Microbial community assemblages of the California Current Ecosystem (CCE) are assessed for abundance and biomass using high-throughput digital epifluorescence microscopy. Samples to estimate the nano- and microplankton (0.2-2.0-µm and 2.0-20-µm size, respectively) are collected, preserved, stained, and filtered onto a membrane filter and mounted on a glass microscope slide in the field. Slides are then frozen at -80°C for subsequent imaging and analysis in the laboratory onshore.


1. Principle

Microscopical assessment of nano- and microplankton: Aliquots of 50- and 500-ml are collected for analyses of nano- and microplankton by digitally enhanced epifluorescence microscopy. Samples for nanoplankton (2-20-µm) biomass and abundance are preserved with paraformaldehyde (0.5% final concentration) and stained with proflavin (0.33% w/v). Samples for microplankton abundance and biomass are preserved with 260 µl of alkaline Lugol's solution followed by 10 ml of buffered formalin and 500 µl of sodium thiosulfate (modified protocol from Sherr and Sherr, 1993), and then stained with proflavin (0.33% w/v). Preserved samples are allowed to fix at room temperature for at least one hour prior to gentle filtration (<100 mm Hg) onto 0.8-µm (nano) or 8.0-µm (micro) pore size 25-mm black Nuclepore filters, placed on top a 20-µm nylon backing filter. During filtration samples are drawn down until ~5 ml remains in the filtration tower, and concentrated DAPI (50 mg ml-1) is then added and allowed to sit briefly (5 s) before filtering the remaining sample until dry. Filters are mounted onto glass slides with immersion oil and cover slip. Slides are stored at -80°C for analysis back on shore.

2. Sampling


Nano- and microplankton are sampled from 3 to 8 depths for each of the 66 CalCOFI stations located in the CCE. Seawater from the Niskin bottle is collected into a 600-ml sample bottle. When sampling, make sure the flow from the valves is low since no sample tubing is used.

3. Analysis

Slides are imaged and digitized with a Zeiss AxioVert 200M inverted epifluorescence microscope equipped with a fully motorized stage and controlled by Zeiss AxioVision software. Digital images are captured with a Zeiss AxioCam HR color CCD digital camera, using the auto-exposure function to prevent over exposure. Slides are viewed at 630X (nano) or 200X (micro), and at least 20 random fields per slide are imaged. Each field image consists of three- to four different fluorescent channels: Chl a, DAPI, FITC (50- and 500-ml aliquots), and phycoerythrin (50-ml aliquots only). The separate channel images for each field are composited into 24-bit RGB images for analysis.

Counting and sizing of eukaryotes of >1.5-µm cell lengths is semi-automated with ImagePro software. Seen as bright spots against a dark background, individual cells are selected and outlined, automated using VBA script within the ImagePro software. All pre-processing steps are performed on the green channel, corresponding to fluorescence of proflavin-stained cell protein, extracted as an 8-bit gray scale image from the original 24-bit RGB image. After cells are automatically segmented from the image background, a manual interaction step is required to split connected objects, delete artifacts, and add cells that were too dim to be segmented from the background automatically.

Cells are typically identified and grouped manually into six plankton functional types (heterotrophic flagellates, autotrophic flagellates, diatoms, heterotrophic dinoflagellates, autotrophic dinoflagellates and prymnesiophytes). Autotrophs are distinguished from heterotrophic cells by the presence of chlorophyll, seen as red autofluorescence under blue light excitation. In addition to functional groupings, all cells are binned into size categories (<2, 2-5, 5-10, 10-20, 20-40 and >40 µm) based on measurement of the longest cell axis.

4. Calculations

Length (L) and width (W) measurements of each cell are converted to biovolumes (BV; µm3) by applying the geometric formula of a prolate sphere (BV = 0.524 L W H). Carbon (C; pg cell-1) biomass is computed from BV from the equations of Menden-Deuer and Lessard (2000): C = 0.216 x BV0.939 for non-diatoms, and C = 0.288 x BV0.811 for diatoms.

5. Equipment/Supplies

  • Sample collection bottles
  • Micropipette & sterile tips
  • Glass microscope slides & #2 cover slips
  • Type DF immersion oil
  • 25-mm black PC filters (0.8- & 8.0-µm pore)
  • 25-mm nylon backing filters (20-µm pore)
  • Zeiss Axiovert 200M microscope equipped for epifluorescence
  • Zeiss Axiocam HRc (color CCD) and MRm (monochrome CCD)
  • ImagePro software (Media Cybernetics)

6. Reagents

  • Paraformaldehyde (10%)
  • Buffered formalin (10%)
  • Alkaline Lugol's
  • Sodium Thiosulfate
  • DAPI (50 mg ml-1)
  • Proflavin (0.33% w/v)

7. References