Enumeration and biomass estimation of planktonic bacteria and viruses by transmission electron microscopy

Bacteria and virus particles were harvested from water samples by ultracentrifugation directly onto Formvar-coated electron microscopy grids and counted in a transmission electron microscope. With this technique, we have counted and sized bacteria and viruses in marine water samples and during laboratory incubations. By X-ray microanalysis, we could determine the elemental composition and dry-matter content of individual bacteria. The dry weight/volume ratio for the bacteria was 600 fg of dry weight microns-3. The potassium content of the bacteria was normal compared with previous estimates from other bacterial assemblages; thus, this harvesting procedure did not disrupt the bacterial cells. Virus particles were, by an order of magnitude, more abundant than bacteria in marine coastal waters. During the first 5 to 7 days of incubation, the total number of viruses increased exponentially at a rate of 0.4 day-1 and thereafter declined. The high proliferation rate suggests that viral parasitism may affect mortality of bacteria in aquatic environments.     

Enumeration and biomass estimation of planktonic bacteria and viruses by transmission electron microscopy.

Bacteria and virus particles were harvested from water samples by ultracentrifugation directly onto Formvar-coated electron microscopy grids and counted in a transmission electron microscope. With this technique, we have counted and sized bacteria and viruses in marine water samples and during laboratory incubations. By X-ray microanalysis, we could determine the elemental composition and dry-matter content of individual bacteria. The dry weight/volume ratio for the bacteria was 600 fg of dry weight microns-3. The potassium content of the bacteria was normal compared with previous estimates from other bacterial assemblages; thus, this harvesting procedure did not disrupt the bacterial cells. Virus particles were, by an order of magnitude, more abundant than bacteria in marine coastal waters. During the first 5 to 7 days of incubation, the total number of viruses increased exponentially at a rate of 0.4 day-1 and thereafter declined. The high proliferation rate suggests that viral parasitism may affect mortality of bacteria in aquatic environments.     

Determination of bacterial number and biomass in the marine environment.

Three techniques for the measurement of bacterial numbers and biomass in the marine environment are described. Two are direct methods for counting bacteria. The first employs an epifluorescence microscope to view bacteria that have been concentrated on membrane filters and stained with acridine orange. The second uses a transmission electron microscope for observing replicas of bacteria that are concentrated on membrane filters. The other technique uses Limulus amebocyte lysate, an aqueous extract from the amebocytes of the horseshoe crab, Limulus polyphemus, to quantitate lipopolysaccharide (LPS) in seawater samples. The biomass of gram-negative (LPS containing) bacteria was shown to be related to the LPS content of the samples. A factor of 6.35 was determined for converting LPS to bacterial carbon.     

Determination of bacterial number and biomass in the marine environment.

Three techniques for the measurement of bacterial numbers and biomass in the marine environment are described. Two are direct methods for counting bacteria. The first employs an epifluorescence microscope to view bacteria that have been concentrated on membrane filters and stained with acridine orange. The second uses a transmission electron microscope for observing replicas of bacteria that are concentrated on membrane filters. The other technique uses Limulus amebocyte lysate, an aqueous extract from the amebocytes of the horseshoe crab, Limulus polyphemus, to quantitate lipopolysaccharide (LPS) in seawater samples. The biomass of gram-negative (LPS containing) bacteria was shown to be related to the LPS content of the samples. A factor of 6.35 was determined for converting LPS to bacterial carbon.     

Successive changes in the epimural bacterial community of young lambs as revealed by scanning electron microscopy.

Scanning electron microscopy was used to determine the time of initial colonization of the rumen epithelium of young lambs and successive changes with time in the morphological composition of the epimural community. Tissue samples were obtained from two groups of lambs at 1, 2, 4, 6, 8, and 10 weeks of age. Comparisons were made with the epimural communities observed at 12 well-distributed sites in the rumen of a mature wether. Epimural bacteria were already present on the epithelium at 1 week of age. The morphological composition of the epimural community changed with age, with the pattern of succession being similar in both groups of lambs. A total of 24 morphotypes were distinguished by scanning electron microscopy; 17 were rod shaped, 4 were cocci, 2 were spiral, and 1 was filamentous. These morphotypes were further subdivided into: (i) those persisting after their initial colonization in young lambs and present in the adult (7 morphotypes), (ii) those seen only in the adult (2 morphotypes), and (iii) those present only in young lambs (15 morphotypes). The seven morphotypes present in both the lamb and the adult could be considered indigenous members of the epimural community. Several morphotypes appeared restricted in their colonization to certain regions of the papillae, suggesting the presence of microhabitats within the epithelial habitat. Two rod-shaped bacteria were repeatedly seen specifically attached to one another, suggesting an interspecific association.     

