Depth Distribution of Bacterial Production in a Stratified Lake with an Anoxic Hypolimnion

The purpose of this study was to determine the depth distribution of bacterial biomass and production in a stratified lake and to test techniques to measure bacterial production in anaerobic waters. Bacterial abundance and incorporation of both [³H]thymidine and [³H]leucine into protein were highest in the metalimnion, at the depth at which oxygen first became unmeasurable. In contrast, [³H]thymidine incorporation into DNA was highest in the epilimnion. The ratios of incorporation into DNA/protein averaged 2.2, 0.49, and 0.95 for the epilimnion, metalimnion, and hypolimnion, respectively. Low incorporation into DNA was not due to artifacts associated with the DNA isolation procedure. Recovery of added [³H]DNA was about 90% in waters in which the portion of [³H]thymidine incorporation into DNA was about 40%. At least some obligate anaerobic bacteria were capable of assimilating thymidine since aeration of anaerobic hypolimnion waters substantially inhibited thymidine incorporation. The depth profile of bacterial production estimated from total thymidine and leucine incorporation and the frequency of dividing cells were all similar, with maximal rates in the metalimnion. However, estimates of bacterial production based on frequency of dividing cells and leucine incorporation were usually significantly higher than estimates based on thymidine incorporation (using conversion factors from the literature), especially in anaerobic hypolimnion waters. These data indicate that the thymidine approach must be examined carefully if it is to be applied to aquatic systems with low oxygen concentrations. Our results also indicate that the interface between the aerobic epilimnion and anaerobic hypolimnion is the site of intense bacterial mineralization and biomass production which deserves further study.     

Estimating Bacterioplankton Production by Measuring [3H]thymidine Incorporation in a Eutrophic Swedish Lake

Bacterioplankton abundance, [³H]thymidine incorporation, ¹⁴CO₂ uptake in the dark, and fractionated primary production were measured on several occasions between June and August 1982 in eutrophic Lake Norrviken, Sweden. Bacterioplankton abundance and carbon biomass ranged from 0.5 × 10⁹ to 2.4 × 10⁹ cells liter⁻¹ and 7 to 47 μg of C liter⁻¹, respectively. The average bacterial cell volume was 0.185 μm³. [³H]thymidine incorporation into cold-trichloroacetic acid-insoluble material ranged from 12 × 10⁻¹² to 200 × 10⁻¹² mol liter⁻¹ h⁻¹. Bacterial carbon production rates were estimated to be 0.2 to 7.1 μg of C liter⁻¹ h⁻¹. Bacterial production estimates from [³H]thymidine incorporation and ¹⁴CO₂ uptake in the dark agreed when activity was high but diverged when activity was low and when blue-green algae (cyanobacteria) dominated the phytoplankton. Size fractionation indicated negligible uptake of [³H]thymidine in the >3-μm fraction during a chrysophycean bloom in early June. We found that >50% of the ³H activity was in the >3-μm fraction in late August; this phenomenon was most likely due to Microcystis spp., their associated bacteria, or both. Over 60% of the ¹⁴CO₂ uptake in the dark was attributed to algae on each sampling occasion. Algal exudate was an important carbon source for planktonic bacteria. Bacterial production was roughly 50% of primary production.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [³H]uridine incorporation into RNA and [³H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10⁻²¹ M). Insulin stimulated the rate of [³H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [³H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [³H]uridine, [³H]thymidine and [³H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis.     

Bacterial production in freshwater sediments: Cell specific versus system measures

Estimates of bacterial production based on total trichloroacetic acid (TCA)-precipitable [methyl-³H]thymidine incorporation and frequency of dividing cell (FDC) techniques were compared to sediment respiration rates in Lake George, New York. Bacterial growth rates based on thymidine incorporation ranged from 0.024 to 0.41 day^–1, while rates based on FDC ranged from 1.78 to 2.48 day^–1. Respiration rates ranged from 0.11 to 1.8mol O₂·hour^–1·g dry weight sediment^–1. Thymidine incorporation yielded production estimates which were in reasonable agreement with respiration rates. Production estimates based on FDC were 4- to 190-fold higher than those predicted from respiration rates.     

