PI: Hugh Ducklow
of: Virginia Institute of Marine Science
dataset: Bacteria abundance, thymidine incorporation
dates: June 30, 1989 to July 04, 1989
location: N: 59.535 S: 59.4733 W: -21.0183 E: -20.8217
project/cruise: North Atlantic Bloom Experiment/Endeavor 198
ship: R/V Endeavor
DMO Note: Bacteria data from the NABE Atlantis II cruise
are reported with the biology dataset for that cruise.
Methodology
BACTERIAL ABUNDANCE & BACTERIAL THYMIDINE
& LEUCINE INCORPORATION (Ducklow, HPEL)
Abundance samples preserved in 1.25% glutaraldehyde and stored at 5�
C until microscopy was performed at Horn Point Environmental Laboratory.
All samples were enumerated according to the Acridine Orange Direct Count
technique of Hobbie et al., (1977) with modifications by Helen Quinby.
Samples were enumerated on a Nikon Optiphot epifluorescence microscope
at 1850x with a 100 watt Mercury lamp.
Thymidine incorporation samples collected from Niskin rosette casts
were immediately processed as described in Ducklow and Hill (1985),
with the following modifications: Samples were incubated with 5 nM 3H-thymidine
(New England Nuclear, sp. act. 81 Ci/mmol) in polycarbonate bottles,
disposable polyproplyene centrifuge tubes or Whirl-Pak bags. Incubations
were terminated with addition of 0.37% formaldehyde, then filtered onto
0.2 �m Nuclepore filters. Extractions were carried out by rinsing
each filter on its funnel support 3 times with 5% ice cold TCA, over
a weak vacuum (<10 in. Hg), then 3 times with 80% ice cold ethanol.
All extracted filters were stored dry in scintillation vials for counting
at Horn Point Environmental Laboratory.
Leucine incorporation samples were treated according to the method
described in Kirchman et al., (1985), with the following modifications:
Samples were incubated with 0.5 nM 3H- leucine (NEN; Sp. Act. 73 Ci/mmol)
and 10 nM nonradioactive leucine, then treated as described for thymidine.
US JGOFS NABE Bacterial Data
Bacterial data were collected on the US JGOFS NABE cruises aboard RV
ATLANTIS II legs 2 and 3 and Endeavor cruise 198 by Hugh Ducklow, David
Kirchman, Helen Quinby and Hans Dam. On cruise 2, only bacterial abundance
was measured. On cruise 3, bacterial abundance, and bacterial thymidine and
leucine incorporation were measured. On Endeavor cruise 198 only bacteria
abundance and thymidine incorporation were measured by the following methods:
Abundance:
Samples preserved in 1.25% glutaraldehyde and stored at 5C until microscopy was
performed at Horn Point. All samples were enumerated according to the Acridine
Orange Direct Count technique of Hobbie et al., (1977) with modifications by
Helen Quinby. Samples were enumerated on a Nikon Optiphot epifluorescence
microscope at 1850X with a 100 watt Mercury lamp.
Thymidine Incorporation:
Samples collected from Niskin rosette casts were immediately processed as
described in Ducklow and Hill (1985), with the following modifications:
Samples were incubated with 5 nM 3H-thymidine (New England Nuclear, sp. act. 81
Ci/mmol) in polycarbonate bottles, disposable polyproplyene centrifuge tubes or
Whirl-Pak bags. Incubations were terminated with addition of 0.37%
formaldehyde, then filtered onto 0.2 um Nuclepore filters. Extractions were
carried out by rinsing each filter on its funnel support 3 times with 5% ice
cold TCA, over a weak vacuum (< 10 in. Hg), then 3 times with 80% ice cold
ethanol. All extracted filters were stored dry in scintillation vials for
counting at Horn Point.
Additional samples were treated to purify the labelled DNA following the method
of Wicks et al., (1987). 95% of the label in the cold TCA extracts was in pure
DNA. (Data available from HWD).
Leucine Incorporation:
Samples were treated according to the method described in Kirchman et al.,
(1985), with the following modifications:
Samples were incubated with 0.5 nM 3H-leucine (NEN; Sp. Act. 73 Ci/mmol) and 10
nM nonradioactive leucine, then treated as described for thymidine.
Biomass/production/systhesis rate conversions:
The bacteria abundance data can be converted into bacterial biomass (ugC or ugN l-1)
as described for example in Lee and Fuhrman (1988). We will be measuring cell
biovolumes for this conversion and have not supplied nominal biomass data at
this time.
The THYINCORP data can be converted into bacterial production rates (ugC or ugN
l-1 hr-1) as discussed in Ducklow and Hill (1985). We performed separate
experiments to determine the conversion factors, and will report the data
separately. The THYINCORP data provide a relative index of differences in
bacterial production in space and time. Data on the biovolume and rate
conversion factors are required for translation into absolute units.
The LEUINCORP data can be converted into bacterial protein synthesis rates (ugC
or ugN l-1 hr-1) as discussed in Chin-Leo and Kirchman, (1988). We performed
separate experiments to determine the conversion factors, and will report the
data separately. The LEUINCORP data provide a relative index of differences in
protein synthesis in space and time. Data on the biovolume and rate conversion
factors are required for translation into absolute units.
References
Chin-Leo, G. And D.L. Kirchman. 1988. Estimating bacterial production in
natural waters from the simultaneous incorporation of thymidine and
leucine. Appl. Environ. Microbiol. 54:1934-39.
Ducklow, H.W., and S.M. Hill. 1985b. Tritiated thymidine incorporation and the
growth of heterotrophic bacteria in warm core rings. Limnol. Oceanogr.
30:260-272.
Hobbie, J.E., R.J. Daley, and S. Jasper. 1977. Use of Nuclepore filters for
counting bacteria by fluorescence microscopy. Appl. Environ. Microbiol.
33: 1225-1228.
Kirchman, D., E. K'nees and R. Hodson. 1985. Leucine incorporation and its
potential as a measure of protein synthesis by bacteria in natural waters.
Appl. Environ. Microbiol. 49: 599-607.
Lee, S. and J.A. Fuhrman. 1987. Relationships between biovolume and biomass of
naturally-derived marine bacterioplankton. Appl. Environ. Microbiol.
52:1298-1303.
Wicks, R.J. and R.D.Robarts, 1987, The extraction and purification of DNA
labelled with methyl-3H-thymidine in aquatic bacterial production studies,
J. Plankton Res. 9:1159-66.
DMO note:
The Data Management Office has changed the units of the parameters
"bact_het_mic" from cells/liter*10^9 to cells/milliliter
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