Distribution of virus-like Particles in an Oligotrophic Marine Environment (Alboran Sea,Western Mediterranean)

Viruses are abundant in a variety of aquatic environments, often exceeding bacterial abundance by one order of magnitude. In the present study, the spatial distribution of viruses in offshore waters of the Alboran Sea (Western Mediterranean) have been studied to determine the relationships between viruses and host communities in this oligotrophic marine environment. Viral abundance was determined using two methods: (i) epifluorescence light microscopy using the dsDNA binding fluorochrome DAPI, and (ii) direct counts by transmission electron microscopy (TEM). The results obtained were significantly different; the highest viral counts were obtained by mean of TEM analyses. In all the samples tested the number of viruses was exceeded by the bacterial concentrations, with a ratio between viral and bacterial titers varying between 1.4 and 20. VLP (virus-like particle) counts were not significantly correlated (p > 0.001) with chlorophyll a concentration or the abundance of cyanobacteria. However, there was a positive and significant correlation with bacterial abundance (p < 0.001). The analysis of size and morphology of viral particles by TEM and the correlation obtained between the numbers of VLP and bacteria suggest that the majority of the viral particles in the Alboran Sea are bacteriophages. None of the indirect evidence suggested that eukaryotic algae or cyanobacteria were important host organisms in these waters.     

In situ detection of starch-hydrolyzing microorganisms in activated sludge

Polysaccharides constitute a significant part of the organic matter in domestic wastewater and their hydrolysis plays an important role in their transformation and nutrient removal in activated sludge wastewater treatment plants. However, there is no information available about the identity, ecophysiology, and abundance of starch-hydrolyzing organisms (SHOs) in these plants. In this study, fluorescence in situ enzyme staining with BODIPY fluorescein-labeled starch was applied and optimized to label SHOs expressing alpha-amylase in activated sludge plants. Fluorescence on the surface of bacteria expressing alpha-amylase activity was clearly visualized. In 11 full-scale nutrient-removing wastewater treatment plants examined, the morphotypes of the dominant SHOs were always cocci in clusters of tetrads, short rods in clusters, and some filamentous organisms. The SHOs were identified by combining in situ enzyme staining and FISH using a range of available oligonucleotide probes. All the SHOs observed were Actinobacteria, and most had the phenotype of polyphosphate-accumulating organisms closely related to the genus Tetrasphaera in the family Intrasporangiaceae. The SHOs were present in most of the wastewater treatment plants examined and comprised, in total, up to 11% of bacterial biovolume and thus formed an important part of the microbial communities.     

Permanent Presence of Grazing-Resistant Bacteria in a Hypertrophic Lake

The size structure of planktonic bacteria from a hypertrophic lake was investigated at 5- to 15-day intervals by means of a semiautomatic image analysis system during 1 year. Characteristic of this bacterial assemblage was the permanent presence of large filamentous bacteria and small cocci with cell sizes of <0.01 (mu)m(sup3). These filamentous bacteria, sometimes longer than 200 (mu)m and with cell volumes of up to 276 (mu)m(sup3), are larger than nanoflagellates (<20 (mu)m) and, even, metazoans living in the lake. Although they account for only 4 to 16% of bacterial abundance, their contribution to total bacterial biovolume was between 45 and 86%. An analysis of the food web structure indicates that this particular bacterial size structure may be the consequence of a strong bacterivory pressure by nanoflagellates and the absence of other larger bacterivores. The persistence of bacterial forms resistant to grazing has important consequences for the carbon flow within the microbial food web.     

