Flow Cytometry Correction Factors for Enumeration of Heterotrophic Bacteria and Phytoplankton

Hector Nolla¹, Julie Kirshtein², Michael Landry¹, David Karl¹, Lisa Campbell³ and David Pence¹

¹School of Ocean and Earth Science and Technology, University of Hawaii, Honolulu, HI 96822

²Institute of Marine Sciences, University of Califomia Santa Cruz, Santa Cruz, CA 95064

³Department of Oceanography, Texas A&M University, College Station, TX 77843-3146

Abstract

Hawaii Ocean Time-series (HOT) samples for analysis and enumeration of heterotrophic bacteria and phytoplankton by flow cytometry have been collected and processed since December 1990 (HOT-22). This analysis is done using a Coulter EPICS 753 flow cytometer equipped with 2 5-watt lasers tuned to emit UV and 488 nm light. A Coulter Corp. Micro-Sample-Delivery-System (MSDS) is used to adjust the sample volume. Flow cytometry measurements are used to determine both individual cell fluorescence and sample cell concentrations. Briefly, cells are hydrodynamically injected into a fluid stream and illuminated by UV and 488 nm laser beams. As cells pass through the laser beams they scatter light and absorb energy which is then emitted as fluorescence. The instrument has enough optical sensitivity to detect particles as small as 0. 3 µm in diameter. These signals are collected by photomultiplier tubes, amplified, converted into a digital output and processed for computer graphical display. Identification of heterotrophic bacteria and phytoplankton populations is done using software like CYTOPC to view the multiparameter graphical content of flow cytometry data files. While flow cytometry offers the advantage of analyzing samples at rates of up to 10,000 cells per second, sample acquisition at higher count rates can result in lower enumeration efficiencies. This inverse relationship between sample acquisition count rates and instrument enumeration correction factors is the result of signal processing and electronic "dead" time. Another factor that can affect enumeration correction factors involves the method for delivering the sample into the fluid stream for analysis. The use of an adjustable volumetric sample delivery system like MSDS allows for the consistent and reproducible delivery of sample volumes (i.e., 100 µl of sample delivered at 50 µl per minute) and thus reduces volumetric variability. In the course of the last 4 years, we have employed 3 different methods for determining flow cytometry accuracy. Two of these methods were derived using 10 µm and 0.98 µm fluorescenct microspheres while the third was calculated using a culture of Escherichia coli stained with Hoechst 33342 for measurement of DNA fluorescence. We will also compare enumeration correction factors derived using two different electronic data acquisition platforms: (1) an older Coulter Corp. MDADS (Multiparameter Data Acquisition and Display System) and (2) a more state-of-the-art Cytomation Inc. CICERO data acquisition system. A comparison between these methods will be presented as well as a discussion of the limitations involved with the use of flow cytometry for enumerating bacterial populations.