Underestimation of DNA Synthesis by [3H]Thymidine Incorporation in Marine Bacteria

A direct comparison of [³H]thymidine incorporation with DNA synthesis was made by using an exponentially growing estuarine bacterial isolate and the naturally occurring bacterial populations in a eutrophic subtropical estuary and in oligotrophic offshore waters. Simultaneous measurements of [³H]thymidine incorporation into DNA, fluorometrically determined DNA content, and direct counts were made over time. DNA synthesis estimated from thymidine incorporation values was compared with fluorometrically determined changes in DNA content. Even after isotope dilution, nonspecific macromolecular labeling, and efficiency of DNA recovery were accounted for, [³H]thymidine incorporation consistently underestimated DNA synthesized by six- to eightfold. These results indicate that although the relationship of [³H]thymidine incorporation to DNA synthesis appears consistent, there are significant sources of thymine bases incorporated into DNA which cannot be accounted for by standard [³H]thymidine incorporation and isotope dilution assays.     

Estimating Bacterioplankton Production by Measuring [H]thymidine Incorporation in a Eutrophic Swedish Lake

Bacterioplankton abundance, [H]thymidine incorporation, CO(2) uptake in the dark, and fractionated primary production were measured on several occasions between June and August 1982 in eutrophic Lake Norrviken, Sweden. Bacterioplankton abundance and carbon biomass ranged from 0.5 x 10 to 2.4 x 10 cells liter and 7 to 47 mug of C liter, respectively. The average bacterial cell volume was 0.185 mum. [H]thymidine incorporation into cold-trichloroacetic acid-insoluble material ranged from 12 x 10 to 200 x 10 mol liter h. Bacterial carbon production rates were estimated to be 0.2 to 7.1 mug of C liter h. Bacterial production estimates from [H]thymidine incorporation and CO(2) uptake in the dark agreed when activity was high but diverged when activity was low and when blue-green algae (cyanobacteria) dominated the phytoplankton. Size fractionation indicated negligible uptake of [H]thymidine in the >3-mum fraction during a chrysophycean bloom in early June. We found that >50% of the H activity was in the >3-mum fraction in late August; this phenomenon was most likely due to Microcystis spp., their associated bacteria, or both. Over 60% of the CO(2) uptake in the dark was attributed to algae on each sampling occasion. Algal exudate was an important carbon source for planktonic bacteria. Bacterial production was roughly 50% of primary production.     

Uptake and Incorporation of Thymidine by Bacterial Isolates from an Upwelling Environment

Thymidine uptake and incorporation by marine bacterial isolates from an upwelling environment were studied. Of 17 isolates each from upwelled and downwelled water, 1 and 6 isolates, respectively, were found to be negative for [³H]thymidine incorporation at a substrate concentration of 19 μM. Strains lacking the ability to take up thymidine were not confined to one genus. The measurable rates of uptake and incorporation by the 34 isolates varied greatly. Studies carried out using starved Vibrio, Pseudomonas, and Cytophaga cells showed that these isolates transported and incorporated thymidine after periods of as long as 5 weeks of nutrient deprivation. This occurred in the absence of any other exogenously supplied nutrients. Overall, these results indicate that not all marine bacteria take up thymidine and that those that do incorporate the nucleoside may do so at very different rates. The assumption that only actively growing or dividing cells incorporate thymidine must be viewed with caution.     

Steroidal and growth factor regulation of [3H]thymidine incorporation by cultured endosalpingeal cells of the bovine oviduct

Summary Cultured cells from the bovine endosalpinx were used to evaluate effects of estradiol-17β, progesterone, epidermal growth factor, and insulinlike growth factors I and II on [³H]thymidine incorporation. Cells were treated with hormones and growth factors when approximately 50% confluent. After 24 h, DNA synthesis was quantified by pulsing cells with [³H]thymidine for 12 h and determining uptake into DNA. Cells prepared by mechanical dispersal incorporated more [³H]thymidine than cells dispersed with collagenase. However, hormonal responses were the same for both types of cells. As compared to plastic, cells on a Matrigel substratum exhibited lower incorporation of [³H]thymidine and were unresponsive to hormones. Estradiol-17β increased [³H]thymidine incorporation slightly at 10⁻¹⁰ mol/liter and higher. Epidermal growth factor, insulinlike growth factor-I, and insulinlike growth factor-II also stimulated [³H]thymidine incorporation. Effects of insulinlike growth factor-I were greater for cells treated with estradiol-17β. In the absence of estradiol, progesterone inhibited [³H]thymidine incorporation at 1, 10, and 100 ng/ml. When estradiol-17β was present, progesterone stimulated [³H]thymidine incorporation at 1 ng/ml and reduced incorporation at 100 ng/ml. In conclusion, [³H]thymidine incorporation by cultured oviductal endosalpingeal cells can be regulated by ovarian steroids and growth factors. These molecules may represent signals through which the ovary, embryo, and oviduct regulate oviductal growth. Work conducted while on a sabbatical leave supported by the Deutsche Forschungsgemeinschaft.     

