
medium without antibiotics in an incubator for quality control (add small
aliquots of serum and all the other fluids) and monitor them regularely,
especially when media etc. are prepared inhouse.
If you think that you need to rescue your cells because they are *really*
valuable (normally, it's better to take a new aliquot from the freezer and
to start over), then wash your cells extensively once or twice a day with
PBS and use appropriate antibiotics at least for some time until your
culture is 'clean for at least a few days'.
Good luck! Wo
Wolfgang Schechinger
---
EK
> dont know for sure:) we just dont like it under microscope:) I had an
> experiance of working with LNCaP human prostate adenocarcinoma cell
> line... that was an interesting one... a lot of debris were floating
> around, and the cells were VERY SATISFIED with that, and when i tried
> to wash the plate ---> their cell cycle slowed down...
Well, I never had any debris floating in my plates with LNCaP. I believe it
is just low quality of serum or other additives that you use that
precipitate during growth.
EK
"Sze Wah Agnes CHAN" <cha...@hkucc.hku.hk> wrote in message
news:401F...@webmaila.hku.hk...
spread over LB plate. You will know tomorrow if there are bacteria in your
cell culture. Also, prepare a dry flame-fixed smear from another aliquot of
the media (5-10 ul) and stain with crystal violet. See under microscope at
high magnification if there is anything resembling bacteria. And by the way,
estimate the size of the particles to be sure it is in the range of
bacteria.
EK
Sell Biology
>
> "Sze Wah Agnes CHAN" <cha...@hkucc.hku.hk> wrote in message
> news:401F...@webmaila.hku.hk...
>> Dear cell culture friends,
>> Have any of you seen these before? If I pay very close attention,
>> with
> only
>> 200X, I saw tiny black particles, some spindle shapes, some more
>> round, vibrating in my cell culture flasks. Are they mere particles
>> in Brownian motion? Or some kind of "living" contamination??
>> Culture medium appears clear and cells seem to be growing OK. Thanks
>> in advance for sharing your experience.
>> Agnes
>>
>> ---
> Take an aliquot of the media from you plates and inoculate into LB, or
> spread over LB plate. You will know tomorrow if there are bacteria in
> your cell culture.
Almost certainly there will be a positive result. But that doesn't mean
that her cultures are contaminated. In a perfect world, we would not
have to include antibiotics (pen/strep for bacteria, rarely
fungizone/Amphotericin B for fungi) in any media for (mammalian) cell
cultures, but we do because sterility is a relative state of mind.
But if the poster wants to do an LB/rich medium plate test, I recommend
that the poster do log dilutions of the medium to quantitate the number
of bacteria per unit volume. The poster should see a characteristic
decrease according to dilution if the contamination is really in the
medium.
The best thing for the poster to do is to look at the cells themselves.
Do they look healthy? Are they viable (trypan blue, etc.)? Are they
proliferating (if they are proliferative)?
If there are coliforms in the culture, they will most certainly bloom and
make themselves apparent without even looking at the microscope. Most
contaminations are of yeast/fungi because antifungals are usually not
included, and they sometimes sporulate. This is especially true if the
shelves of the humidified 37 deg incubator are not regularly cleaned
(they often get media on them and you can see fungal "fuzz"), if the
flasks are allowed to have media on the outside of them without being
wiped clean, or if the water from a warming bath is not cleaned regularly
(and the bottles warmed in them not wiped clean with an 70% ethanol-
soaked rag).
If the poster is concerned about mycoplasma, there are tests to identify
them. One is a Hoechst dye staining technique to look for extranuclear
DNA spots.
Nick Theodorakis
<d...@no.email.thankstospam.net> wrote:
[...]
>No, we don't. During more than 7 years of passaging cells practically
>daily, I've had two instances of contamination and we NEVER EVER used
>ANY antibiotics. Personally, I think using antibiotics when workign
>with established lines long-term is WRONG. Very wrong. If you can't
>keep it sterile w/o them - you probably shouldn't be doing it because
>this is absolutely basic, no-real-skill-required technique. If you
>generally can but occasional slip results in contamination - it's
>always better to discover it early and toss the cells instead of
>confronting latent infection, weeks of lost work or (at its worst)
>publishing screwed up results that nobody else can ever reproduce.
I have to agree here. I don't use any antibiotics with established
cell lines, either, and I don't have any problems. Sterility is an
absolute.
Nick
--
Nick Theodorakis
nick_the...@hotmail.com
nicholas_theodorakis [at] urmc [dot] rochester [dot] edu