Diurnal Variation of Cell Proliferation in Three Bacterial Taxa from Coastal North Sea Waters

Pulse-labeling with bromodeoxyuridine (BrdU) in combination with fluorescence in situ hybridization was applied to quantify the percentage of proliferating cells in coastal North Sea waters. In order to assess diurnal variability, we sampled eight or nine times, respectively, within 3 consecutive days at two seasons. Bacteria affiliated with the Roseobacter, SAR86, and NOR5 lineages constituted on average 19% ± 3%, 8% ± 2%, and 6% ± 1% of all cells in May 2002 and 17% ± 3%, 10% ± 2%, and 11% ± 3% in August. The relative abundances of the three populations either remained stable, or they changed very gradually during the observation periods. On average, 38 and 39% of all Bacteria exhibited DNA de novo synthesis in May and August, respectively. The fractions of proliferating cells in bacteria of the SAR86 (May, 59%; August, 72%) and the Roseobacter (48 and 53%) lineages were significantly above the community average. A substantial cell proliferation of population NOR5 (34%) was only encountered in August, concomitant with a dinoflagellate bloom. Significant short-term fluctuations of DNA-synthesizing cells were observed in Roseobacter during May and in NOR5 during August, hinting at a pronounced (temporal or spatial) mesoscale patchiness of growth rates in these populations. Since the BrdU proliferation assay is susceptible to misinterpretation, we also modeled the expected number of labeled cells at increasing BrdU incubation times in a slowly growing bacterial population. We suggest that the absence of visible DNA synthesis in marine bacterioplankton cells after DNA pulse-labeling must not be interpreted as an indication of cell “inactivity.”     

Identification of North Sea molluscs with DNA barcoding

Sequence‐based specimen identification, known as DNA barcoding, is a common method complementing traditional morphology‐based taxonomic assignments. The fundamental resource in DNA barcoding is the availability of a taxonomically reliable sequence database to use as a reference for sequence comparisons. Here, we provide a reference library including 579 sequences of the mitochondrial cytochrome c oxidase subunit I for 113 North Sea mollusc species. We tested the efficacy of this library by simulating a sequence‐based specimen identification scenario using Best Match, Best Close Match (BCM) and All Species Barcode (ASB) criteria with three different threshold values. Each identification result was compared with our prior morphology‐based taxonomic assignments. Our simulation resulted in 87.7% congruent identifications (93.8% when excluding singletons). The highest number of congruent identifications was obtained with BCM and ASB and a 0.05 threshold. We also compared identifications with genetic clustering (Barcode Index Numbers, BINs) computed by the Barcode of Life Datasystem (BOLD). About 68% of our morphological identifications were congruent with BINs created by BOLD. Forty‐nine sequences were clustered in 16 discordant BINs, and these were divided in two classes: sequences from different species clustered in a single BIN and conspecific sequences divided in more BINs. Whereas former incongruences were probably caused by BOLD entries in need of a taxonomic update, the latter incongruences regarded taxa requiring further investigations. These include species with amphi‐Atlantic distribution, whose genetic structure should be evaluated over their entire range to produce a reliable sequence‐based identification system.     

Bacterial community dynamics during the winter-spring transition in the North Sea

Bacterioplankton dynamics at Helgoland Roads (54 degrees 11.3'N, 7 degrees 54.0'E) in the North Sea over the winter-spring transition were investigated. The bacterial community was analyzed and correlated with phytoplankton community data and abiotic parameters. The community structure was analyzed by ribosomal intergenic spacer analysis (RISA) and by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes followed by DNA sequence analysis. The linkage of abiotic and biotic environmental factors and bacterial community as well as phylotypes (sequenced DGGE bands) was analyzed by the ordination technique of canonical correspondence analysis (CCA). Generally, an influence of temperature and phytoplankton on the bacterial community during the sampling period was observed. Additionally, multivariate analysis by factors revealed an influence on specific bacterial phylotypes of these factors. Overall, results indicate that changes in the bacterial community were caused not only by abiotic factors but also by the phytoplankton community.     

