Mycoplasma will not be seen as black dots under light microscope. If you really want to be sure of it - Hoechst staining would be the easiest to do in the lab. I have attached a photo to compare. Magnification : x 600. The dots you see in the culture could be

(1) cell debri from high cell death in the culture due to poor health of the cells (some form of contamination including mycoplasma / senescence )

(2) residual bacterial contamination - could be due to a Pen/Strep resistant strain or a different strain.

If you get a negative results with Hoechst staining, you can think about using another antibiotic (gentamycin) or as suggested by Deobrat Dixit

I will also consider using another batch of FBS. There is a batch to batch variation and your cells may not be tolerating the endotoxins and growth factors in this batch of serum.

Dear Celine, perhaps is a kind of fungus, you can suplemment the media with amphotericin B for avoid this contamination. For confirm is better to do a culture of wells. With best regards

http://www.invitrogen.com/site/us/en/home/References/gibco-cell-culture-basics/biological-contamination/yeast-contamination.html

http://www-p53.iarc.fr/FP6material/Cell%20culture%20contamination.pdf

Did you test your cell lines for mycoplasma contamination? I know that a mycoplasma contamination is usually not visible via a tissue culture microscope; however, I have (unfortunately) seen similar "black dots" in heavily contaminated cell lines. And, unlike contamination caused by most bacteria, yeast or fungi, mycoplasma typically do not alter the turbidity or pH of cell culture media.

Hi Céline,

Your pictures look similar to my own contamination issues at the moment. Whatever the contamination is, it's important to both kill the contaminating cells in your media and find out where the contamination is coming from, if this is a persistent problem. I second Juan Bueno's suggestion of supplementing the media with amphotericin B. As for the detective work, I would suggest streaking LB plates (no antibiotics) with each reagent that you used in your procedures involving your cells and leading up to when you saw the contamination. If anything grows on a given plate, discard that reagent.

Also, if you want to, post some pictures of the cultures that grow. I've been tracking the culture that I made recently be streaking plates to determine the source of my own contamination and am currently growing yellow colonies that I can't identify. They don't grow like the fungi that I've seen in the past, are definitely not yeast and are resistant to pen/strep, which should kill most bacteria. It could be fun to compare notes. I'm always curious about the things that grow in the lab. =)

Please check your culture room condition..

where is your AC, or properly maintain the room humidity... dust condition..

And check th biosafety canbinet hepa or pre filter condition..

Hi Celine,

Most of human cell lines are contaminated with Mycoplasm contamination try to check your cell lines for that contamination. Also check the FBS or your medium for the contamination.

http://www.minerva-biolabs.com/en/services

It could be mycoplasma. Try treating your cells with Cyproxin at a final concentration of 2mg/ml for a few days. If the dots go away, they're mycoplasma!

You may use plasminogen...for mycoplasma

no matter what contamination it is. now the only thing you have to do is to start it over with a new vial of non-contaminated cells with fresh reagent. I will save you time. go get a new cell line and stop wasting time characterizing your contaminations.

I had a very similar issue... it was indeed mycoplasma in my case. I was able to remove the contamination using plasmocin as some have suggested but be warned that I noticed quite a change in signaling of my cells while treated with this drug. I work on p53 signalling and use a MCF7 cell line. I would highly recommend using a PCR test for mycoplasma to ensure that is the contaminant. If you are suffering from a mycoplasma contamination I would almost suggest starting up new cells in a VERY clean new incubator. Mycoplasma is really difficult to kill mainly due to its ethanol resistance.

incubate your medium without cells and see if you still see these structures to ensure they are not coming from serum or medium. I agree that mycoplasma is strong possibility as these structures reflects that cells are under some stress. I would suggest to treat your cells for Mycoplamsa. Good luck!

I will also agree that It is necessary check your cells for presence of mycoplasma, and If it is not the case of contamination I would like to advise you to check your cells at least 2 times per year (using PCR test for mycoplasma).

