Short-term physiologic effects of mechanical flow sorting and the Becton-Dickinson cell concentrator in cultures of the marine phytoflagellata Emiliania huxleyi and Micromonas pusilla.
BACKGROUND
In contrast to large, high-efficiency cytometers, mechanically sorting benchtop instruments provide a feasible alternative for shipboard cell sorting of oceanic microbial communities. However, sorting efficiency of these instruments is constrained by their maximum sorting rate of approximately 300 cells/s and by constant dilution of sorted samples by sheath flow. These factors often render too low sorted cell concentrations for postsorting experiments of oceanic phytoplankton populations of low natural abundance. A Cell Concentrator module has been marketed to overcome these dilution effects. Postsorting experiments also have to consider potential physiologic effects of cell sorting. Short-term physiologic effects on phytoplankton photosynthetic rates and esterase activities by mechanical flow sorting and cell concentration and on the efficiency of the Cell Concentrator module are evaluated.


METHODS
Increasing numbers of the oceanic phytoflagellates Micromonas pusilla and Emiliania huxleyi were sorted and concentrated, and recovery in the concentrated samples was compared with the sorted-only samples (concentration rate) and the total number of sorted cells (recovery rate). Photosynthetic rates and metabolic activities of sorted and sorted/concentrated cells were compared with unsorted cells. Photosynthetic rates were estimated from 14CO2 uptake experiments and metabolic activity quantified cytometrically after cleavage of fluorescein diacetate.


RESULTS
Irrespective of the total number of sorted cells, concentration rates between concentrated and sorted cells remained mostly below 10-fold and did not increase with the number of concentrated cells. Recovery rates in the concentrated samples amounted to fewer than 10% of total sorted cells, except for forceful resuspension attempts in the Concentrator insert (25-44%), which might be unsuitable for delicate species. Cell sorting resulted in a 24-49% decrease in photosynthetic rates. Metabolic activity within metabolically active cells was not affected by cell sorting, but the share of metabolically active cells decreased by 32-37%. Cell concentration did not affect metabolic activity or the fraction of active cells but did increase photosynthetic rate several-fold compared with unsorted cells.


CONCLUSIONS
Low recovery of concentrated cells, probably due to cell adhesion to the filer bottom of the Concentrator insert, render the Cell Concentrator of limited use to overcome dilution problems of mechanical flow sorting, particularly when results are extrapolated to natural, low-abundance populations. Severe changes in photosynthetic rates also render concentrated cells suspicious for subsequent physiologic experiments. Mechanical sorting alone also exhibited significant physiologic effects on sorted cells, some of which might not be temporary. Comparable effects between mechanical sorting and droplet sorting as previously reported confirm that physiologic effects might be caused predominantly by shear stress and laser exposure during cytometric analysis rather than the sorting process. Sufficient recovery time must be allowed before postsorting experiments, but potential changes in cell physiology from the natural conditions during postsorting recovery must be considered.
Jochem
Frank J
FJ
Marine Biology Program, Florida International University, North Miami, Florida 33181, USA. frank@jochem.net
D002113:Calcification, Physiologic / Q000502:physiology*; D002448:Cell Adhesion / Q000502:physiology; D002478:Cells, Cultured; D056890:Eukaryota / Q000166:cytology*; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods*; D008386:Marine Biology; D013997:Time Factors