Identification of DNA-synthesizing bacterial cells in coastal North Sea plankton

We describe a method for microscopic identification of DNA-synthesizing cells in bacterioplankton samples. After incubation with the halogenated thymidine analogue bromodeoxyuridine (BrdU), environmental bacteria were identified by fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-linked oligonucleotide probes. Tyramide signal amplification was used to preserve the FISH staining during the subsequent immunocytochemical detection of BrdU incorporation. DNA-synthesizing cells were visualized by means of an HRP-labeled antibody Fab fragment and a second tyramide signal amplification step. We applied our protocol to samples of prefiltered (pore size, 1.2 micro m) North Sea surface water collected during early autumn. After 4 h of incubation, BrdU incorporation was detected in 3% of all bacterial cells. Within 20 h the detectable DNA-synthesizing fraction increased to >14%. During this period, the cell numbers of members of the Roseobacter lineage remained constant, but the fraction of BrdU-incorporating Roseobacter sp. cells doubled, from 24 to 42%. In Alteromonas sp. high BrdU labeling rates after 4 to 8 h were followed by a 10-fold increase in abundance. Rapid BrdU incorporation was also observed in members of the SAR86 lineage. After 4 h of incubation, cells affiliated with this clade constituted 8% of the total bacteria but almost 50% of the visibly DNA-synthesizing bacterial fraction. Thus, this clade might be an important contributor to total bacterioplankton activity in coastal North Sea water during periods of low phytoplankton primary production. The small size and low ribosome content of SAR86 cells are probably not indications of inactivity or dormancy.     

Temporal and Spatial Diversity of Bacterial Communities in Coastal Waters of the South China Sea

Bacteria are recognized as important drivers of biogeochemical processes in all aquatic ecosystems. Temporal and geographical patterns in ocean bacterial communities have been observed in many studies, but the temporal and spatial patterns in the bacterial communities from the South China Sea remained unexplored. To determine the spatiotemporal patterns, we generated 16S rRNA datasets for 15 samples collected from the five regularly distributed sites of the South China Sea in three seasons (spring, summer, winter). A total of 491 representative sequences were analyzed by MOTHUR, yielding 282 operational taxonomic units (OTUs) grouped at 97% stringency. Significant temporal variations of bacterial diversity were observed. Richness and diversity indices indicated that summer samples were the most diverse. The main bacterial group in spring and summer samples was Alphaproteobacteria, followed by Cyanobacteria and Gammaproteobacteria, whereas Cyanobacteria dominated the winter samples. Spatial patterns in the samples were observed that samples collected from the coastal (D151, D221) waters and offshore (D157, D1512, D224) waters clustered separately, the coastal samples harbored more diverse bacterial communities. However, the temporal pattern of the coastal site D151 was contrary to that of the coastal site D221. The LIBSHUFF statistics revealed noticeable differences among the spring, summer and winter libraries collected at five sites. The UPGMA tree showed there were temporal and spatial heterogeneity of bacterial community composition in coastal waters of the South China Sea. The water salinity (P=0.001) contributed significantly to the bacteria-environment relationship. Our results revealed that bacterial community structures were influenced by environmental factors and community-level changes in 16S-based diversity were better explained by spatial patterns than by temporal patterns.     

Substrate Utilization Profiles of Bacterial Strains in Plankton from the River Warnow, a Humic and Eutrophic River in North Germany

Bacteria are very important degraders of organic substances in aquatic environments. Despite their influential role in the carbon (and many other element) cycle(s), the specific genetic identity of active bacteria is mostly unknown, although contributing phylogenetic groups had been investigated. Moreover, the degree to which phenotypic potential (i.e., utilization of environmentally relevant carbon substrates) is related to the genomic identity of bacteria or bacterial groups is unclear. The present study compared the genomic fingerprints of 27 bacterial isolates from the humic River Warnow with their ability to utilize 14 environmentally relevant substrates. Acetate was the only substrate utilized by all bacterial strains. Only 60% of the strains respired glucose, but this substrate always stimulated the highest bacterial activity (respiration and growth). Two isolates, both closely related to the same Pseudomonas sp., also had very similar substrate utilization patterns. However, similar substrate utilization profiles commonly belonged to genetically different strains (e.g., the substrate profile of Janthinobacterium lividum OW6/RT-3 and Flavobacterium sp. OW3/15-5 differed by only three substrates). Substrate consumption was sometimes totally different for genetically related isolates. Thus, the genomic profiles of bacterial strains were not congruent with their different substrate utilization profiles. Additionally, changes in pre-incubation conditions strongly influenced substrate utilization. Therefore, it is problematic to infer substrate utilization and especially microbial dissolved organic matter transformation in aquatic systems from bacterial molecular taxonomy.     

