[Show abstract][Hide abstract] ABSTRACT: In Lake Zürich, a deep subalpine mesotrophic lake, phosphate was low or limiting at 0.2 to 1 μM relative to combined nitrogen at 50 μM. Heterotrophic bacteria were responsible for 53% of the observed microbial wet biomass in our depth profile while phytoplankton, largely Planktothrix (Oscillatoria) rubescens, contributed most of the remainder. Most cell carbon was contributed by this carbon-sufficient cyanobacterium. A material balance indicated that most of the phosphate was sequestered by the bacteria due to a higher phosphate content and specific affinity for this nutrient. Size distributions of the heterotrophic bacteria were narrow; 90% of organisms were from 0.06 to 0.06 μ3 in volume. Several subpopulations of bacteria were resolved by flow cytometry, and bivariate fluorescence (DAPI-DNA) and light scatter (cell-size) histogram profiles varied with depth. One or two of these subpopulations appeared to be bacteria with sufficient cytoplasmic constituents to produce a normal light-scatter signal but retained only a small amount of DNA; an apparent content of 200 kbp or 5% of a usual oligobacterial genome. These helped increase the oligobacterial population to 6 × 106 ml−1. Application of published specific affinities and measured nutrient concentrations to formulations of system kinetics led to the conclusion that growth rates of the heterotrophic bacterial fraction were carbon limited with cell size, and thus populations were controlled by grazing. The depth profile indicated that phototrophs affected concentrations in a significant way. Considerations of nutrient uptake kinetics suggested that much potential capacity remained in the dissolved phosphate pool to support additional phytoplanktonic biomass. Computations led to the conclusion that, if phosphate is generally limiting in lakes, then additional mechanisms exist which limit populations of phytoplankton to sufficiently small values to allow phosphate accumulation to observed levels. Bacterial biomass then depends on the organic carbon from these phosphate-controlled organisms.
[Show abstract][Hide abstract] ABSTRACT: A theory for solute uptake by whole cells was derived with a focus on the ability of oligobacteria to sequester nutrients. It provided a general relationship that was used to obtain the kinetic constants for in situ marine populations in the presence of naturally occurring substrates. In situ affinities of 0.9 to 400 liters g of cells(-1) h(-1) found were up to 10(3) times smaller than those from a "Marinobacter arcticus " isolate, but springtime values were greatly increased by warming. Affinities of the isolate for usual polar substrates but not for hydrocarbons were diminished by ionophores. A kinetic curve or Monod plot was constructed from the best available data for cytoarchitectural components of the isolate by using the theory together with concepts and calculations from first principles. The order of effect of these components on specific affinity was membrane potential > cytoplasmic enzyme concentration > cytoplasmic enzyme affinity > permease concentration > area of the permease site > translation coefficient > porin concentration. Component balance was influential as well; a small increase in cytoplasmic enzyme concentration gave a large increase in the effect of permease concentration. The effect of permease concentration on specific affinity was large, while the effect on K(m) was small. These results are in contrast to the Michaelis-Menten theory as applied by Monod that has uptake kinetics dependent on the quality of the permease molecules, with K(m) as an independent measure of affinity. Calculations demonstrated that most oligobacteria in the environment must use multiple substrates simultaneously to attain sufficient energy and material for growth, a requirement consistent with communities largely comprising few species.
[Show abstract][Hide abstract] ABSTRACT: These protocols were developed for the quantitative analysis of DNA in aquatic bacteria and have been used to characterize
both marine and freshwater samples and cultures (1). Apparent DNA content is a valuable tool when used in conjunction with forward light scatter for biomass (2–4) to characterize heterotrophic bacterioplankton, organisms which are too small for observation by light microscopy and difficult
to cultivate (5).
[Show abstract][Hide abstract] ABSTRACT: This unit presents methods for measuring biomass of bacteria by flow cytometry. Such measurements are an integral part of determining growth rates and kinetics. This unit discusses calibration and standardization methods for bacterial studies using flow cytometry and provides a detailed discussion of the components involved. The serious microbiologist will appreciate the advantages flow cytometry brings to many aspects of microbial study, and this unit is no exception.
Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 06/2001; Chapter 11:Unit 11.9. DOI:10.1002/0471142956.cy1109s09
[Show abstract][Hide abstract] ABSTRACT: The distribution of DNA among bacterioplankton and bacterial isolates was determined by flow cytometry of DAPI (4',6'-diamidino-2-phenylindole)-stained organisms. Conditions were optimized to minimize error from nonspecific staining, AT bias, DNA packing, changes in ionic strength, and differences in cell permeability. The sensitivity was sufficient to characterize the small 1- to 2-Mb-genome organisms in freshwater and seawater, as well as low-DNA cells ("dims"). The dims could be formed from laboratory cultivars; their apparent DNA content was 0.1 Mb and similar to that of many particles in seawater. Preservation with formaldehyde stabilized samples until analysis. Further permeabilization with Triton X-100 facilitated the penetration of stain into stain-resistant lithotrophs. The amount of DNA per cell determined by flow cytometry agreed with mean values obtained from spectrophotometric analyses of cultures. Correction for the DNA AT bias of the stain was made for bacterial isolates with known G+C contents. The number of chromosome copies per cell was determined with pure cultures, which allowed growth rate analyses based on cell cycle theory. The chromosome ratio was empirically related to the rate of growth, and the rate of growth was related to nutrient concentration through specific affinity theory to obtain a probe for nutrient kinetics. The chromosome size of a Marinobacter arcticus isolate was determined to be 3.0 Mb by this method. In a typical seawater sample the distribution of bacterial DNA revealed two major populations based on DNA content that were not necessarily similar to populations determined by using other stains or protocols. A mean value of 2.5 fg of DNA cell(-1) was obtained for a typical seawater sample, and 90% of the population contained more than 1.1 fg of DNA cell(-1).
[Show abstract][Hide abstract] ABSTRACT: Expanded specific affinity theory specifies the advantages of small dry mass and dilute cytoarchitecture in impoverished systems. For marine samples, bacterioplankton mean dry mass, according to flow cytometry, was near 24.5 fg cell-1. Conversion to volume with buoyant density gave a cell volume of 0.124 μm3. Total DNA was 2.9 fg cell-1. This compared with the size of a single genome of a small extinction-culture isolate, Sphingomonas sp. RB2256, of 3.96 fg or 3.6 Mb. The genome size of such isolates and other cultures decreased with metabolic simplicity. It was found that bacterioplankton could be exposed to radiolabeled amino acids and then sorted for size and that the specific affinities of the fraction of small organisms were as great as the fraction of large cells. Size, DNA, and metabolic-complexity distributions were concordant with the concepts that cell volume approaches a minimum set by sufficient space for the smallest genome that can provide sufficient information for competitive dissolved- nutrient acquisition, and that space requirements are further alleviated by the expression of few cytoplasmic-enzyme molecules in each of the various pathways and a dilute cytoplasm. Bacterioplankton approached a minimum genome size of 1.7 Mb with a minimum cell volume of about 0.06 μ m3 and a DNA content of 16% dry weight. The property of small dry mass with a low DNA content was common in in situ bacteria but absent from cultivated representatives, which led to speculation that failure to grow in the laboratory is related to missing regulatory information.
Limnology and Oceanography 03/2000; 45(2):499-505. DOI:10.4319/lo.2000.45.2.0499 · 3.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dilutions of raw seawater produced a bacterial isolate capable of extended growth in unamended seawater. Its 2.9-Mb genome size and 40-fg dry mass were similar to values for many naturally occurring aquatic organotrophs, but water and DNA comprised a large portion of this small chemoheterotroph, as compared to Escherichia coli. The isolate used only a few aromatic hydrocarbons and acetate, and glucose and amino acid incorporation were entirely absent, although many membrane and cytoplasmic proteins were inducible; it was named Cycloclasticus oligotrophus. A general rate equation that incorporates saturation phenomena into specific affinity theory is derived. It is used to relate the kinetic constants for substrate uptake by the isolate to its cellular proteins. The affinity constant KA for toluene was low at 1.3 microg/liter under optimal conditions, similar to those measured in seawater, and the low value was ascribed to an unknown slow step such as limitation by a cytoplasmic enzyme; KA increased with increasing specific affinities. Specific affinities, a degreess, were protocol sensitive, but under optimal conditions were 47.4 liters/mg of cells/h, the highest reported in the literature and a value sufficient for growth in seawater at concentrations sometimes found. Few rRNA operons, few cytoplasmic proteins, a small genome size, and a small cell size, coupled with a high a degreess and a low solids content and the ability to grow without intentionally added substrate, are consistent with the isolation of a marine bacterium with properties typical of the bulk of those present.