Also always put aliquots of the medium that you are using and check them as well.

I can tell you that if you have this type of mycoplasma contamination, it is better to start over with new and healthy cells..

Good luck!

I was unable to download the pic files, but from the description, it does seem to be mycoplasma, which is a beast to get rid of. Mycoplasma can come from many different sources; sometimes the FBS itself is the source. I agree with David that the PCR test is a good way to confirm that the contamination is indeed mycoplasma.

If you can, it is best to restart your cultures from frozen stocks using medium prepared with FCS that has been screened for mycoplasma. I would also concur that you should decontaminate the incubator that you are culturing the cells in before restarting your cultures.

Good luck!

Although I don't want to rule out Mycoplasm, I am not entirely sure that it would be that visible under a microscope. We ran into the same problem with SKBR3 cells. I was skeptical as the black dots don't necessarily look like a fungal infection, We started adding Fungizone (Amphotericin B) to the medium, which basically took care of it. I would start over with a new batch and try adding the antifungal and see if it can solve the issue. Depending on the cell line, Amphotericin B can be toxic so check the literature first to determine the correct concentration.

Hi

Check the pdf attached. It might be helpful. I have also experienced such dots specially during selection. I have used puromycin and zeocin.

What is the growth rate of your cells? R they growing normally or slowly?

I have tested my cells several for mycoplasma and always found a negative result. Also they come from calls as medium without cells never have the dots.

Mycoplasma will not be seen as black dots under light microscope. If you really want to be sure of it - Hoechst staining would be the easiest to do in the lab. I have attached a photo to compare. Magnification : x 600. The dots you see in the culture could be

(1) cell debri from high cell death in the culture due to poor health of the cells (some form of contamination including mycoplasma / senescence )

(2) residual bacterial contamination - could be due to a Pen/Strep resistant strain or a different strain.

If you get a negative results with Hoechst staining, you can think about using another antibiotic (gentamycin) or as suggested by Deobrat Dixit

I will also consider using another batch of FBS. There is a batch to batch variation and your cells may not be tolerating the endotoxins and growth factors in this batch of serum.

Undoubtedly be a mycoplasma! Test as soon as your cell, and try to "incubate" appropriate Means.

Good luck!

Test your cell culture for mycoplasma contamination!

For mycoplasma detection we are using the MycoAlert Mycoplasma Detection Kit (LONZA) and for decontamination MycoRAZOR (Biontex). However, as mentioned by several other people, if possible you should restart with contamination-free cells, and don't forget to decontaminate your incubator.

I don't think its fungi or bacteria contamination.

How fast your cell line grows? What is its double time? Maybe cell line is suffering and liberating pieces of plasmatic membrane and cytoplasmic granules. Don’t discard the possibility of mycoplasma. You should test it as a routine in your lab.

Hi

I am as yet not sure about mycoplasma, unless it is very strong contamination. The resolution is very simple either u can use PCR based test (Sigma) or u can just plate these cells on a small 3.5 or 6 cm PD with coverslip in it next day quick fix in ice cold MetOH (3-5min), rehydrate in PBS invert coverslip on drop of DAPI or Hoechst DNA stain. Mount just in PBS or any glycerol based mounting medium. What u will see is on the picture above the cells will be covered with a small blue dots especially dense at the cytoplasm edge.

If that is confirmed u have to decide if the cells are so precious u can not get them anywhere else (unique clones) threat them with anti-mycoplasma antibiotics. Otherwise get them from storage or somebody else. I have a good experience with BM-cyclin from Roche Cat No. 10 799 050 001. Use as is in directions. There will be at least two rounds of treatment and I tend to use 1/2 higher concentration of both antibiotics at the second round to kill those resistats. Cells most of the time do not mind. Always check cells before freezing them. Good Luck

First, I want to thank to all for the very helpful suggestions and tips. Indeed, at this point, we are still not convinced that we have any sort of contamination (mycoplasma or other), mostly because: i) our human cells grow quite well (

Sometimes, we see calcium precipitation from the serum. It may even jiggle a bit due to Brownian motion. The DAPI technique of Vlastimil Srsen is quick and easy.