Selected Nucleic Acid Precursors in Studies of Aquatic Microbial Ecology

The use of radiolabeled nucleosides and nucleic acid bases to estimate the rates of RNA and DNA synthesis in naturally occurring microbial assemblages requires numerous assumptions, several of which are evaluated herein. Comparative time series analyses of the uptake and incorporation, labeling specificity, and extent of catabolism of [2-³H]adenine, [methyl-³H]thymidine, and [5-³H]uridine were performed with pure bacterial and algal cultures, as well as with environmental samples. [³H]thymidine yielded the most variable results, especially with regard to the extent of nonspecific macromolecular labeling. The pathways of [³H]thymidine and [³H]adenine metabolism were further evaluated by isotope dilution methods and by comparing incorporation patterns of thymidine labeled at different sites of the molecule. The advantages, uncertainties, and limitations of the use of radiolabeled nucleic acid precursors in studies of aquatic microbial ecology are discussed and a prospectus for future studies presented.     

Incorporation of tritiated thymidine by marine bacterial isolates when undergoing a starvation survival response

Bacterial DNA synthesis, as measured by the incorporation of [methyl-³H] thymidine, was examined during conditions of decreasing biomass and non-growth of three heterotrophic marine bacteria. High rates of [³H] thymidine incorporation were recorded during the initial phase of starvation and two strains exhibited a net increase in DNA during the first few hours of starvation. The decreased rate of [³H] thymidine incorporation with the time of starvation, was in agreement with the decrease in the percentage of the total population that showed uptake of labelled thymidine, as seen by a combined autoradiography-epifluorescence technique. It is suggested that new rounds of replications were initiated after cells had been starved for times that well exceeded the time for replication of genomes during growing The initial increase in cell numbers upon transfer of growing cells to a starvation regime was inhibited by nalidixic acid, suggesting that DNA synthesis, rather than an excess of nuclear bodies, allow for the fragmentation process in these strains.     

Inorganic Phosphorus Stimulation of Bacterioplankton Production in a Meso-Eutrophic Lake

Experiments were conducted to determine whether production of heterotrophic bacterioplankton in a small meso-eutrophic lake was influenced by the dissolved inorganic phosphorus (DIP) supply. DIP may indirectly limit bacterial production by limiting phytoplankton, which in turn may limit the carbon available to bacteria. Direct DIP limitation of bacteria occurs where the availability of DIP for bacteria is insufficient to maintain growth. This work examined direct DIP limitation of bacteria by removing phytoplankton and incubating flasks with or without added P in the dark. Bacterial production was measured via the rate of incorporation of [³H]thymidine ([³H]TdR) into DNA. Bacterial abundance was followed with epifluorescent direct counts. Rates of [³H]TdR incorporation were significantly greater in flasks with added DIP, and changes in cell abundances generally paralleled increases in [³H]TdR incorporation. Even very small additions of P (0.05 μM) were sufficient to stimulate production. DIP addition to whole lakewater also stimulated [³H]TdR incorporation relative to that in zero-addition controls, but there was not a concurrent increase in bacterial cell numbers. The stimulation of [³H]TdR incorporation after DIP addition to whole lakewater was significantly less than the stimulation due to DIP addition to 1-μm-pore-size-filtered lakewater. In this study, addition of DIP caused as much as an eightfold stimulation of [³H]TdR incorporation.     