Applied and Environmental Microbiology 12/1998; 64(11):4467-76. · 3.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The forward light scatter intensity of bacteria analyzed by flow cytometry varied with their dry mass, in accordance with theory. A standard curve was formulated with Rayleigh-Gans theory to accommodate cell shape and alignment. It was calibrated with an extinction-culture isolate of the small marine organism Cycloclasticus oligotrophus, for which dry weight was determined by CHN analysis and 14C-acetate incorporation. Increased light scatter intensity due to formaldehyde accumulation in preserved cells was included in the standard curve. When differences in the refractive indices of culture media and interspecies differences in the effects of preservation were taken into account, there was agreement between cell mass obtained by flow cytometry for various bacterial species and cell mass computed from Coulter Counter volume and buoyant density. This agreement validated the standard curve and supported the assumption that cells were aligned in the flow stream. Several subpopulations were resolved in a mixture of three species analyzed according to forward light scatter and DNA-bound DAPI (4', 6-diamidino-2-phenylindole) fluorescence intensity. The total biomass of the mixture was 340 &mgr;g/liter. The lowest value for mean dry mass, 0.027 +/- 0.008 pg/cell, was for the subpopulation of C. oligotrophus containing cells with a single chromosome. Calculations from measurements of dry mass, Coulter Counter volume, and buoyant density revealed that the dry weight of the isolate was 14 to 18% of its wet weight, compared to 30% for Escherichia coli. The method is suitable for cells with 0.005 to about 1.2 pg of dry weight at concentrations of as low as 10(3) cells/ml and offers a unique capability for determining biomass distributions in mixed bacterial populations.
Applied and Environmental Microbiology 11/1998; 64(10):3900-9. · 3.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Forward scatter signals (0–20°) from a flow cytometer can be converted into cellular volumes or cellular wet or dry biomasses by the calculations presented here. The results are applicable to bacteria, and including very small bacteria (0.02–0.6 μm3), rods as well as cocci. The physical limitations and the mathematical basis of the calculations are discussed to justify the application of the proposed formula for practical use in microbial ecology and physiology of extremely small aquatic bacteria as well as the more well-studied forms. Although the method was developed for the small bacteria present in natural populations that are difficult to size by alternative methods, it will also be useful for laboratory cultures of bacteria when present in very low concentrations as well as for the characterization of subpopulations and different growth phases. Bacteria, in general, are small enough so that inclusion of the phase shifts of the light passing through the bacteria (the Mie theory) is superfluous. That theory is, however, useful for instrumental calibration with latex microspheres and for some blue-green algae. Even the interference between rays scattered from different parts of the cell (the Rayleigh-Gans theory), although significant, is minor, particularly from elongated particles which are oriented by the fluidics. The computer programs, provided here, correct for intraparticle interference and generate cell volumes (or wet or dry masses) from the forward scatter intensity. The result of the calculations of the light scattered into the collection system of the flow cytometer is presented. From analysis of these values a rule is developed to relate the observed intensities, when scaled by an empirical constant and raised to a power between 0.5 and 0.75, to the cell biomass.
