Modified FlowCAM procedure for quantifying size distribution of zooplankton with sample recycling capacity.

We have developed a modified FlowCAM procedure for efficiently quantifying the size distribution of zooplankton. The modified method offers the following new features: 1) prevents animals from settling and clogging with constant bubbling in the sample container; 2) prevents damage to sample animals and facilitates recycling by replacing the built-in peristaltic pump with an external syringe pump, in order to generate negative pressure, creates a steady flow by drawing air from the receiving conical flask (i.e. vacuum pump), and transfers plankton from the sample container toward the main flowcell of the imaging system and finally into the receiving flask; 3) aligns samples in advance of imaging and prevents clogging with an additional flowcell placed ahead of the main flowcell. These modifications were designed to overcome the difficulties applying the standard FlowCAM procedure to studies where the number of individuals per sample is small, and since the FlowCAM can only image a subset of a sample. Our effective recycling procedure allows users to pass the same sample through the FlowCAM many times (i.e. bootstrapping the sample) in order to generate a good size distribution. Although more advanced FlowCAM models are equipped with syringe pump and Field of View (FOV) flowcells which can image all particles passing through the flow field; we note that these advanced setups are very expensive, offer limited syringe and flowcell sizes, and do not guarantee recycling. In contrast, our modifications are inexpensive and flexible. Finally, we compared the biovolumes estimated by automated FlowCAM image analysis versus conventional manual measurements, and found that the size of an individual zooplankter can be estimated by the FlowCAM image system after ground truthing.