Contamination issue in cell culture - (Nov/16/2004 )
Hey!
I am new here and just experience the same problem most you just have. 
I just thawed cells on Thursday, there SK-N-Sh adherent cell lines passage 4. Today I went into lab to split them which was way too confluent already. After I finish passaging/splitting them, I look at the cells under the microscope and saw all these tiny black dots what it looks like bacteria. Then I took a look back at the original flasks where the thawed cells was diluted and there was black dots too. So I decided to throw everything out. The media I used was newly made today and I made sure everything is sprayed even my gloves with 70% etoh before putting it in the hood. But our media we used is not filtered nor added pen/strep. I then took a look @ other cell lines such as Hela cells and I think there are these dark specks too but out of the T-225 flasks, there is only a few (1 or 2 floating around) which was not noticeable at all.
Even the cells were overly confluent, there was no signs of contamination in the media, no turbidity and no pH color change. I use Cellgro MEM with those 10% FBS Oneshot from Invitrogen. A contamination happened to my supervisor before about a few weeks ago where 5 of her cell lines got contaminated by these dark spots. Its really making me stress out. I am going to thaw a new vial tomorrow and make new media.
I was wondering maybe it was the p1000 pippettor was using when transferring the thawed cells.
Before when I was working at another lab, we put pen/strep and filter our media which contamination was never a problem.
Do anyone have any good suggestions for me?
Thanks,
Irene
Hi
I am going growing caco2 cells in transwell inserts and my culture is getting contaminated as days go by. First in the walls of the transwells some fungus starts growing as days goes the cells too get infected . As I m new in using transwells suggest some possible reasons and give some suggestions so that i could grow the cells without any contamination..
.l Mine are MA-104 clone cells infected with the parasite Neospora, and since we obtain the isolate from mice to further adapt them to in vitro culture I guess some sort of contamination is coming out of this animal model. At the beginning we didn't figure we had this contamination because we usually work with medium containing Pen-Strept-Fungizone, but now we try to keep cultures without antibiotics and we have started to see at this tiny "bugs" with irregular shape, either alone or glued each other and sometimes adhered to the cells. 
At first, cultures look fine and medium has the appropriate pH but suddenly they raise their number and pH drops to 5.5 (amazing yellowish medium), so I must get rid of flasks before contamination affects the rest of what we keep on the incubator. We have run several tests (agar and brot culture, PCR for mycoplasma detection) and nothing shows up. If someone looks at the video and photo we are attaching, I would appreciate any advice you can give. Thanks!!! 
Hi,
I have the same problem. Just wondering if you have found a way to fix your problem. I don't know if I can just throw my cells out since I have a limited supply.
Sometime back I posted asking about MUTZ cells. These were from Germany. They are suspension cells. I had similar problem with those cells. No pH change, no turbidity - and the best thing was they also let the 'good cells - the MUTZ cells - grow. I was not able to identify what the tiny speckles were. But yes I thought like many others in this posting that they were definitely multiplying along the MUTZ cells. They came out mycoplasma negative in my testing and finally - I banked them. But since some scientist expressed a doubt that they might be contamination, nobody ever used them again. End of the story.
I am going growing caco2 cells in transwell inserts and my culture is getting contaminated as days go by. First in the walls of the transwells some fungus starts growing as days goes the cells too get infected . As I m new in using transwells suggest some possible reasons and give some suggestions so that i could grow the cells without any contamination..
Dear Fenila,
99% sure that your contamination is coming from your incubator. The incubator gets contaminated when people do not wipe (70% IMS) their flasks/dishes/plates in and out of the incubator. With filtered topped flask you ahve protection from the fungus. In plates however there is no protection and even if you have fantastic aseptic technique, they will go down with contamination.
Kindest regards
Rhombus
I too had a similar problem in my cell lines......the cells grew fine for a few passages. Then, the cytoplasm became granular followed by large vacuoles. Even penicillin-strepto-amphotericin B combo added at more than twice the usual concentrations didn't helped.
Similar oroblem was observed in primary cells. The problem was solved by changing the lot of DMEM and FBS. Also, since you are isolating primary cells, check out the possible infection in animals or biopsies (whatever you are using). Also check for integrity of HEPA filter.
I really do not know if this is the right forum to ask this question. Our lab does a lot of primary cell culture work like T cells and DC. The last week there has been a problem of contamination.
There are wriggly structures, highly motile, no visible flagella, like tiny specks. It was first thought to be cell debris, but they are motile and seem to have penetrated some of the cells or look like swarming inside the cells. Cells look healthy and no pH change in media or obvious turbidity. I have been reading about mycoplasma contamination, however most references state that mycoplasma contamination is usually invisible and detected by PCR kits.
How slowly does yeast grow on primary cell cultures, as some of the cultures were seven day old and it did not look like the organism had taken over even after 24 hours.
could you please help.
A researcher
I had the same problem!
I also thought they were cell debris but as time went on they were dense and destroy everything.
It is correct to try to identify them but I suggest that you throw then away ASAP and the media you used, clean the hood and incubator. And start over ASAP. I tried to avoid all these steps but the cotamination becamse worse. Mine were not rod shaped but were round circles that just moved everywhere in different directions. They were resistant to PenStep and Puromycin!
Another thing you could do is test your media for now and the incubator while you start over. It is frustrating but there could be a point where you might strat doubting results you get etc...due to the contamination so get rid of it.
I hope it work out.