[Show abstract][Hide abstract] ABSTRACT: Marine bacteria in Resurrection Bay near Seward, Alaska, and in the central North Sea off the Dutch coast were cultured in filtered autoclaved seawater following dilution to extinction. The populations present before dilution varied from 0.11 x 10 to 1.07 x 10 cells per liter. The mean cell volume varied between 0.042 and 0.074 mum, and the mean apparent DNA content of the cells ranged from 2.5 to 4.7 fg of DNA per cell. All three parameters were determined by high-resolution flow cytometry. All 37 strains that were obtained from very high dilutions of Resurrection Bay and North Sea samples represented facultatively oligotrophic bacteria. However, 15 of these isolates were eventually obtained from dilution cultures that could initially be cultured only on very low-nutrient media and that could initially not form visible colonies on any of the agar media tested, indicating that these cultures contained obligately oligotrophic bacteria. It was concluded that the cells in these 15 dilution cultures had adapted to growth under laboratory conditions after several months of nutrient deprivation prior to isolation. From the North Sea experiment, it was concluded that the contribution of facultative oligotrophs and eutrophs to the total population was less than 1% and that while more than half of the population behaved as obligately oligotrophic bacteria upon first cultivation in the dilution culture media, around 50% could not be cultured at all. During one of the Resurrection Bay experiments, 53% of the dilution cultures obtained from samples diluted more than 2.5 x 10 times consisted of such obligate oligotrophs. These cultures invariably harbored a small rod-shaped bacterium with a mean cell volume of 0.05 to 0.06 mum and an apparent DNA content of 1 to 1.5 fg per cell. This cell type had the dimensions of ultramicrobacteria. Isolates of these ultramicrobacterial cultures that were eventually obtained on relatively high-nutrient agar plates were, with respect to cell volume and apparent DNA content, identical to the cells in the initially obligately oligotrophic bacterial dilution culture. Determination of kinetic parameters from one of these small rod-shaped strains revealed a high specific affinity for the uptake of mixed amino acids (a degrees (A), 1,860 liters/g of cells per h), but not for glucose or alanine as the sole source of carbon and energy (a degrees (A), +/- 200 liters/g of cells per h). The ultramicrobial strains obtained are potentially a very important part of picoplankton biomass in the areas investigated.
Applied and Environmental Microbiology 08/1993; 59(7):2150-60. · 3.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dilution culture, a method for growing the typical small bacteria from natural aquatic assemblages, has been developed. Each of 11 experimental trials of the technique was successful. Populations are measured, diluted to a small and known number of cells, inoculated into unamended sterilized seawater, and examined three times for the presence of 10 or more cells per ml over a 9-week interval. Mean viability for assemblage members is obtained from the frequency of growth, and many of the cultures produced are pure. Statistical formulations for determining viability and the frequency of pure culture production are derived. Formulations for associated errors are derived as well. Computer simulations of experiments agreed with computed values within the expected error, which verified the formulations. These led to strategies for optimizing viability determinations and pure culture production. Viabilities were usually between 2 and 60% and decreased with >5 mg of amino acids per liter as carbon. In view of difficulties in growing marine oligobacteria, these high values are noteworthy. Significant differences in population characteristics during growth, observed by high-resolution flow cytometry, suggested substantial population diversity. Growth of total populations as well as of cytometry-resolved subpopulations sometimes were truncated at levels of near 10 cells per ml, showing that viable cells could escape detection. Viability is therefore defined as the ability to grow to that population; true viabilities could be even higher. Doubling times, based on whole populations as well as individual subpopulations, were in the 1-day to 1-week range. Data were examined for changes in viability with dilution suggesting cell-cell interactions, but none could be confirmed. The frequency of pure culture production can be adjusted by inoculum size if the viability is known. These apparently pure cultures produced retained the size and apparent DNA-content characteristic of the bulk of the organisms in the parent seawater. Three cultures are now available, two of which have been carried for 3 years. The method is thus seen as a useful step for improving our understanding of typical aquatic organisms.