Seasonal Bacterial Production in a Dimictic Lake as Measured by Increases in Cell Numbers and Thymidine Incorporation

Rates of primary and bacterial production in Little Crooked Lake were calculated from the rates of incorporation of H¹⁴CO₃⁻ and [methyl-³H]thymidine, respectively. Growth rates of bacteria in diluted natural samples were determined for epilimnetic and metalimnetic bacterial populations during the summers of 1982 and 1983. Exponential growth was observed in these diluted samples, with increases in cell numbers of 30 to 250%. No lag was observed in bacterial growth in 14 of 16 experiments. Correlation of bacterial growth rates to corresponding rates of thymidine incorporation by natural samples produced a conversion factor of 2.2 × 10¹⁸ cells produced per mole of thymidine incorporated. The mass of the average bacterial cell in the lake was 1.40 × 10⁻¹⁴ ± 0.05 × 10⁻¹⁴ g of C cell⁻¹. Doubling times of natural bacteria calculated from thymidine incorporation rates and in situ cell numbers ranged from 0.35 to 12.00 days (median, 1.50 days). Bacterial production amounted to 66.7 g of C m⁻² from April through September, accounting for 29.4% of total (primary plus bacterial) production during this period. The vertical and seasonal distribution of bacterial production in Little Crooked Lake was strongly influenced by the distribution of primary production. From April through September 1983, the depth of maximum bacterial production rates in the water column was related to the depth of high rates of primary production. On a seasonal basis, primary production increased steadily from May through September, and bacterial production increased from May through August and then decreased in September.     

Interferon inhibition of thymidine incorporation into DNA through effects on thymidine transport and uptake

Replenishment of medium after 72 hr of growth of HeLa-S3 cells in dense suspension cultures increased [³H]-thymidine uptake into cells and incorporation into DNA, with the levels reaching a peak ～ 12 hr following medium change; β interferon inhibits the enhanced uptake of [³H]-thymidine and labeling of DNA in a dose-dependent manner. Some reduction in these processes is observed at a concentration as low as 1 u/ml, and ～ 75% inhibition at 640 u/ml. Kinetic analysis has revealed that the rate of labeling of the acid-soluble pool with [³H]-thymidine, measured either at 22°C, or 37°C, is reduced in interferon-treated (640 u/ml, 24 hr) HeLa-S3 cells. At 22°C, the initial rate of thymidine transport at a high (500 μM) thymidine concentration, determined within the first 30 sec of [³H]-thymidine addition was depressed by 44% in interferon-treated HeLa cells. At 37°C, labeled precursors accumulate in acid-soluble material for ～ 8 min after the addition of [³H]-thymidine, after which an apparent equilibrium level is attained. At this temperature, the rate of thymidine uptake and the apparent equilibrium level attained were depressed by 70% in interferon-treated HeLa cells. The reduced incorporation of [³H]-thymidine into DNA in interferon-treated HeLa-S3 cells can be largely explained by interferon inhibition of thymidine transport and phosphorylation.     

Effects of Toxic Substances on Natural Bacterial Assemblages Determined by Means of [3H]Thymidine Incorporation

The effects of 3,5-dichlorophenol, 2,4-dinitrophenol, and potassium dichromate on natural bacterial assemblages were examined by means of [³H]thymidine incorporation into trichloroacetic acid-insoluble material. Results from a large number of coastal marine and freshwater samples suggest the following. (i) The effects of the three toxicants included reductions in the bacterial cell number as well as changes in rates of [³H]thymidine incorporation and in [³H]thymidine incorporation per cell. The concentrations that inhibited [³H]thymidine incorporation by 50% ranged from 3 to 11 mg liter⁻¹ for 3,5-dichlorophenol, 6 to 10 mg liter⁻¹ for 2,4-dinitrophenol, and 21 to 123 mg liter⁻¹ for potassium dichromate, with a tendency to higher values in bacterial assemblages from more eutrophic environments. (ii) The effects of 3,5-dichlorophenol and potassium dichromate determined by [³H]leucine incorporation into bacterial protein were similar or larger than those obtained from [³H]thymidine incorporation. (iii) Two to four hours of exposure to the toxicants was necessary before stable maximum effects were found in [³H]thymidine incorporation. (iv) Storage of natural environmental samples should be avoided, since tests with water stored for 1 to 3 days sometimes produced results different from results obtained from in situ tests. (v) The effects of 3,5-dichlorophenol, 2,4-dinitrophenol, and potassium dichromate on natural bacterial assemblages were relatively constant during periods with different growth rates in the assemblages, during various periods of the year, and between samples from freshwater and marine localities. With some precautions, [³H]thymidine incorporation can be used as a quick and sensitive method for determining the effects of toxicants on aquatic bacterial assemblages from natural environmental samples.     

