Tiny black particles vibrating in my cell culture...Contamination?

D.K. dk at no.email.thankstospam.net
Sat Feb 7 22:20:10 EST 2004

On Sun, 08 Feb 2004 02:03:30 GMT, Sell Biology <stemcells at noaddress.none> wrote:

>"EK" <nobody at elnino.com> wrote in inimitable style:
>> "Sze Wah Agnes CHAN" <chansw3 at hkucc.hku.hk> wrote in message
>> news:401F830B at 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.  

Why is it so? Every time my paranoia took over and I did the test, 
it always came out negative. 

>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.

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. 

>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).

Nothing's wrong with doing things "right", but in my experience even 
when the incubator's shelves are covered with visible sporulating 
Pennicillum and Aspergillus, no contamination results if one does not
open dishes outside the hood or sticks fingers inside. It's wise to 
remember that human hands handling all those dishes and flasks are
invaribly the dirtiest thing in the tissue culture room. 

>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.

Mycoplasma infection does not manifest itself as visible free-floating
particle the orginal poster described.


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