Other bacterial or fungal contaminations make the media turbid or foul smelling, although sniffing the flask compromises their sterility.

Remove media from the culture flask and filter through 0.2 micron filter and make 2 pieces place one piece of filter on nutrient agar plate and another on fungal suitable media plate and incubate to confirm contamination.

If contamination is present rinse the cells with antibiotic solution for few minutes and subculture.

Finally check for mycoplasma.

Dear Celine, Is your "contamination" irregular in shape with sharp edges (cell debris) or spherical. If spherical you could be looking at totipotent stem cells (0.2 to 2.0 microns in size). For pictures see Stout et al., Amer Surg 73:1106-1110, 2007. The real small ones will go through a 0.2 micron filter, so if you follow Chandra Sekhar's suggestion I would propose using a 0.1 micron filter (which will stop the cells) instead of a 0.2 micron filter (which will not stop the cells).

The next question of course, is can you expand the population, whatever it might be. If it is the 0.2-1.0 micron totipotent stem cells, they will grow in suspension culture (or adherent). We use Opti-MEM with Glutamax + beta-mercaptoethanol + 10% heat inactivated serum (Atlas Biologicals, Fort Collins, CO), pH 7.4. When we innoculated an uncoated T-75 flask containing 350-ml medium with 1 billion of the "very small entities" (what we called them at the time), after 7 days in a 37C incubator we had 1 trillion of the same entities. We went on to characterize them [Minerva Biotech 17:55-63, 2005] and discovered a very unique population of cells with some very unique healing properties.

Since you are working with cancer cell lines, you may be culturing the stem cells on the side and not know it. I have seen totipotent stem cells in pictures from multiple journals and the investigators describe them as debris. If the cancer cell lines were isolated by ring cloning, there are probably more different kinds of cell types present, than cells from a single cell progeny.

Cell surface markers for the totipotent stem cells are CD66e, CEA, HCEA, etc., some variation of carcinoembryonic antigen, which is another way to identify them. Also, be forewarned, the totipotent stem cells are Trypan blue positive. That may sound strange because one is always taught in cell culture that Trypan blue negative means live cells and Trypan blue positive means dead cells. The reasoning is that dead or dying cells that have corrupted cell membranes will let in the dye. The actual reasoning is the ability of the cell to pump out the dye, live cells will pump the dye out, while dead cells won't. That holds for most cell types except the totipotent stem cells which do not contain the cellular machinery to pump out the dye. We looked with transmission electron microscopy. The totipotent stem cells are basically a compacted VERY heterochromatic nucleus surrounded with a plasma membrane with enough cytoplasm for 2 small lamellar mitochondria and that is all. But they will differentiate into at least 68 different cell types across all three primary germ layer lineages (ectoderm, mesoderm, and endoderm) as well as form spermatogonia. From this data we changed the name from "very small entities" to totipotent stem cells. We have found the totipotent stem cells in primary isolates from mice, rats, rabbits, cats, dog, sheep, goats, pigs, cows, horses, and humans.

I have observed similar black dots in our cultures (some floating, some attached). All our cultures are mycoplasma negative. One current hypothesis is that they are Achromobacter that may be present in FBS (I'm not sure how they pass through the filtration steps though). There's an article published about the black dots (Biologicals 38 (2010) 273-277). After my inquiry along with a few other inquiries, a cell company did extensive analysis on the cells and claim that the cells are clean of any contamination. The articles' lead author claims that the dots are very difficult to grow on their own. Further, once they did grow, lysing them for DNA identification was even more difficult.

I have tried many antibiotics/antimycotics with no help. It does appear that the contamination level is somewhat cell line specific. Some lines have few dots. Other lines have many dots. I have changed serum lots and companies 3 times already. I've had our biological safety cabinet sterilized along with our incubators. I've ordered new cells with the new serum and still get similar results - dots.

