Radiometric Method for the Detection of Coliform Organisms in Water

A new radiometric method for the detection of coliform bacteria in water has been described. The method is based on the release of ¹⁴CO₂ from [¹⁴C]lactose by bacteria suspended in growth medium and incubated at 37 C. The evolved ¹⁴CO₂ is trapped by hyamine hydroxide and counted in a liquid scintillation spectrometer. The method permits the detection of 1 to 10 organisms within 6 h of incubation. Coliform bacteria suspended in water for several days recover from starvation and may be quantitated by the proposed method. Bacteria from water samples may also be concentrated by filtration through membrane filters and detected by the radiometric assay.     

Improved multiplex-PCR method for the simultaneous detection of food bacteria producing biogenic amines

This study describes a simple and rapid multiplex-PCR method to determine the ability to produce histamine, tyramine and putrescine by bacteria. The assay is an improved method based on an assay designed for lactic acid bacteria. This improved method includes a pair of primers based on sequences from histidine decarboxylases from Gram-negative bacteria. Under the optimised conditions, the assay yielded a 367-bp DNA fragment from histidine decarboxylases of Gram-positive bacteria, 534-bp fragment from histidine decarboxylases of Gram-negative bacteria, 924-bp from bacterial tyrosine decarboxylases, and 1446-bp fragment from bacterial ornithine decarboxylases. The method was successfully applied to several biogenic amine-producing bacterial strains, even when DNAs of several target organisms were included in the same reaction. This simple method could be easily incorporated in food control laboratories to detect potentially biogenic amine-producing bacteria in foods.     

The rate of killing of Escherichia coli by beta-lactam antibiotics is strictly proportional to the rate of bacterial growth

Nongrowing bacteria evade the bactericidal activity of beta-lactam antibiotics. We sought to determine if slow growth rate also alters bactericidal activity. The bactericidal activity of two beta-lactams on Escherichia coli grown in glucose limited chemostats was compared for generation times ranging from 0.7 to 12 h. The degree of killing varied with drug structure and with E. coli strain. However, all killing rates were a constant function of the bacterial generation time: slowly growing bacteria became progressively more phenotypically tolerant to beta-lactam antibiotics as the generation time was extended.     

Predicted steady-state cell size distributions for various growth models

The question of how an individual bacterial cell grows during its life cycle remains controversial. In 1962 Collins and Richmond derived a very general expression relating the size distributions of newborn, dividing and extant cells in steady-state growth and their growth rate; it represents the most powerful framework currently available for the analysis of bacterial growth kinetics. The Collins-Richmond equation is in effect a statement of the conservation of cell numbers for populations in steady-state exponential growth. It has usually been used to calculate the growth rate from a measured cell size distribution under various assumptions regarding the dividing and newborn cell distributions, but can also be applied in reverse--to compute the theoretical cell size distribution from a specified growth law. This has the advantage that it is not limited to models in which growth rate is a deterministic function of cell size, such as in simple exponential or linear growth, but permits evaluation of far more sophisticated hypotheses. Here we employed this reverse approach to obtain theoretical cell size distributions for two exponential and six linear growth models. The former differ as to whether there exists in each cell a minimal size that does not contribute to growth, the latter as to when the presumptive doubling of the growth rate takes place: in the linear age models, it is taken to occur at a particular cell age, at a fixed time prior to division, or at division itself; in the linear size models, the growth rate is considered to double with a constant probability from cell birth, with a constant probability but only after the cell has reached a minimal size, or after the minimal size has been attained but with a probability that increases linearly with cell size. Each model contains a small number of adjustable parameters but no assumptions other than that all cells obey the same growth law. In the present article, the various growth laws are described and rigorous mathematical expressions developed to predict the size distribution of extant cells in steady-state exponential growth; in the following paper, these predictions are tested against high-quality experimental data.     

Denitrification and a numerical modelling approach for shallow waters

A numerical model (`DiffDeni') has been developed to describe the disappearance of nitrate from the water column of 10–200 cm deep waters. The disappearance is caused by bacterial denitrification in the sediments. The model employs the molecular diffusion constant, an acceleration factor describing eddy diffusion, and three bacterial growth constants, viz. the inoculum size, the maximum growth rate and the half saturation constant for the hyperbolic process. The values of these system-constants were varied over a wide range. The curves obtained were compared with the curves for well-defined situations, viz. in which diffusion takes place without any or with a complete, immediate reaction. These cases have analytical solutions, and were simulated closely by the model `DiffDeni', though this model is based on different assumptions. It is shown that, when the bacterial growth rate is above a critical value, a negative exponential curve describes the nitrate disappearance well. On the other hand, a more complicated negative exponential equation can be used to describe the first phase of this denitrification in which bacterial activity is low and nitrate behaves as a conservative compound. The change-over period from phase 1 (no reaction) to phase 2 (complete, immediate reaction) which may vary between <1 and 50 days cannot be described analytically (mathematically correctly). The influence of temperature on denitrification is assessed and it is shown that both bacterial activity and diffusion may influence the denitrification rate.     

