Dataset: bacteria
Deployment: TT049

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

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.

<h2>Hugh Ducklow's notes on methodology</h2>
<pre>
Comments:

Incorporation data are only available for stations 13 - 30 as Omani
customs would not allow our isotopes into the country.

Methods:

Bacteria were enumerated essentially as described in our EqPac data
documentation and the article in the EQPAC I DSR volume. Multiple
images of all samples were acquired, processed and analyzed with Zeiss
software. We used a new algorithm which removed most, but possibly not
all of the prochlorophytes, so these data represent heterotrophic
bacterial counts only. We will be comparing samples with Rob Olson and
David Caron to constrain the bacteria counts. We also include estimates
of the mean volume of cells in each sample.

Bacterial abundance times cell volume yields total biovolume per liter,
an index of bacterial biomass, to which carbon per unit volume factors
can (and will) be applied. All raw images have been archived and can be
made available by separate arrangement.

Bacterial production was estimated by the incorporation of 3H-thymidine
and 3H-leucine into cold, 5% TCA-insoluble extracts, essentially as
described in our earlier JGOFS work, although using the new method of
Smith & Azam (see the Azam data documentation for Process-1).
Production estimates are forthcoming.

Analytical precision for bacterial abundance and cell volume.

We do not routinely perform replicate determinations of bacterial
counts. However, reanalysis of 92 slides from the March series gave
the following coefficients of variation (sd/mean) for bacterial
abundance and cell volume:

property mean st.dev. CV (%) n
- - - - - - - - - - - - -
abundance* 0.53 0.061 11.7 92
cell volume 0.037 0.006 16.4 92
- - - - - - - - - - - - -
*units for abundance are x 10^9 cells/liter. volumes are um3/cell;
these are mean values for 92 replicate determinations of each
property.

Hugh Ducklow tel 804-642-7180
Virginia Institute of Marine Sciences fax 804-642-7293
Box 1346 email duck@vims.edu
Gloucester Point, VA 23062-1346

</pre>