Black dots in cell culture

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Preety
Preety's picture
Black dots in cell culture

Dear All,
I am working on APC 10.1 cell lines. there are small black dots in the culture. The cells are healthy, the cell morphology is normal. And there is absence of bacterial and fungal contamination.
Please, suggest what these black dots are ??????
the other thing is the cell line is very sticky and cell disaggregation is a problem. I am using trypsin for this purpose, but the cells dont detach after normal 2-5 min. There are sheets/ flakes of cells in media after tryp. I am interested in proteomics, so i am worried about prolonged use of trypsin then normal std time.
Any suggestions or comments are very welcome !!
Thanks & regards
Preety
 

hilltrekker
hilltrekker's picture
Please recheck for fungus and

Please recheck for fungus and media, also check for Mycoplasma, cell residues

Midsci
Midsci's picture
You could also use a scraper

You could also use a scraper to remove your cells, which is especially important if you want to look at the cell surface proteins. Once you collect the cells, you could use a small amount of trypsin for a short amount of time to disaggregate the cells. The black dots could be many things, including cell debris from normal cell death or contamination.

Preety
Preety's picture
Hi Hill trekker & Midsci ,

Hi Hill trekker & Midsci ,
Thanks very much , use of scrapper seems really good.
But i was wondering if it wil be fine to use scrapper while splitting cell lines.  
Yeah, i also think so, because there is absence of contamination. i had discarded all the old media ,PBS and trypsin , washed the cells and subcultured them in fresh media. Now there are less dots but still  there are some.
if it is cell debris then rate of cell death seems much higher. The black dots are floating in the media.
Is it possible to have so many black dots/ cell debris due to normal cell death??
Thanks very much for  this valuable input
Preety

Midsci
Midsci's picture
From looking at other blogs

From looking at other blogs from scientists, a few things seem to be common:cell debris, precipitates from the media, very early fungal infection.  Overall, it seems harmless, but you could always do a control with media alone in your incubator for a few weeks to be sure. For example:
http://www.bio.net/bionet/mm/cellbiol/1995-April/001950.html
http://www.protocol-online.org/biology-forums/posts/12608.html
Hope this helps!

Preety
Preety's picture
Thanks a lot for all this

Thanks a lot for all this input,
i am grateful to u for this.  Ah, atleast one thing is clear that others had also experienced such wierd little things. 
but still whenever i see my cultures under microscope and i am curious to know what is it ?? en why in my cell culture only.
i was worried as there was possibility of its being the early stage of fungal contamination. this possibilty was ruled out bec after subsequent sub culturing there was no fungal contamination but those black dots.  I think as Simom did we should ignore them and lets see if it works well.
i was wondering if  someone can tell what it is exactly??
Thanks very  much for ur help,
Cheers!!
Preety

jamew
jamew's picture
I am very interesting for

I am very interesting for your black dots finding.  Could you tell me the size, shape of the black dots?

Preety
Preety's picture
 

 
The black dots are like tiny spores even a bit smaller  en are floating , but definitely they are not spores. they are round or say spherical in shape. i washed my cell lines with PBS 2-3 times and after 5 passages they are almost gone. but some times in some cultures they do appear again, but  this time less in number.

csguy
csguy's picture
I too am having problems with

I too am having problems with black dots in my cultures. I am growing Vero Cells and the media is very clear with no cloudiness or dicolouration, but there are lots of black dots floating in the culture. I passaged them yesterday and already there are lots of dots. They don't seem to be bacteria or fungal and don't look like yeast. I will try and take a photo today
Any clues?

Preety
Preety's picture
Hi csguy,

Hi csguy,

few months ago, there were lot of black dots in my cultures also. it was same like you explained . try to passage cells frequently and wash with PBS at least 5-6 times. if the cell line grows too fast then use less volume while splitting them.

make sure the cells are not under stress,like check your incubator etc.

if it is not any type of contamination then, certainly it will disappear in couple of weeks.

dont worry, just follow these steps and hopefully , you will get rid of this problem.

cheers!

preety

jamew
jamew's picture
Hi Preety:

Hi Preety:
   could you send a picture of your black dots to me?

