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Bacterioplankton in the western Gulf of Mexico: Analysis by epifluorescence microscopy and flow cytometry

by 

Frank J. Jochem

Presentation:
AGU Ocean Sciences Meeting 2000, San Antonio, Texas, January 2000

 

Abstract

The distribution of pelagic bacteria along two onshore - offshore transects in the western Gulf of Mexico was assessed aboard the RV Longhorn in July and October 1999. Cell abundance was estimated by epifluorescence microscopy after DAPI staining and by flow cytometry after DNA staining with SYBR Green. Bacteria abundance in the upper 200 m of the water column ranged from 88x103 to 1350x103 cells ml-1. Bacteria exhibited surface maxima in July but subsurface maxima within the chlorophyll maximum in October. In July, abundance over the continental shelf was up to 2 times higher than at offshore stations. Regional differences were lower in October as were bacteria abundances in general.

Epifluorescence microscopy revealed three morphotypes of bacteria: cocci; rods; spirillae and curved bacteria. Cocci (48-58% of total bacteria) were the most common morphotype. Rods and spirillae/curved bacteria had similar shares, which were higher in October than in July. Flow cytometry revealed 4 bacterial sub-populations distinguished by their DNA content. DNA-types do not seem to reflect morphotypes.

For total bacteria, flow cytometry results correspond to those from epifluorescence microscopy. Analyses of formalin-fixed samples performed immediately after the October cruise exhibited a close to 1:1 relationship between the two methods.

Material and Methods

Samples for bacteria counts were taken by CTD-mounted Niskin-type water bottles from 8-10 depths in the upper 200 m water column at 6 stations on July 21-24, 1999, and at 5 stations on October 21-25, 1999, in the western Gulf of Mexico (Fig. 1). Samples were fixed by 1% (final conc.) formaldehyde and stored at 4C in the dark until analyses. For epifluorescence microscopy, 0.5 to 1 ml of sample was diluted with 0.2 m filtered seawater to a final volume of 5 ml, incubated with DAPI (1 mg l-1 final conc.) for 5 min and filtered onto black 0.2 m Nuclepore filters. Filters were stored frozen until counted under UV excitation on a Zeiss Axioskop microscope. Depending on abundance, 200-500 cells were counted on 60-200 view fields. Cell counts of coccoid bacteria were corrected for Prochlorococcus abundance derived from flow cytometry analyses of unstained samples. Prochlorococcus would also be counted as coccoid bacteria on DAPI-stained filters and can overestimate bacteria abundance (Sieracki et al. 1995).

Fig. 1: Sampled stations during R/V Longhorn cruises in July and October 1999 in the western Gulf of Mexico. The July transect reached 328 km into the Gulf, the October transect 658 km.

For flow cytometry, 1 ml of fixed samples was incubated for 30 min at 37C with 0.1 g l-1 RNAse (1:1 mix of RNAse A and B) prior to staining with SYBR Green I (10-5 dilution of commercial stock; Molecular Probes, Eugene, OR) in the presence of 30 mM potassium citrate (Marie et al. 1997). After 10 min of staining, samples were analyzed on a Becton-Dickinson FACSort flow cytometer. Bacteria cell size was assessed by Side-Angle Light Scatter (SSC). Due to its broad emission spectrum, SYBR DNA- fluorescence was detected both in the green FL1 (53515 nm) and the red fluorescence FL3 (>650 nm) channels. All signals were recorded on a four-decades log scale. Data were analyzed by PC Lysys software (Becton-Dickinson, San Jos, CA). In near-surface samples, where prochlorophytes and bacteria were not separated in the cytometric analyses, bacteria measurements were corrected for prochlorophyte counts from unstained fixed samples measured on the same day.

Bacteria Counts by Microscopy and Flow Cytometry

Three different types of bacteria were identified under the microscope: coccoid, rod- shaped, and curved bacteria (Fig. 2). Most rod-shaped bacteria were small, but some samples contained elongated rods in low numbers. Among curved bacteria, small C-form cells were most abundant. Large C-form and large S-form cells occurred in variable but relatively low numbers. For statistically significant cells counts, all variations in curved bacteria were cumulated.

Fig. 2: Different morphotypes of pelagic bacteria, DAPI staining: Cocci, rod-shaped, and curved cells. Curved cells comprised small and large C-forms and large S-forms.

In flow cytometric analyses, 4 clusters of bacteria were distinguished by DNA content (Fig. 3). All clusters were present in all samples. Clusters B-Ia and B-Ib probably correspond to B-I bacteria as cytometrically identified and termed by Li et al. (1995) and Marie et al. (1997) in samples from the North Atlantic, the Mediterranean and the English Channel. Cluster B-III probably corresponds to B-III of Marie et al. (1997), which was not detected by Li et al. (1995). A cluster termed B-II by Li et al. (1995) and Marie et al. (1997), distinguished by higher DNA content but lower light scatter compared to B-I, was not detected. Instead, a high DNA content and light scatter cluster indicating larger cell size (B-IV) occurred.

Fig. 3: Cytometric analysis of bacteria revealed 4 types of bacteria distinguished by their DNA content. Only population B-IV exhibited higher light scatter (SSC) indicative of larger cell size. B-III was the most abundant DNA type. Cell types named after previously published results (see text). Prochlorococcus were separated from bacteria cells in two-parameter-histograms except for surface-near samples.

Bacteria counts by microscopy and flow cytometry were correlated significantly (Fig. 4). For the July cruise, cytometric analyses were performed 2.5 months later from formalin-fixed samples stored at 4C. Recovery of total bacteria was 65% compared to microscopy. This number compares to the reported loss of bacteria cells in such stored samples over time (Turley & Hughes 1992, Troussellier et al. 1995). In October, microscopic and cytometric analyses were performed immediately after the cruise. The relationship between both estimates of total bacteria abundance is close to 1 (0.903 0.022). Regression analysis showed that none of the cytometrically established DNA-types of bacteria reflected any microscopically identified morphotype.

Fig. 4: Comparison of bacteria abundance derived from microscopic counts and flow cytometry analysis.

Distribution of Bacteria

In July, bacteria exhibited surface maxima of abundance, ranging from ~400x103 cells ml-1 offshore to ~1000x103 cells ml-1 inshore. Only the shallow (60 m) shelf Stn 6 showed a near-bottom maximum of 1350x103 cells ml-1. In October, subsurface maxima of 300-400x103 cells ml-1 occurred within the chlorophyll maximum, and abundance did not increase towards the shelf as recorded in July. Typical profiles of both cruises are depicted in Fig. 5.

Fig. 5: Vertical profiles of bacteria abundance (upper row), fractions of the three morphotypes distinguished by microscopy to total bacteria (middle row), and fraction of the four DNA types established by flow cytometry to total bacteria. Left column: Stn 4 (105 km off shore) of July cruise; right column: Stn 3 (334 km off shore) of October cruise. Profiles of chlorophyll fluorescence from CTD casts (relative units) are overlaid

Cocci were the most abundant morphotype of bacteria, but their dominance was much higher in July (58 7%) than in October (48 6%). Rod-shaped and curved bacteria were equally abundant in October (27 5% and 25 5%), but curved bacteria were a little more abundant than rods in July (18 4% and 24 5%). During cruises, maximum shares of cocci, and minimum shares of rods and curved bacteria, occurred within the lower part of the chlorophyll subsurface maximum (Fig. 5). B-Ia and B-III were the most abundant DNA-types of bacteria during both cruises. B-Ia dominated the upper water column to the bottom of the chlorophyll subsurface maximum, whereas B-III were dominant below the euphotic zone. B-Ib followed the vertical distribution of B-Ia, whereas the larger B-IV bacteria also gained a higher share below the euphotic zone. The vertical pattern of DNA-types depicted in Fig. 5 was consistent at all stations.

Bacteria abundance integrated over 200 m water depth was higher in July than in October (Fig. 6, gray bars). Except for the shallow shelf stations, integrated abundance showed minor regional variation. Despite their high cell numbers per liter, the shelf stations exhibited lower integrated abundances due to their shallow depths. Among the morphotypes, the shares of cocci tend to decrease towards the coast, and those of curved bacteria tend to increase. Among the cytometric DNA-types, larger B-IV bacteria seem to be more important at offshore stations. Despite some variation in the shares of the different DNA-types, no regional trend or major variation is discernible (Fig. 6).

Fig. 6a: Fraction of microscopic morphotypes (upper) and cytometric DNA-types (lower) to total bacteria numbers integrated over 200 m water depth in July 1999. Integrated abundance (1012 cells m-2) is overlaid as gray bars in upper graph.

Fig. 6b: Fraction of microscopic morphotypes (upper) and cytometric DNA-types (lower) to total bacteria numbers integrated over 200 m water depth in October 1999. Integrated abundance (1012 cells m-2) is overlaid as gray bars in upper graph.

Conclusions

  • Cytometric analysis after SYBR Green staining provides a fast and reliable quantification of pelagic bacteria. The method is easy and requires few sample manipulations.
  • Microscopic and cytometric analyses provide different information on bacteria diversity.
  • Cytometric DNA-types do not reflect microscopic morphotypes.
  • Variation in bacteria diversity is higher on vertical than on regional scales. Higher vertical than regional variation in biotic (primary production, grazer communities) and abiotic (nutrients) variables in the western Gulf of Mexico might be the cause.

References

  • Li WKW, Jellett JF, Dickie PM. 1995. DNA distributions in planktonic bacteria stained with TOTO or TO-PRO. Limnol Oceanogr 40: 1485-1495.

  • Marie D, Partensky F, Jacquet S, Vaulot D. 1997. Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the nucleic acid stain SYBR Green I. Appl Environ Microbiol 63: 186-193.
  • Sieracki ME, Haugen EM, Cucci TL. 1995. Overestimation of heterotrophic bacteria in the Sargasso Sea: Direct evidence by flow and imaging cytometry. Deep-Sea Res 42: 1399-1409.
  • Troussellier M, Courties C, Zettelmaier S. 1995. Flow cytometric analysis of coastal lagoon bacterioplankton and picophytoplankton: fixation and storage effects. Estuar Coast Shelf Sci 40: 621-633.
  • Turley CM, Hughes DJ. 1992. Effects of storage on direct estimates of bacterial numbers of preserved seawater samples. Deep-Sea Res 39: 375-394