To generate peritoneal exudate macrophages (PEMs), I inject mice intraperitoneally with Concanavalin A, then euthanize them 3 days later and and flush the peritoneal cavity with DMEM+antibiotics. I have done this several times in the past and never had trouble isolating PEMs.
However, the past 2 times I have attempted to recover PEMs, I have noticed a large quantity of rod-shaped contaminants in the peritoneal wash fluid. The first time this happened, I assumed they were fibers from a pipette tip. The next day, all the cells that I plated were dead, and only these fiber-shaped objects remained.
Today, I performed another peritoneal wash, and noticed these fibers again when attempting to count the cells. As you can see, they vastly outnumber the cells, and some of the rods even appeared to be moving.
I wouldn't think they would be a bacterial or fungal species due to size, shape and motility. It looks more like a protozoal species.
These mice are BALB/c mice purchased from the Jackson Laboratories, and have not been infected with any pathogens or exposed to any infected mice.
The media used to flush the peritoneum has been examined under a microscope, and does not appear to contain any of these contaminants.
So far, I have plated the Concanavalin A, the recovery media, and the contaminated peritoneal exudate on blood agar and emailed our veterinarian.
Does anyone have any idea what this might be, or any tips to figure it out?
Hi Catherine:
We started to work in 2013 with human primary cells to test a powder chemical drug. We had detected since the beginning these transparent and shining structures in our cells culture very similar that yours that must be eliminated.
We used 10% of Penicillin / Streptomycin (P4333-100ml, Sigma Aldrich) in our culture media and that is why we discarded the possibility of bacteria contamination. We tested , however, for bacteria contamination and mycoplasms and the results were negative for both.
We assumed then, that these structures could be either or undissolved powder chemical drug (ethanol as vehicle) or fungi or both. We started to use since 2013 Fungizone R Amhotenicin (15290-026,50ml, Gibco) and the problem was solved.
I hope that you could solve yours as well. Good lock.
Although I have never seen such contamination in the past, but I have had a similar problem. It has been even more striking, because I just melted a frozen tube with a well known and very viable cell line, which we maintain in the lab for a long time. I found square prisms of a size of a cell. The cells were dead by the next day. The prisms changed their size and number (became smaller and increased in number), which might mimic proliferation. To make a long story short: the prisms were NOT a living matter, they were soluble in diluted HCl. They appeared to be Ca-oxalate crystals coming from serum. The crystals dissolved and re-precipitated upon dilution in the medium, and this was mimicking proliferation. Obviously, the high concentration of both Ca and oxalate in the medium was toxic to the cells. Later on I found the crystals in serum itself, which is a likely source of toxic oxalate. Apparently, this lot of serum contained an unusual concentration of it. In most of the cases when the oxalate concentration in serum is lower, and the crystals are dissolved in the medium without re-precipitation, so it goes unnoticed. Your crystals are probably not Ca-oxalate, they are something else. Still, I advise you to examine your serum, preferably cold, before the dilution, and filter it through the membrane filter before adding it to the medium.
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