Proportion of prokaryotes enumerated as viruses by epifluorescence microscopy

It is well known that there are prokaryotes small in size (e.g. ultra-microprokaryotes) that pass through a 0.2-μm filter. As bacterial and viral abundances are determined by epifluorescence microscopy and the differentiation between them is based on particle size, some bacteria can be erroneously enumerated as viruses, namely in marine waters where bacteria are small. However, there is no information on the proportion of prokaryotes that could be misidentified as viruses by epifluorescence microscopy. In this work, we assessed, in water samples collected in the estuarine system Ria de Aveiro (Portugal), the proportion of prokaryotes that could be counted as viruses by the current widespread epifluorescence microscopy and, for the first time, by fluorescence in situ hybridization (FISH). The total number of particles was determined on membranes of 0.2 and 0.02 μm after staining with 4′,6-diamidino-2-phenylindole (DAPI), and the number of prokaryotes (Bacteria and Archaea) was determined by FISH for both pore size membranes. The results show that, in the marine zone of the estuarine system, 28 % of particles enumerated as virus-like particles were prokaryotes, but, in the brackish water zone, only 13 % of the particles counted as viruses were actually prokaryotic cells. Epifluorescence microscopy overestimates viral abundance, and also the ratio viruses:prokaryotes, and this error must be taken into consideration because it can vary significantly within a system. In fact, in the marine zone of an estuarine system, the overestimation of viral abundance can be twice as high as in the brackish water zone.     

Bacterial aggregates in the tentacles of the sea anemone <Emphasis Type="Italic">Metridium senile</Emphasis>

This paper provides first information on organ-like bacterial aggregates in the tentacles of the sea anemone Metridium senile. The specimens were collected from waters near Helgoland (German Bight, North Sea) and the Orkney Islands. Tentacles were prepared for morphological inspection by light and scanning electron microscopy as well as for the phylogenetic analysis of endocytic bacteria. Bacterial aggregates are located in caverns of the tentacles’ epidermis. The aggregates are enwrapped in thin envelopes, which contain coccoid and/or rod-shaped tightly packed bacteria of different division states. Most of the bacterial cells are connected by fine filamentous structures. The phylogenetic determination is based on the sequence data of the 16S rDNA derived from tentacle material. Sequence analysis revealed three different subgroups of intratentacular proteobacteria. The dominant band, detected in all of the samples tested, showed a close relationship (98%) to a gram-negative Endozoicimonas elysicola. Two bands, only detected in tentacles of M. senile from Helgoland were assigned to Pseudomonas saccherophilia (99%), a knallgas bacterium, and to Ralstonia pickettii (100%). The bacteria represent a specific bacterial community. Their DGGE profiles do not correspond to the profiles of the planktonic bacteria generated from seawater close to the habitats of the anemones. The allocation of DNA sequences to the different morphotypes, their isolation, culturing and the elucidation of the physiological functions of intratentacular bacteria are in progress.     

Bloom of filamentous bacteria in a mesotrophic lake: identity and potential controlling mechanism

Ephemeral blooms of filamentous bacteria are a common phenomenon in the water column of oligo- to mesotrophic lakes. It is assumed that the appearance of such morphotypes is favored by selective predation of bacterivorous protists and that filter-feeding zooplankton plays a major role in suppressing these bacteria. The phylogenetic affiliation of the important bloom-forming filamentous bacteria in freshwaters is presently unknown. Here we report the identification of dominant members of a filamentous bacterial assemblage during a bloom of such morphotypes in a mesotrophic lake. By molecular cloning and fluorescence in situ hybridization with specific oligonucleotide probes, up to 98% of filamentous cells in lake water could be assigned to a clade of almost identical (99% similarity) 16S rRNA gene sequence types, the cosmopolitan freshwater LD2 cluster. For a period of less than 1 week, members of the LD2 clade constituted >40% of the total bacterial biomass, potentially favored by high grazing of planktivorous protists. This is probably the most pronounced case of dominance by a single bacterioplankton species ever observed in natural freshwaters. In enclosures artificially stocked with the metazoan filter feeder Daphnia, bacteria related to the LD2 clade formed a significantly larger fraction of filaments than in enclosures where Daphnia had been removed. However, in the presence of higher numbers of Daphnia individuals, the LD2 bacteria, like other filaments, were eventually eliminated both in enclosures and in the lake. This points at the potential importance of filter-feeding zooplankton in controlling the occurrence and species composition of filamentous bacterial morphotypes in freshwater plankton.     