   J

Preety
Preety's picture
Hi J,

Hi J,

Sorry, i didnot take picture of black dots. However, i can tell you about their morphology. the black dots were of varying shapes ,mostly spherical and some a bit elongated. They didnot look like spores either. very small in size, just like dots. 
fortunately , i dont have these dots now .

Hope this helps!!

Are you having the same problem with your cells?????
 
preety

jamew
jamew's picture
 Hi Pretty:

 Hi Pretty:
   Yes, I have the same trouble!  Could yopu tell me how to remove it?

   J

Jith sn
Jith sn's picture
Dear all,

Dear all,
It was intersting to have querries and answers regarding those" little wierd dots".
I have come across the same tiny dark spots in my culture , especially when i was using some suspension cell lines, i strongly believe that those are the cell debris floating in the media, may be due to increased cell death???

I have experienced those dark particle disappears after say 3-4 subcultures and it is common just after the revival od cells from LN.

Thanks n regards
jith

Preety
Preety's picture
Hi J,

Hi J,

i am using adherent cell line. i dont know the exact cause of these black dots.
but i dont feel it was cell debris because media was perfect, passaging was done on time etc.
However, i washed the cells with PBS at least 6-7 times, in initial stages 8 times also.

Second thing to do is to reduce the seeding density.e.g if you use 3ml for splitting then reduce it to half i.e 1.5 ml. if your cells grow too fast then passage them frequently say if you are doing it after 3 days do it 1-2 days. every time wash cells with 6-7 times with PBS(normally we do it 1-2 times), you may increase the number of washes if you feel they are quite a lot in number.

after couple of weeks these will be gone.

try to remind if at any stage you cells were under stress. i know we try to en maintain TC conditions under control, but still it is good to check.

Passage frequently and wash with PBS thoroughly, hopefully your problem will be resolved.

All the best !
preety

calciumcurrent
calciumcurrent's picture
Hi Preety, 

Hi Preety, 
     I think you can try to trpsin them at 37 degree! My cells are also very sticky. At room temperature it is difficult to get cells with trpsin even for 5 min. But when I put them in the incubator, it is easy to get cells within 2 min.  Good luck!

Calciumcurrent

Abhishek Singh
Abhishek Singh's picture
hi the black dot if in higher

hi the black dot if in higher magnification in phase contrast shows a cicular ring sourrounding that then it is mycoplasma

alo check if FBS or DMEM remain there during trypsin addition it counters trypsin mediated detachment of cell so wash with PBS twice or thrice then add typsin and incubate for 30 sec to 1 min at 37 C because at lower temp trypsin didnt work .

Preety
Preety's picture
Hi all,                      

Hi all,                                     

Thanks very much for the input.  I used to trypsinize the cells at 37o C using I X T. 
Now i am using 2x and it is much better now. Still it takes ~10 min or more depending on the confluency of cells. 
These dots were so small that even under high magnification nothing like outline or circular ring was visible.

Thanks
Preety
  

Jason King
Jason King's picture
Just a note about the PBS

Just a note about the PBS that is used to wash cells prior to trypsinizing them. Make sure is does NOT contain calcium. Washing twice with Ca- PBS will allow the trypsin/EDTA to work more efficiently and reduce the amount of time the cells are out of their growth media. This will improve the health of the cells and reduce the amount of black debris "dots" seen in the culture.

