Hello. Using DAPI to strain the cells can not be considered as the criterion of cell contamination. Because we can not wash the superfluous DAPI in the cells, the DAPI will be observed by long exposure. Observation under bright field will be better.

Hello,

Be sure to check all of your staining reagents for contamination as well. As

Li Xiang mentions, you need to assess the quality of your cell culture under brightfield microscope to observe bacteria. Usually, contaminated cultures will have cells that are dying and yours seems to be fine (based on the nucleus staining size). Cells undergoing necrosis or apoptosis will have pyknotic (condensed) or karyorrhectic (segmented) nuclei. This does not appear to be the case, but I would expect some cell death in contaminated cultures (at least those contaminated prior to fixation). Again, check your blocking buffer etc to make sure it is fresh. Contaminated block or PBS used in staining will have a detrimental effect on the stain and could be showing up here.

Also. keep in mind that it is not uncommon to see nonspecific coloration in any wavelength. Endogenous fluorescence can always be an issue, not matter what fluorescent tag being examined, especially if excitation is high (high gain or laser power). The key is to tune the laser power/gain down as to ensure real signal is not overblown (usually you can use the imaging software to tell this) and that extraneous information or endogenous fluorescence isn't picked up. It looks like you have a sense of this "tuning" from your images.