Bloom of Filamentous Bacteria in a Mesotrophic Lake: Identity and Potential Controlling Mechanism

Ephemeral blooms of filamentous bacteria are a common phenomenon in the water column of oligo- to mesotrophic lakes. It is assumed that the appearance of such morphotypes is favored by selective predation of bacterivorous protists and that filter-feeding zooplankton plays a major role in suppressing these bacteria. The phylogenetic affiliation of the important bloom-forming filamentous bacteria in freshwaters is presently unknown. Here we report the identification of dominant members of a filamentous bacterial assemblage during a bloom of such morphotypes in a mesotrophic lake. By molecular cloning and fluorescence in situ hybridization with specific oligonucleotide probes, up to 98% of filamentous cells in lake water could be assigned to a clade of almost identical (99% similarity) 16S rRNA gene sequence types, the cosmopolitan freshwater LD2 cluster. For a period of less than 1 week, members of the LD2 clade constituted >40% of the total bacterial biomass, potentially favored by high grazing of planktivorous protists. This is probably the most pronounced case of dominance by a single bacterioplankton species ever observed in natural freshwaters. In enclosures artificially stocked with the metazoan filter feeder Daphnia, bacteria related to the LD2 clade formed a significantly larger fraction of filaments than in enclosures where Daphnia had been removed. However, in the presence of higher numbers of Daphnia individuals, the LD2 bacteria, like other filaments, were eventually eliminated both in enclosures and in the lake. This points at the potential importance of filter-feeding zooplankton in controlling the occurrence and species composition of filamentous bacterial morphotypes in freshwater plankton.     

Bacterial cell shape regulation: testing of additional predictions unique to the two-competing-sites model for peptidoglycan assembly and isolation of conditional rod-shaped mutants from some wild-type cocci.

The two-competing-sites model for peptidoglycan assembly for bacterial cell shape regulation suggests that in rods, bacterial cell shape depends on the balance between two reactions (sites), one responsible for lateral wall elongation and the other responsible for septum formation. The two reactions compete with each other so that no lateral wall can be formed during septum formation and vice versa. When the site for lateral wall elongation overcomes that for septum formation, long rods or filaments are formed and cell division may be blocked. When the reaction leading to septum formation is hyperactive compared with the other, coccobacilli or cocci are formed. Other bacteria carry only one site for peptidoglycan assembly and can grow only as cocci. The two-competing-sites model predicts that two different types of cocci exist (among both morphology mutants and wild-type strains); one carries only the site for septum formation, whereas the other also carries the site for lateral wall elongation, the former site predominating over the latter. As a consequence of the inhibition (by antibiotics or by mutations) of septum formation in wild-type cocci of various species and in coccoid morphology mutants, some cocci are expected to undergo transition to rod shape and others are not. We have evaluated these predictions and show that they are in agreement. In fact, we found that among wild-type cocci belonging to 13 species, those of 6 species formed rods, whereas the remaining organisms maintained their coccal shape when septa were inhibited by antibiotics. Some coccoid morphology mutants of rod-shaped bacteria underwent coccus-to-rod transition after septum inhibition by antibiotics, whereas others maintained their coccal shape. When a mutation that causes septum inhibition was expressed in a morphology mutant of Klebsiella pneumoniae grown as a coccus, transition to rod shape was observed. A total of 914 mutants unable to form colonies at 42 degrees C were isolated from the coccoid species mentioned above. Between 75 and 95% of the mutants isolated from the species that formed rods when septum formation was inhibited by antibiotics but none of those isolated from the others underwent coccus-to-rod transition upon incubation at the nonpermissive temperature.     

Ingestion of suspended bacteria by fish: a modified approach

The ingestion of suspended bacteria by common carp larvae and juvenile tilapias was quantified by counting of fluorescently labelled bacteria (FLB) in guts of experimental fish under an epifluorescence microscope.     

