Suggestions and Troubleshooting

In our recent work with the FlowCAM, we've identified some common problems and potential solutions. While these suggestions may be largely unique to our projects, they are published here in hopes that they will be useful.

Clogging: When using a 10x objective- even to test apparently clear water- it can easily become clogged. The following signs generally indicate a partly clogged cell:

• A thin brown line at the top of the cell;
• Numerous bubbles in the portion of the tubing after the flow cell. A few irregularly spaced bubbles at the beginning of a run do not always indicate an issue;
• Slow depletion of liquid in the loading vessel.

To solve or prevent clogging, it generally helps to:

• Avoid running visibly dirty samples.
  
• Clean with acetone by running 3-5 mL through at the beginning and end of each run (and whenever a clog occurs).
• If acetone proceeds very slowly through the tubing, run several milliliters in "prime" and "reverse" mode with the tubing end in the acetone sample. This will reverse-elute the reagent, attacking the blockage from the opposite direction.

Image Quality: Use the rough-focus knobs to maneuver the camera onto the flow cell. Edges of the cell shouldn't be visible, and as few particles as possible should be seen stuck to the side of the glass. These particles will be accounted for in image calibration, but if they move even slightly, repeated imaging will occur and recalibration will be necessary. Using the fine focus knob and with sample running through the machine on the intended speed setting, focus the lens on the sample in between the two panes of glass. In our experience, image quality varies with the 10x: some larger particles will be especially sharp and clear, while smaller ones can appear blurry and indistinct. AutoImage Mode (No Save) is useful when the apparatus appears to be focused