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I have thawed Hl-60 cells from liquid nitrogen chamber two times in RPMI 1640 media (containing 10% FBS, L glutamin, penicillin/streptomycin). In the first day the cells remain okay. But when subcultured from the first flask to new flasks (cell solution : media= 1:3) all the flasks became contaminated on the 2nd day of culture. Cloud was found in both time. It can be mentioned that I used sterile one-time usable pipettes and uncoated flask for growth. All the transfers to other flasks and media addition were done very carefully and done inside clean bench. Before using clean bench, it was sprayed by 70% EtOH. 

Has anyone experienced like this problem?  Please share and if have, please give some valuable suggestions or references.

Thanks

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The single most likely reason for the contamination is that your frozen stock is contaminated. Assuming you've been properly trained in cell culture, you're working in a correctly-maintained biosafety cabinet, your reagents are all sterile (has your media been sterile-filtered after adding FBS, etc.?), and the incubator is clean (water bath pan has been autoclaved, sterile water with biocide/fungicide added to it, entire incubator wiped down and all removable parts autoclaved periodically, etc.), then stock contamination is the most reasonable answer. Try thawing earlier passages than you have been working with, or order a new vial or two from ATCC or whoever your cell vendor is. Make sure your pipetors are clean, and you're using sterile filtered pipet tips.

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Avoid passing your hands through the openings of the flasks/tubes since skin originated bacteria are the major source of contamination. It's safe to briefly burn the caps and the opening of the tubes before opening or capping. If you maintained good aseptic techniques through thawing and subcultures, it's possible that the frozen cells were contaminated previously. Try thawing a new vial of frozen cells and see if there's improvement.

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There is a simple reason for this "cloudy" cultures. HL-60 is a suspension cell line (see ATCC on this), so most of the cells stay in the liquid phase and do not adhere to the plastic surface of the culture flask. Some may, but usually they don't adhere very good and come off when moving the flask or gently tapping it.

Suspension cultures look different from bacterial contamination, they are less dense and you can see the cells (which are much bigger than bacteria) in the microscope when you look at the culture. Bacterial cultures often of build up deposits of bacteria at the border of the medium. And the media of bacterial contaminated cultures are almost always yellow, as the metabolism products make the media acidic. An image would be helpful here.

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  • $\begingroup$ Under inverted microscope cluster of very tiny colony were observed along with the cells. It was clearly distinguished between the unwelcoming materials and cells. The color of the media was not so changed, it was the same like normal media. @Chris $\endgroup$ – M Rahman Dec 7 '16 at 11:09
  • $\begingroup$ Ok, then please post some images of the culture. And if possible microscopic as well. $\endgroup$ – Chris Dec 7 '16 at 11:17
  • $\begingroup$ Sorry to say, they were discarded, and thinking to thaw new vial of HL-60 again. That is why searching for suggestion or advice. $\endgroup$ – M Rahman Dec 7 '16 at 11:23

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