Incorporation of Glucose under Anoxic Conditions by Bacterioplankton from Coastal North Sea Surface Waters

It has been hypothesized that the potential for anaerobic metabolism might be a common feature of bacteria in coastal marine waters (L. Riemann and F. Azam, Appl. Environ. Microbiol. 68: 5554-5562, 2002). Therefore, we investigated whether different phylogenetic groups of heterotrophic picoplankton from the coastal North Sea were able to take up a simple carbon source under anoxic conditions. Oxic and anoxic incubations (4 h) or enrichments (24 h) of seawater with radiolabeled glucose were performed in July and August 2003. Bacteria with incorporated substrate were identified by using a novel protocol in which we combined fluorescence in situ hybridization and microautoradiography of cells on membrane filters. Incorporation of glucose under oxic and anoxic conditions was found in α-Proteobacteria, γ-Proteobacteria, and the Cytophaga-Flavobacterium cluster of the Bacteroidetes at both times, but not in marine Euryarchaeota. In July, the majority of cells belonging to the α-proteobacterial Roseobacter clade showed tracer incorporation both in oxic incubations and in oxic and anoxic enrichments. In August, only a minority of the Roseobacter cells, but most bacteria affiliated with Vibrio spp., were able to incorporate the tracer under either condition. A preference for glucose uptake under anoxic conditions was observed for bacteria related to Alteromonas and the Pseudoalteromonas-Colwellia group. These genera are commonly considered to be strictly aerobic, but facultatively fermentative strains have been described. Our findings suggest that the ability to incorporate substrates anaerobically is widespread in pelagic marine bacteria belonging to different phylogenetic groups. Such bacteria may be abundant in fully aerated coastal marine surface waters.     

Large Plankton Enhance Heterotrophy Under Experimental Warming in a Temperate Coastal Ecosystem

Microbes are key players in oceanic carbon fluxes. Temperate ecosystems are seasonally variable and thus suitable for testing the effect of warming on microbial carbon fluxes at contrasting oceanographic conditions. In four experiments conducted in February, April, August and October 2013 in coastal NE Atlantic waters, we monitored microbial plankton stocks and daily rates of primary production, bacterial heterotrophic production and respiration at in situ temperature and at 2 and 4°C over ambient values during 4-day incubations. Ambient total primary production (TPP) exceeded total community respiration (< 200 µm, TR) in winter and fall but not in spring and summer. The bacterial contribution to ecosystem carbon fluxes was low, with bacterial production representing on average 6.9 ± 3.2% of TPP and bacterial respiration (between 0.8 and 0.2 µm) contributing on average 35 ± 7% to TR. Warming did not result in a uniform increase in the variables considered, and most significant effects were found only for the 4°C increase. In the summer and fall experiments, under warm and nutrient-deficient conditions, the net TPP/TR ratio decreased by 39 and 34% in the 4°C treatment, mainly due to the increase in respiration of large organisms rather than bacteria. Our results indicate that the interaction of temperature and substrate availability in determining microbial carbon fluxes has a strong seasonal component in temperate planktonic ecosystems, with temperature having a more pronounced effect and generating a shift toward net heterotrophy under more oligotrophic conditions as found in summer and early fall.     

Diversity of Oligotrichia and Choreotrichia Ciliates in Coastal Marine Sediments and in Overlying Plankton