I have seen the picture you have attached here. But I need to know, when you see these cells under microscope do you see the black spot as a "spot with a fine tail attached to dense head" . I too have faced the same problem, but nothing worked, so I threw all the stuff, cells, media, trypsin, FCS, and Flasks too. This things, for me were visible in adherent cells as I tried using the same media in my suspension cells. They were fine. If you try to plate you culture supernatant on Agar plate, will not get anything. Three things worked for me, one I changed the trypsin, second used other older frozen stock and 3rd before doing it I fumigated the whole cell culture room and everything.

I hope this helps you.

do post the result.

All the Best

It might be some contamination but more pronouncly it looks like uncontrolled division of cells indicating some type of cancer growth.

It doesnt be any fungal contamination because it takes much longer time for its growth .

It may be mycoplasma me to observing such type of partiacals im my cancer culture flasks try to cheeck for mycoplasma contamination by PCR

An additional method to test if the dots you see are cells versus debris versus contamination is to run FACS analysis on a released culture of your cells with propidium iodide. Set your flow at essentially 0.00 - 10^2 side scatter versus 0.00 - 10^2 forward scatter and look at the lower left box. If you have the 0.2 to 2.0 micron cells they will be PI+ and be located in that area.

You can also stain unfixed cells with CD66e (if human) or CEA (if non-human) bound to phycoerythrin (PE). Run that in conjunction with the PI. Co-localization of PI+ and PE+ staining denotes the cells.

Additionally, the 0.2 - 2.0 micron cells in the unfixed state autofluoresce in the FITC emmision wavelength range . Set your sheer fluid as background and run unfixed, unstained tissue culture contents through your flow cytometer and look for entities auto-fluorescing in the FITC channel. Anything positive and you have the cells.

Hi,

I am also experiencing the same problem in some adherent cell lines. I have observed that these DOTS are only adhered to cells for first 2 days after passaging, but these dots then start to become visible in the media also. The number of dots increase after 3-4 days if cells are not sub-cultured.

Cell lines grow properly, no change in pH of media or turbidity.

Changed FBS, Trypsin, Media etc. i also thawed frozen vials but the problem still persists.

After washing 4-5 times at 500 RPM the number of dots decreases significantly but after culturing the dots start to appear again.

We did DAPI for mycoplasma but it came Negative.

i am working on cell signaling and i used to get good and reproducible results before these dots came from no were, and now because of these i am not able to repeat the results.

So kindly suggest some possible reasons.

With so many suggestions and hypotheses, it looks to me that is best to simply get a new stock of cells than attempting to identify/struggle with the problem... But then again, if someone finds what these mysterious dots are, we will all be happy to know!

Arjan,

While I can't comment on Celine's cells and what was done to them. I have never used polybrene nor performed viral infections with my cells and have noticed these dots for several years. Newly purchased cells also acquire these dots (this assumes they were dot-free when I bought them). This is why I (and others) believe the source of these dots is the FBS. However, as mentioned above, I've tried serum from 4 different suppliers with mixed results. I've spoken with several company reps that sell serum. While they didn't know anything about 'dots', they did say that there are only a few wholesale sources of serum. If the companies are just repackaging the serum under their brand, then most of the serum may contain black dots.

Hi Salwa,

Please, I would like to see the video.

Hi Celine,

1) Discard any kind of contamination (many methods have been aforementioned). As you wrote, some tests results seems to indicate that the cells are not contaminated.

2) If this is the case, the dots could be salt or protein precipitates. The pictures you have attached, remind a similar problem I had when testing serum free media for our cell lines. In our case the conclusion was that they were salt precipitates due to pH acidification of de cell culture medium.

Although my post is two months delayed I hope it will help you.

Has anyone figured out what these black dots really are?? I would really like to know. Thanks!

