Phytoplankton Enumeration and Cell Sorting

Phytoplankton are found in most aquatic environments (freshwater and marine systems) and SRLs are sometimes asked to detect, enumerate and sort these cells for various purposes. Phytoplankton produce photosynthetic pigments that fluoresce and can be detected on most standard flow cytometers. The most common fluorescent pigments found in phytoplankton (both cyanobacteria and other eukaryotic plankton) are chlorophyll a & b as well as phycoerythrin and phycocyanin (phycobiliproteins). All of these pigments can be excited with blue light (standard 488 nm laser as well as custom lasers), however different pigments will produce different emissions. Chlorophyll (a & b) is best detected using a band pass filter around the emission maximum of (660-670 nm). Standard flow cytometric instruments will use a red filter (e.g. 692/30 BP) or 670 LP. Phycoerythrin can also be excited/detected using blue excitation (488 nm) however, will be optimally excited with green light (532 – 550nm) with an fluorescence emission maximum around 575nm (e.g. 575/30 BP). For freshwater systems

Phytoplankton range in size, however, in most aquatic communities they are dominated by small pico-nano sized cyanobacteria and other small eukaryotic cells ranging in size from 0.5-20 μm (and larger) – Figure 1.

Image KEY – Coastal Ocean Sample:

• Synechococcus - cyanobacteria = Red
• Small PicoEukaryotes = Blue
• Cryptophytes = Green (identified by dual red and orange fluorescence)

• (Note: X- axis = Side Scatter Y- axis = Relative Red Fluorescence (670/LP)

Sample Preparation

Samples can originate from a variety of aquatic environments and as with most samples running samples without preservation is preferred (primarily due to pigment loss and cell disruption during preservation). However, most aquatic samples are collected in the field and returned to the laboratory for analysis. Sample density ranges from 104 to 105 in most cases so no sample dilution or concentration is required.

For sample preservation, aldehydes are recommended (glutaraldehyde or formalin). For the Center for Aquatic Cytometry we use the following aldehyde SOP preparation for most phytoplankton samples – however, others are available and can be used when preserving for phytoplankton, bacteria and viruses (Marie et al. 2005 and Marie et al 2014).

For preservation for down-stream molecular work (post cell sorting) the following preservatives are recommended: glycine betaine and glycerol/TE (see links for SOPs)

Instrument Modifications and Setup

Fluidics – Most standard flow cytometers use commercial sheath fluids that are designed for use with biomedical samples. For aquatic and environmental these fluids can be used for some samples – however, alternatives are recommended.

• Analyzers – For detection and enumeration of phytoplankton the ideal sheath fluid would contain no additives – Deionized water is recommended. In the case of marine samples, the use of deionized water or augmented deionized water with salt (NaCl 8-30 ppt) can be used. It is best to keep all additives (surfactants and preservatives – out of the sheath system) – as seawater has been known to precipitate in commercial sheath fluids. It is important to clean the sheath lines frequently and replace the inline sheath filter – in order to disinfect and keep the sheath lines clean in order to prevent growth.
• Cell Sorters – Fluidics need to be particle free, unlike traditional flow cytometers in a biomedical setting, the Center for Aquatic Cytometry does not use inline sheath filters on its cell sorters. The fluidics lines and tank are cleaned prior to use and the sheath fluid entering the tank is 0.2 μm filtered. A primary source of small particle contamination can originate from the inline sheath filter including extraneous DNA contamination.

References:

Lomas, M. W., Steinberg, D. K., Dickey, T., Carlson, C. A., Nelson, N. B., Condon, R. H., and Bates, N. R.: 2010 Increased ocean carbon export in the Sargasso Sea linked to climate variability is countered by its enhanced mesopelagic attenuation, Biogeosciences, 7, 57–70, https://doi.org/10.5194/bg-7-57-2010

Marie D., N. Simon and D. Vaulot. 2005. Phytoplankton Cell Counting by Flow Cytometry. In: Algal Culture Techniques Ed. R. A. Andersen. Elsevier. p. 253.

Marie D., F. Rigaut-Jalabert, D. Vaulot. 2014. An improved protocol for flow cytometry analysis of phytoplankton cultures and natural samples. Cytometry A, 85(11): 962-968 doi: 10.1002/cyto.a.22517