Elucidating the relationship between ciliate communities in the benthos and the plankton is critical to understanding ciliate diversity in marine systems. Although data for many lineages are sparse, at least some members of the dominant marine ciliate clades Oligotrichia and Choreotrichia can be found in both plankton and benthos, in the latter either as cysts or active forms. In this study, we developed a molecular approach to address the relationship between the diversity of ciliates in the plankton and those of the underlying benthos in the same locations. Samples from plankton and sediments were compared across three sites along the New England coast, and additional subsamples were analyzed to assess reproducibility of methods. We found that sediment and plankton subsamples differed in their robustness to repeated subsampling. Sediment subsamples (i.e., 1-g aliquots from a single ∼20-g sample) gave variable estimates of diversity, while plankton subsamples produced consistent results. These results indicate the need for additional study to determine the spatial scale over which diversity varies in marine sediments. Clustering of phylogenetic types indicates that benthic assemblages of oligotrichs and choreotrichs appear to be more like those from spatially remote benthic communities than the ciliate communities sampled in the water above them.Planktonic ciliates provide a critical trophic link between the microbial and macroscopic components of the pelagic food web, and the subclasses Choreotrichia and Oligotrichia are the most abundant ciliate groups in this environment (46). One key to understanding the diversity and ecology of Choreotrichia and Oligotrichia is the relationship between benthic and planktonic forms. While the ciliates in these two groups are predominantly swimmers (54), there is crossover between benthic and pelagic environments for many species. Some taxa are described as epibenthic, living in the layer of water just above the sediment (16, 54), some have the capacity to live attached to sediment particles for a period and then become free-swimming (21), and a large number of taxa within these two groups spend a portion of their life cycles in dormancy, persisting in the sediments in cyst form (22, 23, 25, 35, 36, 39, 40, 41, 43, 49, 51). An accurate assessment of ciliate dynamics in the plankton requires careful study of both benthic and pelagic environments and the extent of coupling between the two environments.The role of the cyst in the life cycle of marine planktonic ciliates is particularly critical for understanding their distribution, evolutionary history, and ecology (6) as cysts provide a mechanism for dormancy during periods of poor environmental conditions. Relatively few marine ciliate species have been directly studied to determine conditions for encystment and excystment, period of dormancy (22, 23, 25, 26, 43), and role of the encystment cycle in the ecology of the organism (36). Moreover, studies on the conditions related to encystment and excystment in ciliates reveal different patterns and potential causes depending on the species (22, 23, 25, 26, 36, 43). While some data link the cycle of encystment with environmental factors such as light (23), temperature (23, 25, 26), and presence of food (22), other data suggest a temporal/seasonal cycling independent of external environmental conditions (26, 36, 43).A further factor limiting our understanding of the role of cysts in the life cycle of ciliates is identification based on the limited morphological features of the cysts, which are highly convergent (4, 17). In the case of ciliates that encyst within a lorica, as in the tintinnids, this is less of a problem (45), but for aloricate species, identification is not certain without direct observation of excystment (41, 48). Hence, morphological surveys of ciliates in benthic environments frequently capture members of the Oligotrichia and Choreotrichia (19, 31, 52, 53, 54) but are often limited to identification at the genus level using morphological approaches.More is known about planktonic ciliates, where morphology provides a wealth of data (11) and where molecular studies have revealed tremendous diversity, with many rare haplotypes (10). We define distinct sequences at the small-subunit (SSU) ribosomal DNA (rDNA) locus as haplotypes to remain conservative in our approach to identifying operational taxonomic units (OTUs) because ciliates have an unusual genome structure with high chromosome copy number, which potentially could generate multiple sequence types for the same locus within an organism or within a species. Planktonic ciliates show high molecular diversity at the SSU rDNA locus (10, 24), and primer sequences have been developed to detect ciliates from environmental samples within the subclasses Choreotrichia and Oligotrichia (10). Ciliates from these subclasses sampled across three coastal locations comprised distinct assemblages, with a few ubiquitous and abundant haplotypes (10) and many singletons (haplotypes unique to a particular sample).This study lays the groundwork for an alternative to morphological methods for analyzing benthic assemblages of oligotrichs and choreotrichs and comparing them to assemblages in the overlying water. Our goal was to compare levels of genetic diversity between sediment and plankton samples as a means of assessing the potential of methods for monitoring exchange between these two communities. There are two main questions addressed in this study: (i) are the two environments, plankton and sediment, comparable in robustness to repeated sampling using PCR, cloning, and sequencing and (ii) what is the relationship between genetic diversity of oligotrich and choreotrich ciliate communities sampled in marine sediments and in the plankton?To investigate the first question, we designed resampling experiments in plankton and sediment collections to test spatial heterogeneity as well as the robustness of repeated PCR cloning and sequencing for capturing diversity. Using two plankton samples collected by different means from the same time and place, we compared the similarity of subsamples in this environment to the similarity between separate subsamples of sediment collected at the same time and place. Additionally, we resampled DNA extracted from each of the two environments and investigated the reproducibility of repeated PCR cloning and sequencing between environmental types.To investigate the second question, we compared the diversity in sediment samples collected in the Gulf of Maine and Long Island Sound in May 2005 to previously published data from plankton samples collected at the same times and locations (10). Cluster analyses of the communities in sediment and plankton were used to determine the degree of coupling between the benthic and pelagic forms of Oligotrichia and Choreotrichia. The predicted result would be that the ciliate community observed in the plankton represents a subset of the diversity found in the benthic community, including cysts, beneath it. While the community in the plankton for many oligotrichs and choreotrichs would change depending on prevailing environmental conditions, predation, and chance, the benthic community, which includes encysted planktonic forms, should represent the longer-term diversity in a given region.     

