particle counting

"In the lab I made counting comparisons with our CoulterCounter_III (MIII). I have attached the outcome in an excel file. I trust the counting performance of the CoulterCounter wich have been compared with manual counts in the past. As you can see in the attached file the CytoSense is performing outstanding quantitatively. This is a really pleasant observation. Also keep in mind that the MIII data are triplicates. If you sum the triplicate counts from the CytoSense, then the slope of the line is even further improved. Moreover it turns out that the CytoSense is able to count cells at much lower concentrations than the MIII. This is due to background noise generated by dust and bacteria in the MIII. These noise particles are easily disregarded due to their lack of fluorescence in the CytoSense. At the moment I have two students working with the unit and it works flawlessly. In particular we are impressed by the instruments stability. While we often experience clogged orifices while using the MIII when analysing natural samples, we have never experienced such issues in the CytoSense."

Hans H Jakobsen Research Associate Technical University of Denmark Danish Institute for Fisheries Research, Dept. of Marine Ecology and Aquaculture

Phytoplankton counting can be done effectively in many cases by combining the advantages of the microscope for its identification power and the flow cytometer for its speed. This requires also knowledge of the weak points of both methods in order to obtain the most efficient synergy. Comparison with traditional phytoplankton cell counting of cultures is easy since the species is known and even if contamination occurs since that is easily detected. Counting of field samples is more difficult since it depends on the exclusive recognition of the target cells in large numbers of other cells and debris (this may be difficult!) on the one hand and on good sample preparation and optical separation in the microscope specimen on the other hand (may be difficult). Such comparisons may show large discrepancies at first which can be minimized by optimizing the flow cytometric data analysis but also by critically evaluating the microscopical preparation and counting protocol used sofar. Often the flow cytometer counts more small cells because these are hard to see with a traditional microscope. The cytometer counting is less affected by some of the limitations associated with traditional microscopicy protocols such as Utermohl microscopy. We advise to use fluorescence microscopy. For example, validation trials were performed in the early nineties in The Neherlands (Ministry of Public Works and Transportation) basing on microscopical counting (following standardized protocols) of the group of relatively large and easy recognizable Rhodomonas cells in natural North Sea samples and flow cytometric analysis (the OPA instrument - the predecessor of CytoSense) of the same samples and counting the Rhodomonas type cells from the data. Initially, large discrepancies between the flow cytometer and microscope results (top panel) were observed. In the following year it was decided to keep the flow cytometric protocol the same but to use fluorescence based microscopy instead of the standard transmission light microscopy. As a result of these adaptations in the microscopy protocol, the results between microscopy and flow cytometery showed a much better and consistent match (lower panel) afterwards.