Dataset: Microbiology
Deployment: 61TG_3052

See SOIREE Preliminary Voyage Report

Bacteria enumeration
Bacterial abundance was determined by Flow Cytometry using a FACScan (15mW air-cooled
argon-ion laser fixed at 488 nm) instrument (Becton Dickinson, Mountain View, Calif.)
with CellQuest Vers. 3.1f software. The sheath fluid was 0.1 µm filtered sea water and
the analysed volume was calculated using TrucountTM (Becton Dickinson, Mountain View, Calif.)
beads as a tracer. The samples were frozen in liquid nitrogen and stained using SYBRII stain
(Molecular Probes Inc.) at a concentration of 10-4 of stock solution and then incubated in
the dark for 10-15 mins before being analysed following the methods described in Lebaron et. al., (1998).
Just prior to analysis, 100ml of TrucountTM beads were added to each sample as a tracer.
Samples were run on the low flow setting (~12 µl/min) with a minimum of 10,000 counts per sample.
The bacterial population was identified using a FL1 (green light, 530/30 BP filter, log scale)
vs FSC (Cell size, linear scale) scatter plot. FL1 was set to a voltage of 520 with an amp gain of 1.0.
FSC was set to the E02 voltage setting with an amp gain of 7.37. Cell carbon for bacteria was estimated
to be 12.4 fg C per cell as reported by Fukuda et .al., 1998). Flow cytometric counts of both bacteria
were verified by direct counts using epifluorescent microscopy following Hobbie et. al., (1977).
The variations between cytometric counts and microscopy counts were within the range reported by both
Lebaron et. al., (1998) and Del Giorgio et. al., (1996).

Eukaryotic picophytoplankton enumeration
Eukaryotic picophytoplankton abundance was determined by Flow Cytometry using a FACScan
(15mW air-cooled argon-ion laser fixed at 488 nm) instrument (Becton Dickinson, Mountain View, Calif.)
with CellQuest Vers. 3.1f software. The sheath fluid was 0.1 µm filtered sea water and the
analysed volume was calculated using TrucountTM (Becton Dickinson, Mountain View, Calif.)
beads as a tracer. The samples were frozen in liquid nitrogen (Lebaron et. al., 1998) and
were thawed immediately before counting and 50ml of TrucountTM beads were added as a tracer.
Samples were run on the FACScan at Hi flow setting (~60 µl/min) with a minimum of 1500 counts
per sample following methods based on Corzo et. al. (1999) and Li (1989). Eukaryotic picophytoplankton
numbers were then determined under FL1 vs FL2 (orange/red light, 585/42 BP filter). FL1 was
set to a voltage of 520 with an amp gain of 1.0. FL2 was set to a voltage of 420 with an amp
gain of 1.0. To remove background electronic noise FL2 was also set at a threshold of 52.
Cell carbon for <2 µm eukaryotic picophytoplankton was determined by assessing average spherical
diameter by direct microscope observation using epifluorescence. This was then converted following
Booth (1988) conversion for small phytoplankton (<4 µm) using 220 fg C µm3 to yield a factor
of 920 fg C per eukaryotic picophytoplankton. Flow cytometric counts of eukaryotic picophytoplankton
were verified by direct counts using epifluorescent microscopy following Hall (1991). The variations
between cytometric counts and microscopy counts were within the range reported by both Lebaron
et. al., (1998) and Del Giorgio et. al., (1996).

Bacterial Production
Heterotrophic bacterial productivity was measured using (methyl-3H) thymidine as detailed in
Smith & Hall (1997). Incubations were conducted at in situ surface temperatures. The extraction
procedure followed Wicks & Robarts (1987) modified TCA precipitate method, which involved rinsing
the TCA precipitate with phenol-chloroform followed by ethanol. Tritium incorporation was determined
with a liquid scintillation counter (LKB, 1217 Rackbeta) using OptiPhase HiSafe 3 (Wallac) as the
scintillation fluor. Counts were corrected for quench by external standards. In order to estimate
bacterial production we converted mol thymidine to g C using Fuhrman & Azam's (1982) conversion
factor of 2.4 x 1018 cells per mol thymidine incorporated, and Lee & Fuhrman's (1987) estimate of
20 x 10-15 g C cell -1.