Applied and Environmental Microbiology 03/1993; 59(3):881-91. · 3.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Turnover times for toluene in Resurrection Bay after the Exxon Valdez grounding were determined to be decades, longer than expected considering that dissolved hydrocarbons were anticipated to drift with the current and stimulate development of additional hydrocarbon-utilizing capacity among the microflora in that downcurrent location. These turnover times were based on the recovery of 14CO2 from added [14C]toluene that was oxidized. The concentrations of toluene there, 0.1 to 0.2 microgram/liter, were similar to prespill values. Oxidation rates appeared to be enhanced upstream near islands in the wake of the wind-blown slick, and even more within the slick itself. Specific affinities of the water column bacteria for toluene were computed with the help of biomass data, as measured by high-resolution flow cytometry. They were a very low 0.3 to 3 liters/g of cells.h-1, indicating limited capacity to utilize this hydrocarbon. Since current-driven mixing rates exceeded those of oxidation, dissolved spill components such as toluene should enter the world-ocean pool of hydrocarbons rather than biooxidize in place. Some of the floating oil slick washed ashore and permeated a coarse gravel beach. A bacterial biomass of 2 to 14 mg/kg appeared in apparent response to the new carbon and energy source. This biomass was computed from that of the organisms and associated naphthalene oxidation activity washed from the gravel compared with the original suspension. These sediment organisms were very small at approximately 0.06 microns 3 in volume, low in DNA at approximately 5.5 g per cell, and unlike the aquatic bacteria obtained by enrichment culture but quite similar to the oligobacteria in the water column.(ABSTRACT TRUNCATED AT 250 WORDS)
Applied and Environmental Microbiology 02/1992; 58(1):243-51. · 3.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The two primary kinetic constants for describing the concentration dependency of nutrient uptake by microorganisms are shown to be maximal rate of substrate uptake and, rather than the Michaelis constant for transport, specific affinity. Of the two, the specific affinity is more important for describing natural aquatic microbial processes because it can be used independently at small substrate concentrations. Flow cytometry was used to evaluate specific affinities in natural populations of aquatic bacteria because it gives a convenient measure of biomass, which is an essential measurement in the specific-affinity approach to microbial kinetics. Total biomass, biomass in various filter fractions, and the specific affinity of the bacteria in each fraction were determined in samples from a near-arctic lake. The partial growth rate of the pelagic bacteria from the 25 micrograms/liter of dissolved amino acids present (growth rate from the amino acid fraction alone) was determined to be 0.78 per day. By measuring activity in screened and whole-system populations, the biomass of the bacteria associated with particles was computed to be 427 micrograms/liter.
[Show abstract][Hide abstract] ABSTRACT: Selected features of Alaria marginata Postels et Ruprecht, Laminaria saccharina (L.) Lamouroux and Cymathere triplicata (Postels et Ruprecht) J. G. Agardh meiospores were described using flow cytometry. The relative sizes (forward scatter), chlorophyll contents (red fluorescence) and DNA contents (blue fluorescence) were measured on living, fixed, Hoechst and DAPI (4,6-diamidino-2-phenylindole) stained and unstained cells. The meiospores were similar among species and the frequency distributions of the monitored features were essentially normal. Meiospores sorted for low blue fluorescence departed significantly from the expected 1:1 male to female ratio in favour of the male. Cells sorted for high blue fluorescence did not result in a departure from the expected 1:1 ratio. We suggest that our ability to sexually sort meiospores is a result of a differential nonstoichiometric fluorescence in DNA, which possibly reflects a greater DNA compactedness and/or nuclear protein content in the male meiospores.
[Show abstract][Hide abstract] ABSTRACT: Flow cytometry offers a rapid method for characterizing aquatic populations according to the properties of individual cells. This technology has been extended to aquatic bacteria by using high-intensity UV excitation, condensing the laser beam onto a small area, using blemish-free flow cells, optimizing organism staining protocol, segregating the optical signal produced with high-transmittance optical filters collecting the signal with sensitive photomultipliers, and expanding the range of data displayed from individual samples with calibrated circuitry. Bacteria could be counted according to event frequency, and populations agreed with direct counts by epifluorescence microscopy. Forward scatter intensity was a linear function of volume for bacterial cells between 1.3 and 0.25 μm3 as calibrated by coulter impedance. Plastic spheres down to 0.014 μm3, 0.3 μm in diameter, were resolved. Aquatic bacteria 0.05 μm3 in volume were clearly resolved according to DNA content by staining with DAPI. The observed signal was DNA-dependent because DNase treatment eliminated most fluorescence.