Calculation of Cell Production from [3H]Thymidine Incorporation with Freshwater Bacteria

The conversion factor for the calculation of bacterial production from rates of [³H]thymidine incorporation was examined with diluted batch cultures of freshwater bacteria. Natural bacterial assemblages were grown in aged, normal, and enriched media at 10 to 20°C. The generation time during 101 growth cycles covered a range from 4 to >200 h. The average conversion factor was 2.15 × 10¹⁸ cells mol^-1 of thymidine incorporated into the trichloroacetic acid (TCA) precipitate (standard error = 0.29 × 10¹⁸; n = 54), when the generation time exceeded 20 h. At generation times of <20 h, the average conversion factor was 11.8 × 10¹⁸ cells mol^-1 of thymidine incorporated into TCA precipitate (standard error = 1.72 × 10¹⁸; n = 47). The amount of radioactivity in purified DNA increased with decreasing generation time and increasing conversion factor (calculated from the TCA precipitate), corresponding to a decrease in the percentage in protein. The conversion factors calculated from purified DNA or from the TCA precipitate gave the same variability. Conversion factors did not change significantly with the medium, but were significantly higher at 20°C than at 15 and 10°C. A detailed examination of the [³H]thymidine concentrations that were needed to achieve maximum labeling in DNA was carried out 6 times during a complete growth cycle. During periods with low generation times and high conversion factors, 15 nM [³H]thymidine was enough for the maximum labeling of the TCA precipitate. This suggests that incorporation of [³H]thymidine into DNA is probably limited by uptake during periods with generation times of <20 h and that freshwater bacterioplankton cell production sometimes is underestimated when a conversion factor of 2.15 × 10¹⁸ cells mol^-1 of thymidine incorporated is used.     

Lack of effect of butyrate on S phase DNA synthesis despite presence of histone hyperacetylation

The effects of sodium butyrate on [³H]thymidine incorporation and cell growth characteristics in randomly growing and synchronized HeLa S₃ cells have been examined in an attempt to determine what effects, if any, butyrate has on S phase cells. Whereas 5 mM sodium butyrate rapidly inhibits [⁵H]thymidine incorporation in a randomly growing cell populations, it has no effect on incorporation during the S phase in cells synchronized by double thymidine block techniques. This lack of effect does not result from an impaired ability of the S phase cells to take up butyrate, since butyrate administration during this period leads to histone hyperacetylation that is identical with that seen with butyrate treatment of randomly growing cells. Furthermore, the ability to induce such hyperacetylation with butyrate during an apparently normal progression through S phase indicates that histone hyperacetylation probably has no effect on the overall process of DNA replication. Temporal patterns of [³H]thymidine incorporation and cell growth following release from a 24-h exposure to butyrate confirm blockage of cell growth in the G1 phase of the cell cycle. Thus, the inhibition by butyrate of [³H]thymidine incorporation in randomly growing HeLa S₃ cell populations can be accounted for solely on the basis of a G1 phase block, with no inhibitory effects on cells already engaged in DNA synthesis or cells beyond the G1 phase block at the time of butyrate administration.     

Metabolic Fate of [3H]1-β-d-Arabinofuranosyl-5-[(E)-2-bromovinyl]uracil in Herpes Simplex Virus Type 1-Infected Cells

The metabolic fate of 1-β-d -arabinofuranosyl-5-[(E)-2-bromovinyl]uracil (BV-araU) in herpes simplex virus type 1-infected cells was studied using tritium-labeled BV-araU. [³H]BV-araU was selectively taken-up by infected cells. Approximately 10% of the total uptake of [³H]BV-araU was recovered from the acid-insoluble fraction at any time post-infection. Both cellular uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction increased with increasing incubation time through 8 hr post-infection. Uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction also increased proportionally to the duration of exposure to [³H]BV-araU. An alkaline sucrose gradient sedimentation analysis revealed that the radioactive DNA obtained from cells pulse-labeled with [³H]BV-araU were small DNA fragments which remained at the top following a chasing period in isotope-free medium, whereas that pulse-labeled with [³H]thymidine was chased to a fraction of high molecular weight DNA. Nuclease P₁ digestion reduced 99% of the [³H]BV-araU-labeled DNA extracted from infected cells to a low molecular weight. Following digestion of [³H]BV-araU-labeled DNA with micrococcal nuclease and spleen exonuclease, all of the radioactivity was recovered as [³H]BV-araU 3′-monophosphate. Thus, BV-araU strongly inhibits the elongation of viral DNA strands as demonstrated by the alkaline sucrose gradient sedimentation analysis, whereas at least a portion of the [³H]BV-araU is incorporated inside viral DNA strands in infected cells.     