Nanoflagellate enumeration
Samples collected for nanoflagellate enumeration were size fractionated through a 20 µm nylon mesh.
The filtrate was then fixed 1:1 with ice cold glutaraldehyde (2% final concentration) for 1 hour
(Sanders et. al., 1989). Fixed samples were filtered onto prestained 0.8 µm black Nuclepore filters,
stained for five minutes with 2 ml primulin, rinsed with 2 ml Tris HCL, mounted on slides and stored
rozen (Bloem et. al., 1986). Nanoflagellates were counted under UV excitation using a Leica compound
microscope (BP 450-490 nm excitation, LP 520 barrier filter, FT 510 dichromatic beam splitter).
Nanophytoflagellates were differentiated by chlorophyll a fluorescing red under blue light excitation
(BP 450-490 excitation, LP 515 barrier filter, RPK 510 dichromatic beam splitter). Forty randomly
selected fields were counted per filter. Nanoflagellate biovolumes were calculated using dimensions
and approximated geometric shape (Chang 1988). Biovolumes were calculated from measurements on a
minimum of 200 cells, collected at 20 m for each sampling. Cell carbon for phyto and heterotrophic
nanoflagellate biomass was estimated to be 0.24 pg C per µm-3 as reported by Verity et. al., (1992)
for nanophytoflagellates.

Ciliate enumeration
Samples for enumeration of ciliates were preserved in 1% Lugol's iodine. Samples were left to settle
for 48 h and the supernatant removed. The remaining sample was transferred to a 25 ml Utermohl chamber.
The microzooplankton were identified to genus where possible and enumerated using a Wild inverted
microscope (James & Hall, 1995) but with no differentiation of plastidic ciliates. Ciliate biomass
was estimated from dimensions of 10 to 20 randomly chosen individuals of each taxon. The volumes
were estimated from approximate geometric shapes and were converted to carbon biomass using a factor
of 0.19 pg C µm -3 (Putt & Stoecker 1989). The use of Lugol's iodine for preservation may have resulted
in an underestimation of biomass due to cell shrinkage.

Microzooplankton grazing rates and phytoplankton growth rates
Microzooplankton grazing rates and phytoplankton growth rates were determined using methods based on
the dilution technique of Landry & Hassett (1982), modified following the experimental protocols
described in Gallegos et. al., (1996). Water for dilution was gravity filtered at 0.2 µm through a
pre-rinsed Gelman SuporCapTM 100. Filtration of the dilution water required about 1 h. The total
standing time between water collection and the start of the experiments was a maximum 2 h. In a
set of 12 acid washed, 2.4-litre polycarbonate bottles, <200 µm screened water was diluted with
0.2 µm filtered water to concentrations of ~10%, 40%, 70% and 100% (i.e. undiluted). The screening
of the water at 200 µm removed up to 40% of the phytoplankton particularly towards the end of the
experiment when chains of large diatoms had formed. All bottles were then placed in an on deck
incubator, with continuous sea water supply and covered with shade cloth that transmitted ~40% of
the incident light, simulating ambient conditions. Incubations for all experiments were conducted
in triplicate bottles for 24 h.

The dilution factor for each bottle was calculated by taking sub-samples for <200 µm chlorophyll a at
T0 and measuring the actual percentage of <200 µm chlorophyll a in each treatment. Size fractionated
chlorophyll a and picophytoplankton sub-samples were also taken from experiments at T24 from all dilutions,
but only from 100% undiluted water at T0. To determine growth and grazing rates on phytoplankton, size
fractionated chlorophyll a and picophytoplankton at T0 were then estimated for each dilution using the
calculated dilution. In addition, to determine growth rate of grazers, we measured heterotrophic
nanoflagellate and ciliate in only 100% undiluted water at T0 and T24.

Chlorophyll a was measured by filtering 500 mls of sample through a Whatman GF/F filter. Size
fractioned chlorophyll a samples were collected using prefiltration at 20 and 2 µm. The filters
were stored frozen until analysed spectrofluorometrically on a Perkin-Elmer LS 50B (Strickland & Parsons 1972).

BCO-DMO Processing Notes
Generated from original file Microbiology.xls provided on the
Deep-Sea Research II 48 (2001) accompanying CD-Rom

BCO-DMO Edits
- parameter names modified to conform to BCO-DMO convention
- date reformatted to YYYYMMDD
- Station Number changed to station
- added 'T' to CTD Station number for compatibility with events in other spreadsheets
- blank rows in original sheet removed
- 'nd' added to blank cells or cells with '-' in original file
- added Patch Location column and removed individual rows with 'Outside' and 'Inside'
- date_UTC, time_UTC, date_local, time_local, timezone, lon and lat added from CTD sampling spreadsheet