Good luck and check cell lines that were frozen down. Sometimes bad cell lines get frozen down and when you use it this is what happens.
One of my fellow colleague (both of us sharing the same cell culture lab) got these creatures in his primary neuronal cultures. Same wriggling creatures (about 1 micron big at times), without flagella but still highly motile. Also had a capsule around them. At low magnification they appeared to be as if two spherical structures are joined together (like a diplococcus) but at higher magnification they were found to be rod-like with a slight depression around the middle. They showed several kinds of movements: spinning when one half sits one on the other vertically, spinning and horizontal movement, horizontal movement using the two halves (like pseudopodia is used by several mammalian cells). They moved really fast - typically they remained in microscope field for 1-5 seconds at 10X objective.
Check your media and FCS by incubating for 1-2 days. If using primary cells, animals may be a source of problem. Check if, while excising the tissue, the intestine is not damaged. Inoculate the cells in another medium to check if media or tissue is the source.
We tried the Strep-PenG cocktail, gentamycin, Amphotericin B but in vain. Culture media appeared to be culprit. The problem may have appeared during media preparation or from DMEM. The animals may also have been the source.
The organisms also formed colonies/aggregates at high concentration, settled at the bottom the centrifuge when kept undisturbed for around 1 hour. The medium was yellow and turbid and also slightly stnking at times but not always. We performed sensitivity studies with several antibiotics commonly used in cell culture but none of them worked out.
The only option is to wash the culture several times with PBS/HBBS/DMEM/saline or any other medium/salt solution. Washing the cells with a medium/salt solution several times (5 times may be sufficient but confirm in microscope) will remove the extracellular bacteria. Then, incubate the cells with sodium azide (its a blocker of ATP production. I dont remember the conc. used but u will easily find it in literature) for 30 min. Then wash cells again to remove sodium azide. Culture in presence of doxycycline, quiniodochlor, gentamycin, Strep-PenG-Amphotericin B cocktail.
Hope that helps.
We have the same type of artefacts, problem is we get them as soon as lymphocytes are recovered from the ficoll after processing fresh blood samples. Different donors, different lab stocks of PBS, different Ficolls, different heamocytometer, different microscopes, different centrifuges, different vacutainers...I have ad it with these things...i am starting to think it is my biological safety cabinet, perhaps the HEPA filter? Perhaps my pipettman...haven't changed the filter on that thing since months...God this is frsutrating...I feel like drinking acrylamide with my beer tonight.
I`ve been giving it more than a months thought, could it be our automatic pipettors are to blame, perhaps bacteria growing on the filter discs which avoid the pipettor's mechanism from getting contaminated from over-aspirating media are instilled into otherwise sterile media/cells. Perhaps our biological safety cabinets HEPA filters are to blame. I also believe these things are airborne as we get the right from the beginning from our human lymphocyte preps carried out according to medical aseptic technique. I think we have also ruled out operators (technicians or researchers) being the culprits as we were a complete BSL-3 outfit in the tissue culture room (gown, gloves, shoe-covers, particle respirators, etc).
Well, Well, Well. Finally pinned it down, sorted the contaminant out and learnt A LOT about the biology of these contaminants. As mentioned in previous posts we ran into these bacteria(yeast)-like objects immediately after extracting mononuclear cells from fresh blood. Although we first encountered these things in our lymphocyte cultures, our investigations traced these things far back to upstream procedures....as way back as the initial peripheral blood (human) mononuclear cell extraction. These things exhibited motion (although the lymphocytes remained still). These things did not behave like bacteria nor yeast. They did not grow fast enough nor seemed to rapidly divide (if at all), the culture media was not cloudy, no pH shift. These things did not look like budding yeast, they could not be clensed with ficoll prep and were obviously growing in penstrep. Weirdest of all was their motion, Brownian-like motion (text-book brownian actually). We tried to look for these things in the RPMI, in our PBS, in the ficoll. We tried different bacthes of all solutiones, tried different pipette tips and automatic pipettors, nothing. We feared our centrifuge had died or that our rotor was inducing vibrations...A different centrifuge (and rotor) told us otherwise. We were about to crack the Biological Safety Cabinet fearing a ruptured HEPA filter and perhaps a big mold growing on it...After several (weeks full of internet searches for Brownian motion in blood cells and ficoll contaminats) I ran into this webpage: http://www.microscopy-uk.org.uk/dww/home/hombrown.htm In this webpage there is a small video of particles with Brownian motion reflecting EXACTLY what we were seeing under the inverted microscope. PubMed then revealed two interesting articles (Chang HN, Robertson CR. Platelet aggregation by laminar shear and Brownian motion. Ann Biomed Eng. 1976 Jun;4(2):151-83. and Mody NA, King MR. Influence of Brownian motion on blood platelet flow behavior and adhesive dynamics near a planar wall. Langmuir. 2007 May 22;23(11):6321-8.). In the end, it turns out we might have been doing our spins at more than the required Gs...apparently this conditioned the penetration of PLATELETs (normally resident of the upper, plasma phase of ficoll-preps) into the buffy coat and into our lymphocyte isolates. And it now seems quite normal for these things to exhibit Brownian motion. I'd be more thah happy to send out a copy of our pictures or movies that demnstrate this. I am very pleased (and yes, self satisfied) at finally having resolved this issue. I can now die with a grin on my face and knowing that (once again) UFOs have a a very logical explanation which sometimes simply resides in our ignorance. My very best to you all. 