Also, as I understand it, it is not possible to see mycoplasma using a phase contrast microscope and the lenses commonly used in TC labs.....or does someone else know better? If so please supply a reference, as this helps reduce the number of "old wives tales" etc that circulate.

samm
samm's picture
Short of PCR, there does not

Short of PCR, there does not seem to be a fully reliable method to detect Mp. The cover-slip-for-3-days followed by DAPI probably is next best, but you need to know what you are looking for (the even blue dots surrounding, but some way out of the eukaryotic nuclei). I don't trust any of the other light/phase contrast microscopy stuff mentioned here.

maheshsdhar
maheshsdhar's picture
Hi all, The black dots that u

Hi all, The black dots that u witness are nothing but  apoptotic cells debris (apoptotic bodies). The tend to move and some times look like bacterial movement, but that is brownian movement and nothing else. As u examine ur culture closely for some days u will see that there is no cloudiness. There could be toxicity that is killin the cells. Best of Luck

gapa
gapa's picture
HELLO PREETY,

HELLO PREETY,
I HAVE JOINED NEW TO THE FORUM. I AM WORKING ON CERVICAL CANCER AND I AM ALSO FACING SAME BLACK DOTS PROBLEM WITH MY CELL LINE. ALTHOUGH, THE CELLS GROW FINE FOR 3 DAYS WITH DOTS FEW IN NUMBER, AFTER SUBCULTURING THESE BLACK DOTS INCREASE IN NUMBER OUT GROWING CELLS IN NUMBER AND THUS INCREASE IN GRANULATION. AFTER THIS THE CELLS(FEW) MORPHOLOGY STARTS DETEORATING SPECIALLY THEIR CELL MEMBRANE(OUTER PERIMETER OF THE CELLS ARE NOT CLEAR AND DISTINCT)CANNOT BE DISTINGUISHED. ANTIBIOTICS PEN/STREPTO WAS USED. MEDIA WAS CHECKED FOR CONTAMINATION AND FOUND TO BE FREE FROM CONTAMINATION. ALSO THE MEDIA FROM THE FLASKS WAS USED FOR MICROBIAL TESTING AND FOUND TO BE FREE FROM ANY SORT OF FUNGAL CONTANMINATION. ALTHOUGH FOR BACTERIAL TESTING THE MEDIA SHOWED SOME TURBIDITY AFTER 8 DAYS INCUBATION. ONE MORE OBSERVATION FOR YOU PEOPLE IS THAT WHEN ANTIBIOTIC FREE MEDIA WAS USED THE THINGS MENTIONED ABOVE APPEARED WITHIN 2 DAYS ALSO THIS TIME A LARGE MESHY NETWORK OF ROPE LIKE STRUCTURE INTERMINGLED TIGHTLY WAS SEEN WITH CHANGE IN PH OVERNIGHT ALONG WITH WHITE CLUMPS FLOATING IN THE MEDIA(POSSIBLY OF THE MESHY NETWORK) AFTER CHANGING THE MEDIA THE THINGS REPEATED IN SAME MANNER WITH CELLS START DETACHING AFTER 3 DAYS.

KINDLY HELP ME TROUBLESHOOT THIS PROBLEM. BECAUSE AFTERDOING STERILISATION FOR ALL THE STUFFS THIS PROBLEM RESURFACES AFTER A GAP OF EVERY ONE AND A HALF MONTHS AND IT IS HINDERING THE PROGRESS OF OUR WORK.

THANKS AND REGARD
GAPA(GAURAV PARASHAR)

samm
samm's picture
Hi GP, just as a little aside

Hi GP, just as a little aside, its considered rude to post messages in all-CAPS (its the physical equivalent of yelling). Also, please do not post the same message multiple times: all posts are indexed, and will show up in the appropriate category.
As to your problem, it does appear that you have intermittent contamination. If you are sure its not your techniques or media, that leaves equipment. Is your hoods' HEPA filter fresh? If you are using glassware (pipettes etc) - are they sterile? Do you share equipment with anyone else - are they following proper aseptic protocol?
-s

