NOAA - Great Lakes Environmental Research Laboratory
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Samples for enumerating bacteria and picophytoplankton (cyanobacteria and eukaryotic phytoplankton ‹3 µm) were collected from CTD or peristaltic pump while underway. They were immediately preserved in 1% (final concentration) formaldehyde and shock-frozen in liquid nitrogen. In the laboratory, bacteria were quantified by flow cytometry after DNA staining (Marie et al. 1997; Jochem 2001). One milliliter of sample was incubated for 30 min at 37oC with 0.1 g l-1 RNAse (1:1 mix of RNAse A and B) before staining with SYBR Green I (Invitrogen) in the presence of 30 mM potassium citrate. After 30 min of staining, samples were analyzed on a Becton Dickinson FACSort flow cytometer with FACS Loader autosampler at a sample flow rate of 0.2 ?l s-1. Measured sample volume was estimated from measurement time based on repeated weight calibration of flow rates. Data were analyzed by WinMDI software. Cyanobacteria and picoeukaryotic algae were analyzed in separate subsamples based on autofluorescence of their photopigments without DNA staining.
Jochem FJ (2001) Morphology and DNA content of bacterioplankton in the northern Gulf of Mexico: analysis by epifluorescence microscopy and flow cytometry. Aquat Microb Ecol 25: 179-194.
Marie D, Partensky F, Jacquet S, Vaulot D (1997) Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the nucleic acid stain SYBR Green I. Appl Environ Microbiol 63: 186-193
Peter J. Lavrentyev
Department of Biology
University of Akron
Akron, OH 44325-3908
phone: 330-972-7922
email:
