We investigated the report of Zweifel and Hagstriim that only a portion of marine bacteria contain nucleoids --the DNA-containing regions of procaryotic cells-and that such bacteria correspond to the active or viable fraction of bacterioplankton.In Oregon coastal waters, 21-64% of bacteria had visible nucleoids; numbers of nucleoid-visible (NV) bacteria were greater than numbers of metabolically active bacteria, based on cells with active electron transport systems (ETS) and intact cell membranes. During log growth of a marine isolate, proportions of NV and ETS-active cells approached 100%. In stationary growth phase, the fraction of ETS-active cells decreased rapidly, while that of NV cells remained high for 7 d. When starved cells of the isolate were resupplied with nutrient (50 mg liter-l peptone), total cell number did not increase during the initial 6 h, but the proportion of NV cells increased from 27 to lOO%, and that of ETS-active cells from 6 to 75%. In an analogous experiment with a bacterioplankton assemblage, a similar trend was observed: the number of NV cells doubled during the initial 6 h prior to an increase in total cell counts. These results show that some bacteria without visible nucleoids are capable of becoming NV cells, and thus have DNA in a nucleoid region not detectable with the method used here. Enumeration of bacterioplanktonby staining with the fluorochromes acridine orange or 4',6-diamidino-2-phenylidole (DAPI) and then counting via epifluorescence microscopy is one of the most common methods used in aquatic microbiology. Zweifel and Hagstriim (1995) presented evidence that only a fraction of the cells counted with these methods appear to be living bacteria. They based this observation on the presence or absence of a nucleoid-a region packed with condensed DNA in the bacterial cell. The standard direct count method based on DAPI staining of aldehyde-preserved marine bacteria (Porter and Feig 1980) was shown to be nonspecific for DNA; a modification of the method resulted in efficient binding of DAPI to DNA and removal of most nonspecific DAPI staining of other cell components (Zweifel and Hagstrijm 1995). In seawater taken from the Baltic Sea, the North Sea, and the Mediterranean Sea, Zweifel and Hagstriim reported that from 2 to 32% of bacterioplankton appeared to contain nucleoids. They inferred from their results, which included laboratory culture experiments, that cells with nucleoids corresponded to the portion of the bacterial assemblage that is active or viable and that cells without nucleoids were "ghost" cells that did not contain DNA and were therefore incapable of growth.Acknowledgments

The majority of bacteria suspended in seawater do not appear to be metabolically active or in good physiological condition as assessed by various methods. We tested the idea that a large fraction of 'inactive' bacterial cells can become 'active' with respect to detectable cell-specific electron transport system (ETS) activity, determined by the ability of cells to reduce the fluorogenic tetrazolium salt, CTC, when incubated for periods of time with or without additional substrate. Aliquots of 1.0 pm filtered seawater were amended with mixed antibiotics to inhibit DNA synthesis and thus cell division, and incubated at in situ (12.8 and 16.4"C) temperature or at 20°C. Additions included: phosphate (0.83 mM P, 5.3 mgP 1-'), ammonium (1.67 mM N, 23.4 mgN l-'), and organic carbon as glucose, mixed amino acids or yeast extract (8.33 mM C, 100 mgC 1-l). At 2OoC, the addition of mixed amino acids and yeast extract resulted in a large increase of % ETS-active cells (CTC-positive [CTC+] cells), from 1.9-2.4 % at 0 h to 55-87 % CTC+ cells by 21 to 28 h. At in situ temperature, the increase in % CTC+ cells was less, and the glucose addition caused the greatest increase in % CTC+ cells. Under conditions of increased temperature and high concentration of organic substrate, a large proportion of the apparently 'inactive' bacteria can become highly ETS-active within a day, suggesting that these cells are in fact alive, and are capable of attaining significant metabolic activity. The different response patterns of the bacterial assemblages at 20°C compared to those at 12.8 and 16.4"C suggests that temperature can be an important factor in bactenoplankton response to increase in substrate concentration.

The effects of fixation on the cell volume of marine heterotrophic nanoflagellates and planktonic ciliates were investigated. Decreases in cell volume depended on the combination of the protozoan taxa and the particular fixative. For a particular fixative and protozoan species, degree of shrinkage was independent of physiological state. The volume of fixed cells was found to be approximately 20 to 55% lower than the cell volume of live organisms. For the heterotrophic microflagellates, the fixatives ranked, in order of decreasing effect on cell volume, as glutarialdehyde, formaldehyde, acid Lugol's solution, and modified van der Veer solution. With oligotrichous ciliates and a tintinnid ciliate, formaldehyde caused less shrinkage than glutaraldehyde or acid Lugol's solution. With the aldehyde fixatives, the microflagellates were found to shrink more than the ciliates. Differential effects of fixation on cell volumes may result in an underestimation of the biomass of certain protozoan taxa in natural samples. Protozoa are major consumers of bacterioplankton (14, 31, 36) and phytoplankton (11, 19; D. A. Caron, Ph.D. thesis, Massachusetts Institute of Technology, Cambridge, 1984) in marine planktonic ecosystems. In addition to their role as grazers, heterotrophic protozoa, rather than bacteria, may also be the major nutrient remineralizers in open ocean (17, 45). The microbial food web is considered to be an important source of metabolic activity in open ocean (30, 35, 46); thus, data on the abundance and activity of heterotrophic protozoa are crucial to understanding the Plow of energy and material in the ocean. Heterotrophic microflagellates and ciliates usually dominate the protozoan biomass; however, because they overlap

Two different psychrophilic types of the heterotrophic nanoflagellate Paraphysomonas imperforata were isolated from Newfoundland coastal waters and the Arctic Ocean. When fed bacteria without food limitation, both isolates were able to grow at temperatures from-1.8 to 20°C, with maximum growth rates of 3.28 day-' at 15°C and 2.28 day-l at 12.3°C for the Newfoundland and the Arctic isolates, respectively. Ingestion rates increased with temperature from 14 to 62 bacteria flagellate-l h'for the Newfoundland isolate and from 30 to 99 bacteria flagellate-' h-1 for the Arctic isolate. While temperature did not affect cell yields (number of protozoa produced divided by number of bacteria consumed), it affected flagellate sizes. This differential effect of temperature on cell yield and cell size resulted in a changing gross growth efficiency (GGE) in terms of biovolume; colder temperatures favored higher GGEs. The comparison of Qlo values for growth rates and ingestion rates between the isolates shows that the Arctic isolate is better adapted to extremely cold temperature than the Newfoundland isolate. At seawater-freezing temperature (-1.8°C), the estimated maximum growth rates and maximum ingestion rates are 0.81 day-' and 30 bacteria flagellate-1 h-1 for the Arctic isolate and 0.54 day-' and 12 bacteria flagellate-' h-' for the Newfoundland isolate. Our findings about psychrophilic nanoflagellates fit the general characteristics of cold-water-dwelling organisms: reduced physiological rates and higher GGEs at lower temperatures. Because of the large and persistent differences between the isolates, we conclude that they are ecotypes adapted to specific environmental conditions.

ABSTRACT. Ingestion rate of Paraphysomonas imperforata was found to be a hyperbolic function of prey density. But the same flagellate clone had multiple ingestion responses to prey density, depending on its physiological state and physical stress it suffers. The flagellates in a physiological state of higher growth tended to have higher maximum ingestion and clearance rates than ones in a physiological state of lower growth. The same trend was observed for volume‐specific maximum ingestion and volume‐specific clearance rate. In response to changing prey density, the growth rate did not change as quickly as the ingestion rate, suggesting imbalance between the two. The tested physical stresses, including shaking, centrifugation, and filtration, also resulted in reduction of ingestion parameters of the flagellates. But half‐saturation constants did not show any trend in response to either physiological state or physical stress. In light of the dynamic nature of protistan ingestion response to prey abundance, short incubation, which minimizes the physiological change, and careful handling, which prevents the possible physical stress, should be employed in order to avoid underestimation of in situ ingestion rates. Previously reported ingestion parameters of lab‐cultured protists, which are thought to be unrealistic in natural conditions, may represent only one of multiple ingestion responses, probably prey‐rich condition.