Possible contamination of leukemic cell culture - debris, growth inhibition, cell aggregates (May/29/2010 )

Hello,
I have a bit of a problem with my soon (2 months) ending diploma thesis: a culture I received for my experiments this monday, after it had been cultured for one month with always surprisingly bad growth (usual doubling time ~24h), to me seems contaminated. Now I would like to hear some other opinions on this, anyway I´m grateful for any idea concerning this culture. It´s a promyelocytic cell line (~9-15µm in size, leukemia, HL-60) that can be easily differentiated along the neutrophilic lineage and is somewhat a suspension culture model system for cytoskeletal organization what is central to the thesis.
I first thought what I see would be bacteria/mycoplasms, but for sure it may also be debris, on the other hand this does not explain the overall bad growth and unusual appearance.
In fact HL-60 responds to terminal differentiation with apoptosis, and when differentiated gets polarized by LPS and activated by fMLP, so the spots may be debris and the cells may show terminal differentiation and activation due to altered gene expression (mycoplasma induced?) and stimulation by bacteria specific motifs, respectively; after one month in culture this would also explain the amount of apoptotic bodies and the few "cells". Do the "cells" look like conglomerates of activated or normal cells to you, do you know if neutrophils form conglomerates when cultures are contaminated?
Otherwise density may have been low (had not been counted previously), I still hope to get into the proposed range over the weekend though they don´t divide when differentiated, additionally I could imagine that the medium may have only contained half the concentration of serum, though this seems unlikely.
Do you think I could wash the debris out with PBS, what centrifugation speed would you suggest and how long should I centrifugate?

Here are some pictures (40x objective):

a picture of how the culture actually looks:
http://i780.photobucket.com/albums/yy85/My...a1/IMG_0687.jpg

the cells should look similar to this other line I still got (thanks the lord!):
http://i780.photobucket.com/albums/yy85/My...a1/IMG_0699.jpg

these pictures show resuspended "pellet":
medium from flask:
http://i780.photobucket.com/albums/yy85/My...a1/IMG_0666.jpg

pure culture medium:
http://i780.photobucket.com/albums/yy85/My...a1/IMG_0705.jpg

a nightmare:
http://i780.photobucket.com/albums/yy85/My...a1/IMG_0672.jpg

two different foci:
http://i780.photobucket.com/albums/yy85/My...a1/IMG_0680.jpg
http://i780.photobucket.com/albums/yy85/My...a1/IMG_0683.jpg

Some pictures from activated migrating neutrophils from the web:
http://media.wiley.com/CurrentProtocols/CB...0004-1-full.gif

And the picture of what I would have had to expect according to ATCC:
http://www.atcc.org/attachments/1761.jpg

Has anyone seen something like the pictures in his cultures or knows someone who works with leukocytes?
I would like to try DAPI on monday and order antibiotics to culture some vials we have deep frozen some weeks ago, what do you think about this? And do you have the impression that there are still viable cells visible?

Thanks for your replies!

Well I guess after cultivating the debris for a further week, that the culture was already destroyed when I received it. We will start a new culture tomorrow. My professor didn´t see the necessity to do DAPI stain as she was convinced to be able to see mycoplasms due to her experience with cell culture, so I can´t tell if there was contamination or not. Anyway thanks to everyone who had a look on this post.

Mycoplasma are very small and mostly intracellular, they are very hard to see with conventional light microscopy unless the contamination is very very heavy.

bob1 on Jun 2 2010, 04:18 PM said:

You´re right especially with a 20x objective its somekind of supernatural to reach the "Bragg limit" (small mycoplasma strains have a diameter of 0,3µm=300nm=lamda/2) ;-), but anyway a boss is a boss is a boss...