Methods for the bacterial component of US JGOFS Arabian Sea Process 1 and Process 7 Cruises

Sampling

Seawater samples (250 ml) were taken from the Niskin bottles mounted on
the CTD rosette and transferred directly into brown high-density
polyethylene bottles.  The bottles were maintained within 2 degrees C of
the in situ temperature until the start of the incubations or fixing.

Bacterial Abundance

Bacterial abundance was determined by epifluorescence microscopy of
acridine orange- stained bacteria on Nuclepore filters ( Hobbie et al.
1977 ). Samples of between 50 to 100 ml of seawater for bacteria counts
were fixed with 0.2 m-filtered, EM-grade glutaraldehyde (1.25% final
concentration; Ted Pella).  Subsamples of 2 to 50 ml were vacuum
filtered (< 150 mm Hg) onto 0.2 m pore size, black, polycarbonate
membrane filters (Nuclepore).  During filtration, when a sample volume
had been reduced to 2 ml, 200 l of an 0.2 m-filtered acridine orange
working solution (0.05%) was added to the sample in the tower for a
final concentration of 0.005%.  Filtration rate was kept low enough to
allow at least 5 minutes of contact with the stain before filtration
was complete.  Blanks were 0.2 m-filtered seawater or deionized,
Milli-Q-treated water fixed and processed in the same manner as the
samples.  After filtration, filters were transferred quickly (while
still moist) onto fogged glass slides.  Before the filters could dry, a
coverslip with a drop of paraffin oil on the underside was placed over
the filter.  Slides were stored at approximately 4 degrees C for less
than 1 day, then transferred to a -20 degrees C freezer for longer term
storage.  Bacteria were counted at 1250' magnification by
epifluorescence microscopy with an oil immersion objective.  Each
sample was counted until either 20 fields or 200 cells had been
enumerated.


Bacterial Production

Bacterial production was estimated using both the 3H-thymidine (
Fuhrman and Azam 1980; Fuhrman and Azam 1982  ) and the 3H-leucine
(Kirchman et al. 1985 ) incorporation methods as modified for
processing by microcentrifugation by  Smith and Azam (1992) .  Aliquots
of seawater (1.7 ml) were incubated with 20 nM 3H-leucine or
3H-thymidine in the dark.  The length of incubation ranged from 1 to 6
hours depending on the depth from which the sample was collected.  The
incubations were terminated with the addition of trichloroacetic acid
(TCA; 5% v/v final).  Samples which received the addition of TCA prior
to the radioisotope served as blanks.  The samples were centrifuged at
16000 x g for 7 min and the supernatant was removed.  The pellet was
washed once with 5% TCA and once with 80% ethanol.  Liquid
scintillation cocktail was added directly to the centrifuge tube which
were then radioassayed.

References

 Fuhrman, J. A. and F. Azam 1980. Bacterioplankton secondary production
     estimates for coastal waters of British Columbia, Antarctica and
     California. Appl. Environ.  Microbiol. 39: 1085-1095.

 Fuhrman, J. A. and F. Azam 1982. Thymidine incorporation as a measure
     of heterotrophic bacterioplankton production in marine surface waters:
     evaluation and field results. Mar.  Biol. 66: 109-120.

 Hobbie, J. E., R. J. Daley and S. Jasper 1977. Use of nuclepore
     filters for counting bacteria by epifluorescence 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
     aquatic systems. Appl. Environ.  Microbiol. 49: 599-607.

 Smith, D. C. and F. Azam 1992. A simple, economical method for
     measuring bacterial protein synthesis rates in seawater using
     3H-leucine. Mar. Microb. Food Webs. 6: 107- 114.