Identification of jellyfish from Continuous Plankton Recorder samples

Previous analysis of the Continuous Plankton Recorder (CPR) long-term data set for the presence of ‘coelenterate’ tissue revealed changes in the frequency of jellyfish occurrence in the North Atlantic Ocean and North Sea; however, the identities of the jellyfish were unknown, causing uncertainty in interpreting these findings. To improve the utility of the ‘coelenterate’ data from the CPR, 62 CPR samples were selected for re-analysis from an area where the widespread occurrence of the holoplanktonic scyphomedusan, Pelagia noctiluca (Forsskål, 1775), was previously documented from net tows to test if the CPR sampled P. noctiluca. Examination of the samples revealed smears (<1 mm) of golden gelatinous tissue on the sampling mesh. Subsequent microscopic examination and measurement of nematocysts identified most of the smears as P. noctiluca. Indeed, P. noctiluca was identified on 53% of the CPR samples from the area that best coincided with the documented bloom area. Although hydrozoans were also identified in the samples, the characteristic golden colour of P. noctiluca and its distinct suite of nematocysts allowed identification to species level. The identification of other jellyfish may be more complicated due to the labour-intensive examination of samples and unclear delineation of nematocyst types when multiple similar species are present. Nevertheless, re-analysis of CPR samples holds great promise for identifying the underlying reason for increases in jellyfish occurrence in the CPR records.     

Coastal peat-beds and peatlands of the southern North Sea: their past,present and future

Peat layers are well represented in the Holocene coastal deposits of the southern North Sea and provide evidence as to the extent and nature of the fens and bogs that occupied the region in the mid and late Holocene. While natural processes contributed to their demise, without human interference extensive areas of peatland would remain. We review the characteristics of the vegetation of these peatlands along with the processes that influenced their development. Spatial and temporal trends are explored through the use of palaeogeographic maps from three areas: the East Anglian Fenland, the Romney Marsh area and the Netherlands. The palaeoecological evidence indicates that eutrophic vegetation promoted by rising relative sea level (RSL) dominated in the mid Holocene, with a trend towards the development of oligotrophic and ombrotrophic vegetation in the late Holocene as the rate of RSL rise declined. Nevertheless, areas of eutrophic vegetation appear capable of long-term stability with areas of fen woodland and herbaceous fen persisting at some locations for several thousand years in the mid and late Holocene. Areas of active peat growth in the region are now largely confined to small remnants within agricultural settings. To retain their characteristic biodiversity these remnants have been managed using traditional practices, although their small size and fragmented distribution limits their biodiversity value. Biodiversity concerns and the ecosystem services peatlands provide, notably carbon sequestration and flood attenuation, underlie recent restoration projects. These efforts are likely to receive additional impetus as a consequence of rising water levels, given projected rates of RSL rise. Future large-scale restoration can be informed by a greater understanding of the processes that formed and sustained coastal peatlands in the past. We identify advances in palaeoenvironmental research that could enhance restoration efforts and help maximise the ecosystem services delivered through such projects.     

珠江口横琴岛海域春季海洋浮游生物的初步研究

根据2008年5月在珠江口横琴岛海域的调查采样,本文对该海域的叶绿素a、初级生产力(C)、浮游动、植物进行了初步的研究,分析了浮游动、植物的种类组成、群落结构、数量和生物量等.结果表明,叶绿素a浓度和初级生产力(C)均值分别为5.27 mg·m^-3和123.56 mg·m^-2·d^-1.浮游植物共有111种,以硅藻为主,绝对优势种为中肋骨条藻Skeletonema costatum,调查海域浮游植物平均细胞丰度为6832.75×10⁴cell·m^-3,以近海广布种为主要类群,多样性指数和均匀度均值分别为2.29和0.45.浮游动物共有41种,暖水种沿岸类群种类占大多数,以中华异水蚤Acartiella sinensis为绝对优势种,平均丰度和生物量分别742.25 ind·m^-3和131.12 mg·m^-3,多样性指数和均匀度均值分别为2.54和0.67.     