Investigations into the number of respiring bacteria in groundwater from sandy and gravelly deposits

Samples were collected from organically polluted and unpolluted groundwater of sandy and gravelly deposits. After filtration onto polycarbonate filters (0.2m pore size) the number of respiring bacteria was recorded by microscopically counting cells containing red INT-formazan spots, which characterize respiring bacteria. The total number of bacteria was simultaneously recorded by epifluorescence microscopy after staining with acridine orange. The number of respiring bacteria in the groundwater samples (55–490×10³/cm³) is within the range of values for other aquatic biotopes, but as the total number of bacteria in groundwater was usually higher, the proportion of respiring groundwater bacteria (0.66–7. 4%) was lower. Mainly larger bacteria, rods, and bacteria on particles could be identified as being active, whereas hardly any respiratory activity could be detected among small cocci and free interstitial bacteria. If the supply of dissolved organic matter (DOM) is adequate, the biomass of respiring bacteria correlates well with oxygen concentration, but there is no direct correlation between DOM concentration in groundwater and active bacterial biomass. Nor could any relationship be observed between the biomass of total and respiring bacteria, or between the quantity of respiring bacteria and heterotrophic bacterial activity.     

Estimates of protozoan- and viral-mediated mortality of bacterioplankton in Lake Bourget (France)

1. We performed three, 1‐week in situ experiments in March‐April (expt 1), May (expt 2) and August (expt 3) 2003 in order to assess protozoan and virus‐induced mortality of heterotrophic bacteria in a French lake. Viral and bacterial abundances were obtained using flow cytometry (FCM) while protozoa were counted using epifluorescence microscopy (EFM). 2. A dilution approach, applied to pretreated grazer‐free samples, allowed us to estimate that viral lysis could be responsible for 60% (expt 1), 35% (expt 2) and 52% (expt 3) of daily heterotrophic bacterial mortality. Flagellate (both mixotrophic and heterotrophic) grazing in untreated samples, was responsible for 56% (expt 1), 63% (expt 2) and 18% (expt 3) of daily heterotrophic bacteria removal. 3. These results therefore suggest that both viral lysis and flagellate grazing had a strong impact on bacterial mortality, and this impact varied seasonally. 4. From parallel transmission electron microscopy (TEM) analysis, we found that the burst size (i.e. the number of viruses potentially released per lysed cell) ranged from nine to 25 (expt 1), 10 to 35 (expt 2) and eight to 25 (expt 3). The percentage of infected heterotrophic bacteria was 5.7% (expt 1), 3.4% (expt 2) and 5.7% (expt 3) so that the calculated percentage of bacterial mortality induced by viruses was 6.3% (expt 1), 3.7% (expt 2) and 6.3% (expt 3). 5. It is clear that the dilution‐FCM and TEM methods yielded different estimates of viral impact, although both methods revealed an increased impact of viruses during summer.     

Sedimentation counting and morphology of Mycoplasma

Clark, Harold W. (The George Washington University School of Medicine, Washington, D.C.). Sedimentation counting and morphology of Mycoplasma. J. Bacteriol. 90:1373-1386. 1965.-The sedimentation technique for counting viral particles was applied to the quantitation and morphological identification of Mycoplasma in broth cultures. Mycoplasma, apparently in their native form, firmly adhered to the surface, when sedimented on glass cover slips or onto electron microscope grids. The sedimented cover slip preparations stained with crystal violet could be readily counted in the light microscope. The cultures sedimented onto electron microscope grids were readily counted at low magnification and provided excellent preparations for morphological examination at higher magnifications. It was found that air-dried Mycoplasma particles were enlarged considerably because of excessive flattening. Fixation of sedimented Mycoplasma particles in diluted OsO(4) prior to air drying yielded a more realistic morphology, with various sizes and shapes in the stages of the growth cycle exhibited. A new technique of differentially staining Mycoplasma colonies on agar plates was developed to facilitate the quantitation of viable colony-forming units for comparison with total counts. The use of plastic or Parafilm gaskets for dry mounting was developed to facilitate the handling and examination of the stained cover slip preparations. The results of this investigation indicated that the growth cycle of some Mycoplasma species includes a stage of hexadic fission with the cleavage of minimal reproductive units (less than 100 mmu) containing a limited deoxyribonucleic acid genetic coding molecule (approximately 4 x 10(6)).     