These procedures are suitable for direct analysis of the bacteria in marine and freshwater samples without interference from algae, sediment, or most DNA-free organic particles. Cytograms indicated one or more clearly resolved subpopulations of bacteria of substantially smaller size and DNA content than the laboratory organisms typically classified.
[Show abstract][Hide abstract] ABSTRACT: The kinetics of concentration-dependent toluene metabolism were examined by evaluating each term in the second-order rate equation. Marine and freshwater pseudomonads were used. Uptake for Pseudomonas sp. strain T2 was characterized by a completely saturatable system with small transport constant (K(t) = 44 mug/liter) and large specific affinity. Kinetics for Pseudomonas putida PpF1 were similar. Induction had little effect on K(t), but it caused the specific affinity to increase from about 0.03 to 320 liters/g of cells per h. The level of induction depended on the time of exposure, the concentration of inducer, and the initial level of induction. If loss of the inducible system was not severe, toluene caused a linear increase in specific affinity with time, and the maximal value achieved at intermediate times (1 to 3 days) was hyperbolic with concentration when K(ind) was 96 mug/liter (A. T. Law and D. K. Button, Appl. Environ. Microbiol. 51:469-476, 1986). As repression became complete, specific affinities were greatly reduced. Then induction required higher toluene concentrations and longer times, and the shape of the specific-affinity curve became sigmoidal with concentration. Cell yields (0.10 to 0.17 g of cells per g of toluene used) were low owing to liberation of organic products: 2-hydroxy-6-oxohepta-2,4-dienoic acid, toluene dihydrodiol, 3-methylcatechol, acetate, formate, and possibly pyruvate, which in turn caused lower rates of growth. Michaelis constants for the reaccumulation of products exceeded those for toluene, but specific affinities were lower and maximal velocities were higher, so that recycling was favored in cultures with high toluene concentration. Although these kinetics predict deviation from the linear relationship between uptake rate and biomass, we could detect none. Effects of saturation and induction were incorporated into the basic specific-affinity relationship. The result appears to be an improvement in the equation used for describing the kinetics of uptake and growth.
Applied and Environmental Microbiology 10/1987; 53(9):2193-205. · 3.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The continuous addition of toluene as a solute of treated ballast water from oil tankers into a well-defined estuary facilitated the study of the dynamics of dissolved hydrocarbon metabolism in seawater. Most rates of toluene oxidation were in the range of 1 to 30 pg/liter per h at 0.5 mug of toluene per liter. Near the ballast water injection point, a layer of warm ballast water, rich in bacteria, that was trapped below the less-dense fresh surface water was located. Toluene residence times were approximately 2 weeks in this layer, 2 years elsewhere in Port Valdez, and 2 decades in the surface water of a more oceanic receiving estuary adjacent. Mixing was adequate for a steady-state treatment which showed that 98% of the toluene was flushed from Port Valdez before metabolism and gave a steady-state concentration of 0.18 mug/liter. Total bacterial biomass from direct counts and organism size data was usually near 0.1 mg/liter, but ranged up to 0.8 mg/liter in the bacteria-rich layer. The origin of bacteria in this layer was traced to growth in oil tanker ballast during shipments. The biomass of toluene oxidizers in water samples was estimated from the average affinity of pure-culture isolates for toluene (28 liters per g of cells per h) and observed toluene oxidation kinetics. Values ranged from nearly all of the total bacterial biomass within the bacteria-rich layer down to 0.2% at points far removed. Because the population of toluene oxidizers was large with respect to the amount of toluene consumed and because water from a nearby nonpolluted estuary was equally active in facilitating toluene metabolism, we searched for an additional hydrocarbon source. It was found that terpenes could be washed from spruce trees by simulated rainfall, which suggested that riparian conifers provide an additional and significant hydrocarbon source to seawater.
Applied and Environmental Microbiology 11/1981; 42(4):708-19. · 3.67 Impact Factor