Dr. Patrick's answer is v. interesting...What about His, Phe? Do they also polymerize in presence of free radicals triggered by Riboflavin? one usually avoids B-Mercaptoethanol in media. I guess the in vivo cellular environement is more reduced than we realize..??

did they move? few year ago, I also had the same "contamination" until now I still have no answer

this is link which I posted it on Youtube

http://www.youtube.com/watch?v=N7nMqgm-lNs

I use E'MEM 10%FBS for cell cuture

but how about the black particles on the cells? I mean in my case the particles appear in the medium (but medium no change color) and on the cell.

Again, Dr. Patrick's answers are v. interesting.. I wish I ould do these experiments! But, i do not have this option at present. I still wonder about the redox state in vivo. Barring mitochondria, what is known about the in he redox state in vivo in the rest of the cell ?? Afterall, tumor cells behave very differently in normoxic versus hypoxic environments in vitro. When I worked with ARPE cells (retinal pigment epithelial cells), I found they were quite resistant to certain toxins, and their levels of total GSH hardly changed. Some data on Cigarette smoke also found this lack of effect on total GSH. Lots more to do-I guess!

Great input from Dr. Patrick! Thanks

I've only ever used RPMI-1640. Filtration did not remove the dots.

The RPMI we use (#22400, Invitrogen) contains glutathione, phenol red, and HEPES buffer. I believe we are speaking of different dots. I can see them when cells are stained with trypan blue. These dots have motility as well (beyond normal Brownian motion).

Hi! I have the same problem, I I believe you have a contamination by mycoplasma:

http://mbiolika.blogspot.pt/2010/11/detection-of-mycoplasma-in-cell.html

the paper:

http://www.sciencedirect.com/science/article/pii/0141022994000753

use specific antibiotics to remove mycoplasma, and test it by PCR:

http://vri.cz/docs/vetmed/50-6-262.pdf

Fernando,

I don't have mycoplasma. We screen regularly. We also test by fluorodeoxyuridine sensitivity. Mycoplasma causes resistance to the anticancer drug.

Patrick,

Apparently, you need to grow these dots on blood agar plates. Without the presence of other cells, they (dots) don't grow well. More details can be found in this article .

Title: Got black swimming dots in your cell culture?, Biologicals(2010) 38,273.

http://www.sciencedirect.com/science/article/pii/S1045105609001304

Their frequency seems to vary with serum lots which is why they may be coming from the serum. However, I haven't identified a dot-free batch of serum. Their frequency also varies between cell lines (grown in the same RPMI medium) suggesting an unknown cell factor that may stimulate/support dot growth. If it was strictly a medium/serum contaminant, they should be uniformly present in all cell lines.

John Schmitz, even if that is not mycoplasma, in my lab I have two Vero cultures one with that "comtamination" and another without that! and the culture medium is the same.

Even if he culture in blood agar that contamination eventually could not growth without the mamalian cells that can excrete some compounds to the medium...

Send your samples to Multiplexion, they check your cells for many possible cell culture contaminations!

http://www.multiplexion.de

Mostly, it's mycoplasma, you just stain it by DAPI, if it can be stained by DAPI and showed in blue, which means that it's mcoplasma not debris.

Dealing with tissue culture is a big task and needs fine handling. Before going for contamination problem you have to check the origin of your cell line, is it melanoma or any other cell line that can produce special pigments. Then if you focus on contamination problem you have to check for bacterial, fungal (including yeast) and mycoplasma contamination by using conventional method for detection. Using antibiotics(strep or gentamicine with penicillin and with antifungal like amphotercine B or Nystatin may cure your culture. If you will not see any change you can make several dilutions for your cells and try to culture even single cell in 96 multi-wells culture plate with double dose antibiotic you then may able to get clean germ free culture.

What cell line are you using?