SEASONALITY OF MICROBIAL FOULING ON ASCOPHYLLUM NODOSUM (L.) LEJOL., FUCUS VESICULOSUS L., POLYSIPHONIA LANOSA (L.) TANDY AND CHONDRUS CRISPUS STACKH1

The nature and seasonal extent of microbial fouling on Ascophyllum nodosum (L.) LeJol., Fucus vesiculosus L., Polysiphonia lanosa (L.) Tandy and Chondrus crispus Stackh. were observed by scanning electron microscopy. Bacterial filaments and smaller rod and coccoid forms dominated the fouling communities on all species, with pennate diatoms constituting a minor component in contrast to results with plastic substrates on which pennate diatoms dominated and preceded bacterial colonization. The total percent microbial coverage on the surfaces of all four seaweed species was determined by monthly stereological analyses of representative composite micrographs. These showed a simultaneous decline between April and May which could represent the die-off of the cold water bacterial flora when water temperature increased past the threshold for obligate psychrophiles. Microbial colonization patterns were directly correlated (P = 0.005) with maximum coverage in April and November–December and reduced levels from May to October. Significant inverse (P < 0.041) correlations between total percent coverage and water temperature indicate distinct seasonal cycles, however, the patterns of dominance by filamentous bacteria and rod and coccoid forms were markedly different. Total coverage patterns of both rhodophytes showed no apparent seasonal cycle and were not related to water temperature. Rod and coccoid bacteria were apparently suppressed year round on P. lanosa relative to the other species. These interspecific differences in seasonal fouling patterns are discussed in light of possible modes of regulation, especially algal antibiosis.     

Determination of bacterial cell number and cell volume by means of flow cytometry, transmission electron microscopy, and epifluorescence microscopy

Comparative measurements of bacterial total counts and volumes of flow cytometry (FCM), transmission electron (TEM), and epifluorescence microscopy (EFM), were undertaken during a four week mesocosm experiment. Total counts of bacteria measured by TEM, EFM, and FCM were in the range of 1 · 10⁶−6 cells ml⁻¹, 1 · 10⁶−3 · 10¹⁶ cells ml⁻¹, and 5 · 10⁵ cells ml⁻¹ respectively. The mean volume of the bacterial community, measured by means of EFM and TEM, increased from 0.12–0.15 μm³ at the start of the experiment to 0.39–0.53 μm³ at the end. Generally, there was good agreement between the two methods and regression analyses gave r = 0.87 (p < < 0.01) for cell volume and r = 0.97 (p < < 0.01) for cell number. DAPI stained bacteria with volumes less than 0.2 μm³ were not detected by flow cytometry and these were generally an order of magnitude lower than counts made by TEM and EFM. For samples where the mean bacterial cell volume was longer than 0.3 μm³, all three methods were in agreement both with respect to counts and volume estimates.     

Transient development of filamentous Thiothrix species in a marine sulfide oxidizing, denitrifying fluidized bed reactor

In this study, microscopic and molecular microbial analyses were integrated to characterize rapidly developing white filamentous tufts in a fluidized bed reactor used for nitrate removal from a marine recirculating fish culture system. Formation and rapid elongation of the tufts (often exceeding 50 mm day (-1)) was strongly correlated to transient elevated sulfide concentrations (>50 microM) in the reactor. The dominant bacterial constituents of these tufts were filamentous gram-negative bacteria with densely packed intracellular sulfur granules. Using 16S rRNA gene analysis and fluorescence in situ hybridization it was found that these filamentous bacteria represented a novel Thiothrix phylotype closely related (97% sequence identity) to a previously identified Thiothrix strain endogenous to the marine crustacean Urothoe poseidonis. In addition to filamentous morphotypes, rosette-shaped morphotypes of Thiothrix were also detectable within the tufts.