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

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

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.

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

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!

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

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

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.

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?

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

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

   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

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

   J

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

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