Actually I have been working with different cancer cell lines but I have established three long term cancer cell lines one of them was colorectal cancer cell line called NYT27 . I followed its karytypic evolution from primary culture till passage 50. I found clear change in the cells subclones and each passage showed evolutionary subclones. My thesis in Sheffield Univrrsity Medical School in UK explained that task clearly. Now I am in Iraq and I am trying to make electronic version for my thesis and if I finished I will send you the chapter concerning the karyotypic evolution of in vitro cancer cell in culture.

Are you using heat inactivated serum? If not, try that. Some cancer cell lines already secret huge amount og rowth factors, so using HI serum may help.

@Swati: Heat inactivating only inactivates (at least mostly) factors of the complement system.

Sometimes you can find some extrange debris even in just-prepared culture media. It can be due to precipitated serum proteins or little particles coming from plastic pipetes etc. As long as everything is sterile, it should be no problem. If you are sure that there are no micoplasms in your culture and bacterial/fungal contamination is discarded (for example, incubate cell-free culture media for a cuple of days and check it; fungus are usually very evident, and bacteria make the medium to change color and adquire turbidity) then just don't worry.

You can culture the detached cells again to assess their viabilty and their capacity for growth. Before that you can use trypan blue method for viabilty checking as most detached cells either they are over confluent active cells or they are dead cells due to something wrong in culture conditiins.

I am sure that it is nothing but contamination although not sure with what, cell is not fine, the cell membrane disrupted, you can not make subculture with it. you have to discard this flask and thaw a new cell line.

It is mycoplasma only. You can test with mycoplasma detection kit..

Dear Celine, i have seen your images. I think this a contamination and the best way to prove this is to cultivate the centrifuged supernatant medium on BHI agar plate. I suggest to change your Antibiotic with Gentamycin. But i must say that if you want to use these cells for gene expression profiling, at least 21 days must pass after contamination eradication. These contamination seems to be bascilus. I have seen this portrait in a flask contaminated with lactobascilus (as a probiotic). Do you smell any thing unusual like yoghurt or something like that? In contamination with mycoplasma, almost always you will see lower proliferation rate (increment of doubling time) and intracytoplasmic vaculization. I didn't see intracellular vaculization . But all the cells are completely under stress and something is absolutely wrong.

It looks like mycoplasma contamination only. did you try with fresh reveval of new vial and does it look same? You can check with PCR in concentrated media for mycoplasma contamination, as sometimes you wont see anything if you are changing your media every day.

Other thing you can use alternative to L-glutamine, with increased stability that improves cell health, like Gluta Max from Invitrogen which minimizes toxic ammonia build-up.

It didnt look like bacterial contamination , but you cant overllok the possibility of mycoplasma contamination though. Morever its kind of some debris. I believe trying new vials as mentioned above or even centrifuging the FBS(not at higher rpm) may help the cause. You have to try all alternatives.

Let the cells reach a near confluent state, then trypsinize but not to the point of dislodgement, rinse twice and then observe under a microscope. If the spots persist, then that is an issue of concern.

In the picture, it seems to me that definitely your cell culture got contaminated. But the question is what type of contamination? Mycoplasma contamination is not detectable visually under light microscope. To make sure mycoplasma contamination you need to do mycoplasma test using PCR kit. However, bacterial, fungal or similar other type of contaminations are visually detectable under light microscope. I had similar kind of problem like you while I was culturing human cell line. In your uploaded picture, I just saw all the black dots which are the symptoms of bacterial contamination. In fungal contamination, you will see almost round shaped cells with budding and little bit bigger than the black dots in the culture media which I did not notice in your uploaded picture.

You told that you used antibiotic in your culture media and that does not mean that you will not have bacterial contamination. Usually whatever concentration of antibiotic we use in culture media is not enough to kill large amount of bacterial contamination. Once your cell culture got highly contaminated by anyhow, it is very hard to get rid of them. You can increase the antibiotic concentration, but that can affect your cell line.

What I am trying to say that for successful cell culture you need to stick to proper aseptic cell culture technique and there is no alternative than that.