Plankton metabolism in the Greenland Sea during the polar summer of 2007

The polar summer metabolism of the planktonic communities in the Greenland Sea was surveyed in July 2007. Planktonic metabolism showed great variability across the studied area, with on average, higher metabolic rates in the Fram Strait-Svalbard region than along the Greenland Current. A significant fraction (47%) of the planktonic communities in the Fram Strait-Svalbard region were net heterotrophic, suggesting that increased respiration rates with further warming may lead the planktonic communities at this region to act as net CO₂ sources. The thresholds gross primary production for metabolic balance (i.e., gross primary production = community respiration) was much higher in the European sector of the Arctic than reported for the Southern Ocean, suggesting that heterotrophic metabolism is more prevalent in the European sector of the Arctic than in the Southern Ocean, indicating high allochthonous inputs in the Arctic region.     

Direct Detection of Vibrio cholerae and ctxA in Peruvian Coastal Water and Plankton by PCR

Seawater and plankton samples were collected over a period of 17 months from November 1998 to March 2000 along the coast of Peru. Total DNA was extracted from water and from plankton grouped by size into two fractions (64 μm to 202 μm and >202 μm). All samples were assayed for Vibrio cholerae, V. cholerae O1, V. cholerae O139, and ctxA by PCR. Of 50 samples collected and tested, 33 (66.0%) were positive for V. cholerae in at least one of the three fractions. Of these, 62.5% (n = 32) contained V. cholerae O1; ctxA was detected in 25% (n = 20) of the V. cholerae O1-positive samples. None were positive for V. cholerae O139. Thus, PCR was successfully employed in detecting toxigenic V. cholerae directly in seawater and plankton samples and provides evidence for an environmental reservoir for this pathogen in Peruvian coastal waters.     

Diseases in North Sea fishes

Prior to the studies reviewed here, only lymphocystis and skeletal deformities of a variety of fish species and certain diseases of eel were known to occur in the German Bight (North Sea). From 1977 until now, 9 externally visible lesions on North Sea fishes were observed; in addition to those mentioned before, they comprise: fin rot, ulcerations, epidermal papilloma, hyperplasia, pseudobranchial tumour, eye diseases and gill swellings. With the exception of information on changes in frequencies of vertebral deformities of herring from the 1950's to the 1970's, there are no long-term data characterizing changes in frequencies of the diseases under study. For pseudobranchial tumours of cod and epidermal papilloma of dab, information is provided on occurrence and abundance. The distribution pattern of cod afflicted with pseudobranchial tumours is strongly influenced by the migratory behaviour of the fish. Epidermal papillomas of dab were more frequently found at stations within the inner German Bight than in neighbouring areas. The Bight is used for dumping of wastes from titaniumdioxide production. Further disease hot spots are areas off the Humber estuary and the British coast. Analysis of chromium in dab from the German Bight revealed elevated concentrations in epidermal tissues of specimens from the dumping area compared with that found in dab from neighbouring localities. Particulate iron was demonstrated to occur in mucous cells of dab from the dumping area. From increased levels of heavy metals with cancerogenic potential in sensitive target tissues and from increased prevalences of diseased fish in the dumping area it is concluded that these phenomena are possibly causally linked. In the vicinity of the Humber estuary high disease rates were encountered and areas with high prevalences of dab afflicted with epidermal papilloma extended over regions shown to be transport routes for persistent pollutants such as radioactive materials. It is therefore suggested that the long-range distribution of fish diseases in the southern North Sea might reflect the long-range transport of persistent pollutants.     

Killing of Cells in Bacterial Colonies

Bacterial colonies grown on membrane filters and transferred to plates of inhibitor-containing medium decrease in number of total viable colony-forming cells. The decrease in viable cell count occurs in direct proportion to the concentration of inhibitor present and the length of time the colonies are exposed to the inhibitor, suggesting that the kinetics of cell kill approximate a first-order chemical reaction.