Vickite
Vickite's picture
Hi everyone,

Hi everyone,

I know this topic has been covered quite extensively before, and I read your posts with interest. We have the same problem in our TC room. There are two of us with suspension cell lines, and two with adherent. None of us use antibiotics at the moment in our cultures. Just before Christmas, I noticed tiny 'fidgeting' black dots and also larger, more spherical objects that almost appear to spin in multiple directions. Usually all assays are done in flasks so I hadn't noticed this before, you can only see them when you plate out the cells after one or more days on the highest magnification.
After I saw this, everyone else checked and the other neutrophil like cell line was crawling with them, but the two adherent cell lines only had minimal numbers. We all use different media and FCS and have plated these out and they are clean.
What confuses me is how the suspension cell lines appear to carry such higher numbers than the adherent, we have tried growing the bugs in multiple broths and plates but nothing grows. It does not turn our media cloudy.
As we use neutrophils like cell lines we cannot do our assays as they are quite happily munching up whatever the black dots are, I need some advice as to how to get rid of this problem??
Thanks

shorty
shorty's picture
can u tell me wat the black

can u tell me wat the black dot is in a cell cause i need to know

Posted By shorty
on 10/3/2010 10:04 AM   

   

just can u tell me wat a black dot is in a cell

Elmabsout
Elmabsout's picture
Yes , the black dots you saw

Yes , the black dots you saw in culture media are contamination, and is recently known bacteria from environment is called  Achromobacter  and its resistance to conventional antibiotics , you can get rid of them by using combination of ciprofloxacin and piperacillin.

aashya
aashya's picture
 hii....... I am also facing

 hii....... I am also facing the same problem with Vero E6 cell line.........and its also there in the other cell lines in the lab and the other labs too...... what if the antobiotics suggested by you leads to its resistance....... since we had tried using ciplox but they became resistant in about two months and the problem is back.
it would be really gud if someone could give a helpful suggestion :)

uet
uet's picture
dear

dear
with profound regads i am also seeing the black dots in cell culture and cell are sticky. what remedy you had taken at that time
thanks

narcissus
narcissus's picture
Dear GAPA,

Dear GAPA,
I am also facing the same problem with cell culture as detailed by you. I strongly believe that it is some sort of contamination and not a part of cell (apoptotic or cell debris). I also feel that this contamination is coming from serum as when I keep pure serum in the incubator, it does lead to huge vivid growth forming a scum like thing eventually.
I would be happy to know whether the problem you were facing has vanished or is there some way to get rid of it.

mitt29
mitt29's picture
 Hello,

 Hello,

What company FBS are you all using? I am using GIBCO and a friend form another lab has the same problem using gibco... I am not sure it GIBCO is the problem but since FBS came up in the discussion and also the 2010 article suggests the problem source is the FBS although I do not remember reading what brand of FBS they we using...

I am worried because I ham having issues knocking some genes down with siRNA and atlast one of them I have knocked down pretty well earlier (1 year ago) and now problems.... Maybe the siRNA aliquots have degraded but could this bacterial contamination be a reason. I have tried Ciprofloxacin Piperacillin by the way ...the problem does not go away completely....

Thanks

g a
g a's picture
 Hi Mitt29

 Hi Mitt29

All batches of FBS are different and have lot to lot variations. THough there is a possibility that your siRNA had degraded, but you have to confirm it. 
We tried a rule during such experiments that which so ever batch and lot works for 1 experiment ...... keep it safe and never change it...... and success rate was 100% for subsequent experiments.

aashya
aashya's picture
hi Argerine,

hi Argerine,
         yeah thats true in my case too, we are now using Piperacillin with Ciplox and the problem has decreased but its not getting cured completely. But if you are giving PBS washes and media change to it everyday, the cells are actually showing a speedy recovery. Once you keep the media for more time, the blacks dots are coming back.

aashya
aashya's picture
hi Mitt,

hi Mitt,
 I dont personally feel that the problem is being caused by FBS alone since we use FBS from PAN now and still the problem is there. We filter sterilioze everything even before adding to the cells but still the problem comes back if we are not giving PBS washes and media change everday.
     

mitt29
mitt29's picture
 Thanks Guys,

 Thanks Guys,

I think the repeated PBS wash is a very good suggestion that I am going to try from now on. Only problem is with the whole bunch of stable clones and everything its going to take a huge amount of media for changing everyday....