Measuring bacterial production via rate of incorporation of [3H]thymidine into DNA

Thymidine (TdR) incorporation into DNA as a measure of bacterial production in environmental samples relies on assumptions about what organisms incorporate exogenous thymidine, extent of dilution of labelled thymidine by internal and external pools, and analytical methods for recovery and purification of bacterial DNA. We have examined these assumptions with regard to the feasibility of using [³H]TdR incorporation in the water column and sediments of a blackwater river. The extent of dilution of added [³H]TdR may be determined with isotope dilution plots (Moriarty and Pollard, 1981 and 1982) and these indicate a wide range of degree of participation of added [³H]TdR. Previously described methods for extracting DNA from sediment bacteria may lead to underestimates and we described a more efficient recovery scheme.     

Effect of 5-Fluoro-2′-Deoxyuridine on [3H]Thymidine Incorporation by Bacterioplankton in the Waters of Southwest Florida

The effect of 5-fluoro-2′-deoxyuridine (FdUrd) on [methyl-³H] thymidine incorporation by bacterioplankton populations in subtropical freshwater, estuarine, and oceanic environments was examined. In estuarine waters, intracellular isotope dilution was inhibited by FdUrd, which enabled us to estimate both intracellular and extracellular isotope dilution. In 2 of 10 cases, extracellular isotope dilution was significant. At low concentrations of [methyl-³H]thymidine or [6-³H]thymidine, FdUrd completely inhibited incorporation of radioactivity into protein and RNA. At high concentrations of [³H]thymidine, however, FdUrd had little effect on labeling patterns. The dihydrofolate reductase inhibitors amethopterin and trimethoprim had no effect on macromolecular labeling patterns. These results suggest that thymidylate synthase is not involved in nonspecific labeling and that FdUrd inhibits nonspecific labeling by blocking some other enzyme involved in thymidine catabolism. In oligotrophic oceanic and freshwater samples, FdUrd did not inhibit intracellular isotope dilution or [³H]thymidine labeling of protein and RNA, but caused some inhibition of [³H]thymidine incorporation into DNA. The ability of FdUrd to inhibit nonspecific macromolecular labeling during [³H]thymidine incorporation was significantly correlated (r = 0.84) with total thymidine incorporation (in picomoles per liter per hour). The results are discussed in terms of applications of FdUrd to routine bacterial production measurements and the general assumptions of [³H]thymidine incorporation.     

Bacterial Standing Stock, Activity, and Carbon Production during Formation and Growth of Sea Ice in the Weddell Sea, Antarctica

Bacterial response to formation and growth of sea ice was investigated during autumn in the northeastern Weddell Sea. Changes in standing stock, activity, and carbon production of bacteria were determined in successive stages of ice development. During initial ice formation, concentrations of bacterial cells, in the order of 1 × 10⁸ to 3 × 10⁸ liter^-1, were not enhanced within the ice matrix. This suggests that physical enrichment of bacteria by ice crystals is not effective. Due to low concentrations of phytoplankton in the water column during freezing, incorporation of bacteria into newly formed ice via attachment to algal cells or aggregates was not recorded in this study. As soon as the ice had formed, the general metabolic activity of bacterial populations was strongly suppressed. Furthermore, the ratio of [³H]leucine incorporation into proteins to [³H]thymidine incorporation into DNA changed during ice growth. In thick pack ice, bacterial activity recovered and growth rates up to 0.6 day^-1 indicated actively dividing populations. However, biomass-specific utilization of organic compounds remained lower than in open water. Bacterial concentrations of up to 2.8 × 10⁹ cells liter^-1 along with considerably enlarged cell volumes accumulated within thick pack ice, suggesting reduced mortality rates of bacteria within the small brine pores. In the course of ice development, bacterial carbon production increased from about 0.01 to 0.4 μg of C liter^-1 h^-1. In thick ice, bacterial secondary production exceeded primary production of microalgae.