As your cell culture already got high contamination, my recommendation is that if you have back up cell line in liquid nitrogen, chuck this culture and go for new culture and stick to proper aseptic cell culture technique and use antibiotic as well. Change media every 2 or 3 days interval as Pen/Strep is stable at 37 C for not more than 3 days. I usually change media every 2 days interval. But before go for new culture, find out whether the prepared media or any part of your media anyhow got contaminated or not. Once you confident that everything is alight, then go for new cell culture. Good luck !

I am having same problem in tissue culture,, I am trying to make stable cell line and using antibiotics for selection... I think, in my case its just because of antibiotic stress resulting in production of debris... I am still not sure though... Try testing your media. It can be something in your media.. I am not sure though as I am having same problem, but I think its not the contamination, its just debris. My colleagues work on prostate cancer cell line and they have same issue and they actually tested their cell lines and found, it was just the debris.

I do not think this is contamination, try to reduce trypinization time and wash the cells with pbs spin down1000rpm@ 1.5 min and seed them back,

I have an query.. If our cell lines are contaminated with mycoplasma will it affect the adherency of cells or not?

I had tested the cells used in my lab for mycoplasma contamination and every cell line tested positive. I think it is a general situation that most cell lines in academic research are contaminated to some extent with HeLa cells and mycoplasma. In in vivo mouse studies, the cells are treated with antimycoplasma agents and this may bring changes to the cultured cells' behavior. It is essential to keep in mind the limits of our model systems. From my experience, it was nearly impossible to rid the culture system of mycoplasma since not all users of the common facility agreed to treat their own cultures. Regarding HeLa cells, there is little control over the contamination of established cell lines. ATCC tested their stocks and determined that a large proportion of their bank was contaminated with HeLa cells. These are limits to in vitro studies and must be kept in mind when conclusions are drawn.

Jean-Charles

here is the youtube link showing bacterial contamination-

http://www.youtube.com/watch?v=lo96fcXfcVs

re: John Schmitz,

Ah, so thats what that stuff is. I always have wondered, over the years, why we see it once and awhile.

Guys check co2 levels of your incubator. I had the same issue and noticed it was co2 level in my incubator was very high, although the reading was 5%. It's just because the sensor was broke and actual Co2 level was very high. Changed incubator and no black dots now.. Hope this will work for u.

Hi,

I discussed with a Fisher Scientific technician a while ago and he told me that the CO2 detectors only work properly in humidity saturated air. If your incubator is running out of water, the detectors are off and then the CO2 injections won't maintain a 5% CO2 although it might still read 5% CO2. Hope this helps.

Jean-Charles Neel

Hi,

I think they are bacterial contamination. To sure you should pasage your cells at least three times without antibiotics and then try a blood agar culture.Hope this helps.

Parvaneh Farzaneh

Hi,

To me this does not seem to a bacterial contamination. Just observe the cells and the structure under the microscope. If they move they are bacteria otherwise not. It appears that your cells are facing some kind of cytotoxicity or stress. The main reason could be improper trypsinisation or stress due to imabalance in pH and nutrient. Remember once cells are stressed it might take take few passages to recover. Dont trypsinise the cells even they are confluent for one two days. Just change media. This might help them from recovering the trypsinisation shock.

If it is mycoplasm you can remove or mitigate with plasmocin. It is true there are huge amounts of mycoplasm contamination in academia but it must be handled. you can use plamsocin to cure myco or prophetically..

The blackish specks on the images are unlikely to be bacterial contamination, but that doesn't mean that the cells are mycoplasma or contamination free. The specks look to be protein aggregates and the cells appear stressed and looks like they are secreting a lot of something. We have some cell lines that secrete so much protein into the media in response to a growth factor that it changes the viscosity of the media, and in some cases it causes protein aggregation and precipitation.

Also yes, regular use of antibiotics masks contamination, and poor sterile technique. Every lab should regularly test for mycoplasma, and antibiotics should only be used for critical cell lines.