Thanks guys!

narcissus
narcissus's picture
Dear people

Dear people

I recently did a simple experiment where I just plated serum alone and incubated the same in the CO2 incubator and to my surprise and assumption I found humongous growth of the same culprit particles (nightmare of cell culture). I certainly believe that the problem is chiefly because of serum FBS we use, since when I tried yet another batch of Gibco serum, the particle load was quite less.
Also as suggested by people, It seems there exists a sort of balance between the live particle contaminants and your cells, as soon as the health of cells is compromised because of any small insult like transfection or you have not changed the media for long, these live particle contaminants LPCs aggravate and subjugate the culture to the extent that the cells become completely sick.
So the rule of the thumb is to keep cells as healthy as possible, by giving recurrent PBS washes or media change to keep off these LPCs.
Indeed the use of piperacillin and ciprofloxacin also works to some extent.

Saniya
Saniya's picture
I just wanted to add yet

I just wanted to add yet another report of "small black dots". I am culturing primary myoblasts from mice. My first 2 cultures back in July were perfectly clean and good. The next 6-7 attempts all yielded various amounts of black dot contamination, starting as soon as the next day after plating and as late as about 3 days after plating. It's hard to believe this is cell debris. I have run all sorts of controls with media. My plating media generally stays clean in the incubator for at least a week. But after keeping the media int he incubator for about 3 weeks, I did get "small black dots" all over. I have tried Pen/Strep, Pen/Strep/Amphotericin, and Pen/Strep/Amphotericin/Sparfloxacin (like I said, lots of controls). The various antibiotic agents wither make no difference at all, or reduce the "black dots" for a couple of days, after which they come back. Washing adherent cultures definitely helps. Unfortunately, I grow up satellite cells in suspension in a dish before I differentiate them to adherent myocytes, so I cannot wash them for about a week.

Sal_Sal
Sal_Sal's picture
Dear All,

Dear All,

I was trying to investigate this  problem for  the last 2 years!

It’s for sure a hidden contamination not easy to notice or detect…. Not easy to culture on any type of agar or broth!
 
Its is very tricky to stain it’s which I found after long time of trying…
 
We manage to film it on a domestic camera since we didn’t have a digital camera attached to out microscope!
 
The video is perfect showing very interesting behavior of the bacteria
Here is the link…. I welcome any suggestion or comment…
 http://www.youtube.com/watch?v=lo96fcXfcVs
 
Regards
 
Sal 

Sal_Sal
Sal_Sal's picture
Dear Saniya,

Dear Saniya,

I was in your place for the last 2 years..... and I did reach to some results in te way for publication!!!!

I was through all sort of Scenario.... I will mention some of them...... in the near future!

First we did stop working in the labs and fumigates the labs and order new materials
 new cells line and media....

Start over again

then next day after culture the new vial we found the black dots in the culture.....?!!!

This was a big problem...... we thought that contamination is in the lab still!
One of the students went and checks the main campus labs and found to black dots still there!!!

now what I found the people in my work start to worry and freaks out even IT people stop coming to the lab to fix our computers....

the cell contamination story spread in the institute and people who don't work in cell culture relay don’t know what is going on and I assume they think about it as some sort of bacterial breakout....

there is still  more to say but  have to go now!

Regards

Sal

imran.altaf@uva...
imran.altaf@uvas.edu.pk's picture
take following steps to solve

take following steps to solve the problem of black dots in cell culture

Use pencilline+genta+strepto+kana+cipro+tylo+amphotericine-B in cell culture media. though it will put the cell under stress.
try to change the media after every 24 hours.
repeat this till the elimination of black dots
after the elimination of dots, add 5ml of 0.5% glucose in media along with 1ml of non essential amino acid per 100 ml to take the cells out of stress.
try to use gamma radiated cell culture reagents.
With the grace of "GOD ALMIGHTY" the problem will be solved with in 20-25 days.

The reson of problems
1. Achromobacter
2. Mycoplasma
3. Deficient media

Sal_Sal
Sal_Sal's picture
Dear imran,

Dear imran,

Thanks for your replay. We did try different types of antibacterial drugs so far but no luck in removing the black dots?!

We will find them if we look very carefully in the flask in some of the cells!

The thing is this is not Achromobacer or Mycoplasma ......we run many mycoplasma test and they came negative! We did send the cells to Mycoplasma Expert Ltd in UK and the results were Negative!!!

Remember all this work takes a lot of time..... And I feel pity for student who have to stop their work during our investigating..

Thanks again

Sal

uet
uet's picture
if some one found the

if some one found the solution of this contamination please inform

Sal_Sal
Sal_Sal's picture
Hi every one,

Hi every one,

Can each one of you please who is facing the problem of the blackdot in their cells verify their  county and from where they bought the cell ( company, location) or if it’s from the tissue culture bank of their University or department... no need to much details … need to so make a map of the spread of this issue!
 
Thanks for your teamwork!
 
Sal_Sal

bingbing75
bingbing75's picture
Hello,

Hello,

I am performing analysis onCaco-2 cells. I have had a lot of problems with bacterial contamination. This has been solved by use of antibiotic supplemented media and regular disinfection of incubators and less people using them. Now after many weeks of good culture there are small black dots. When confluent these dots appear to be more pronounced around cell junctions. They are non-motile, small and the tissue culture seems OK (apart from the dots). My thoughts are : (1) a response to antibiotics
(2) to disinfectants (hydrogen peroxide based), (3) cell death and debris or (4) mycoplasma (which I don't know much about ...size, susceptibility to antibiotics). (5) abiotic debris from media (6) extensive use of 70% ethanol maybe penetrating into the flasks from sterilisation of external surfaces.

Any help would be much appreciated firstly what this might be and secondly if it will affect the metabolism/immune response of the cells.

After ensuring and making every effort to avoid contamination, this is frustrating-it is unaffected by streptomycin and penicillin. The media looks clear, where in other contamination issues, the initial indicator is cloudiness of the media used.

any help would be appreciated.

Thanks

Saniya
Saniya's picture
 Country- USA, Johns Hopkins

 Country- USA, Johns Hopkins Medical School. I make primary myocyte cultures from mice, so this is not a cell line contamination problem. I am also starting to think that it is cell debris or even fat droplets moving by brownian motion. The reason is that my healthier mice (with fattier muscles) seem to give cultures with more dots. My other hypothesis is that it is protein precipitating out of my high-serum media. So maybe my black dots aren't everyone else's black dots. (Yes, I know, this just makes things more complicated.)

shankaranarayanan.v
shankaranarayanan.v's picture
dear all..

dear all..
I am also having certain problems with the black spots in a few cell lines I have been using. the PBS wash certainly helps. I even tried trypsinizing cells n giving it a pbs wash at low spin. it helped reduce the black spots. but it didn't help remove it..
Has anyone been successful in eliminating the problem. if yes, i would like to know about.

regards,
Shankar

Sal Salem
Sal Salem's picture
Hi Every one,

Hi Every one,

is there any update in relation to this issue

Thanks

Nidhi Sharma Sharma
Nidhi Sharma Sharma's picture
 Hello everyone,

 Hello everyone,

I'm working on CHO, A549 and HUVEC cell lines currently. Since, last month I'm constantly seeing swimming black dots in the culture flasks. I tried thawing cells again and again, but these dots keep appearing. 

Need help!

Mobi_Dot
Mobi_Dot's picture
Dear Nidhishaa,

Dear Nidhishaa,

Please clarify if the black dots you observe in the cells in the cytoplasm of cells or in the culture medium?

If you throwing from frozen you would expect a percentage of dead cells!
Try to wash cells next day after culture and centrifuge at minimum speed as it possible and get rid from supernatant.

Seed cells again and observe if these dots still here?

Good luck