Inferences about human demography based on multilocus analyses of noncoding sequences

Data from 10 unlinked autosomal noncoding regions, resequenced in 15 individuals from each of three populations, were used in a multilocus analysis to test models of human demography. Each of the 10 regions consisted of approximately 2500 bp. The multilocus analysis, based on summary statistics (average and variance of Tajima's D and Fu and Li's D*), was used to test a family of models with recent population expansion. The African sample (Hausa of Cameroon) is compatible with a constant population size model and a range of models with recent expansion. For this population sample, we estimated confidence sets that showed the limited range of parameter values compatible with growth. For an exponential growth rate as low as 1 x 10(-3)/generation, population growth is unlikely to have started prior to 50,000 years ago. For higher growth rates, the onset of growth must be more recent. On the basis of the average value of Tajima's D, our sample from an Italian population was found to be incompatible with a constant population size model or any simple expansion model. In the Chinese sample, the variance of Tajima's D was too large to be compatible with the constant population size model or any simple expansion model.     

Comparison of methods for measurement of bacterial growth rates in mixed batch cultures.

Eight methods of assessing growth rate constants of bacteria were compared in batch cultures of 3-micrometers-filtered estuarine water from the Skidaway River in Ga. Mixed assemblages of bacteria were grown under four nutrient regimes of added yeast extract ranging from 0 to 100 mg/liter. Linear and exponential growth rate constants were computed from changes in cell densities, biovolumes, and ATP concentrations. Exponential growth rate constants were obtained from the frequency of dividing cells and RNA synthesis as measured by [3H]adenine uptake. Rate constants obtained during lag, exponential, and stationary growth phases depended largely on the method used. Constants calculated from changes in cell densities, frequency of dividing cells, and adenine uptake correlated most closely with each other, whereas constants calculated from changes in ATP concentrations and biovolumes correlated best with each other. Estimates of in situ bacterial productivity and growth vary depending on the method used and the assumptions made regarding the growth state of bacteria.     

Liquid Scintillation Vial for Cumulative and Continuous Radiometric Measurement of In Vitro Metabolism

A two-compartment vial is described in which suspensions of bacteria, cells, or tissues may be cultured and their growth and metabolism measured radiometrically by using a liquid scintillation counter. The device consists of a scintillation vial lined with a cylinder of scintillating paper into which is placed a sterilized inner culture vial containing a carbon-14 substrate. The assembled device can be carried by the sample transport systems of conventional liquid scintillation counters. Evolved (14)CO(2) is collected and measured cumulatively and continuously. The device can be constructed simply and economically from readily available reagents and glassware. Data are given on relative sensitivity and on the effect of the color and transparency of the inner vial. A pilot experiment with bacteria (Escherichia coli) is described.     

Chemosensory Responses of Acanthamoeba castellanii: Visual Analysis of Random Movement and Responses to Chemical Signals

A visual assay slide chamber was used in conjunction with time-lapse videomicroscopy to analyze chemotactic behavior of axenically grown Acanthamoeba castellanii. Data were collected and analyzed as vector scatter diagrams and cell tracks. Amebas responded to a variety of bacterial products or potential bacterial products by moving actively toward the attractant. Responses to the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP), lipopolysaccharide, and lipid A were statistically significant (P≤ 0.03), as was the response to fMLP benzylamide (P≤ 0.05). Significant responses to cyclic AMP, lipoteichoic acid, and N-acetyl glucosamine were also found. Chemotactic peptide antagonists, mannose, mannosylated bovine serum albumin, and N-acetyl muramic acid all yielded nonsignificant responses (P > 0.05). There was no single optimal concentration for response to any of the attractants tested, and amebas responded equally over the range of concentrations tested. Pretreatment of amebas with chemotactic peptides, bacterial products, and bacteria reduced the directional response to attractants. Amebas that had been grown in the presence of bacteria appeared more responsive to chemotactic peptides. Treatment of amebas with trypsin reduced the response of cells to chemotactic peptides, though sensitivity was restored within a couple of hours. This suggests the ameba membrane may have receptors, sensitive to these bacterial substances, which are different from the mannose receptors involved in binding bacteria to the membrane during phagocytosis. The rate of movement was relatively constant (ca. 0.40 μm/s), indicating that the locomotor response to these signals is a taxis, or possibly a klinokinesis, but not an orthokinesis. Studies of the population diffusion rate in the absence of signals indicate that the basic population motility follows the pattern of a Levy walk, rather than the more familiar Gaussian diffusion. This suggests that the usual mathematical models of ameboid dispersion may need to be modified.     

The action of sodium deoxycholate on Escherichia coli

Sodium deoxycholate is used in a number of bacteriological media for the isolation and classification of gram-negative bacteria from food and the environment. Initial experiments to study the effect of deoxycholate on the growth parameters of Escherichia coli showed an increase in the lag time constant and generation time and a decrease in the growth rate constant and total cell yield of this microorganism. Cell fractionation studies indicated that sodium deoxycholate at levels used in bacteriological media interferes with the incorporation of [U-14C]glucose into the cold-trichloroacetic acid-soluble, ethanol-soluble, and trypsin-soluble cellular fractions of E. coli. Finally, sodium deoxycholate interfered with the flagellation and motility of Proteus mirabilis and E. coli. It would appear then that further improvement of the deoxycholate medium may be in order.     

Adaptation of adhesion test using Caco-2 cells for anaerobic bacterium<Emphasis Type="Italic">Pseudobutyrivibrio xylanivorans</Emphasis>, a probiotic candidate

Pseudobutyrivibrio xylanivorans strain Mz5(T), an anaerobic bacterium (originating from the rumen of a Holstein-Friesian cow), has some attributes that make it a possible probiotic strain (very active hydrolases, bacteriocin and conjugated linoleic acid production). For the estimation of its adhesion ability, the adhesion test on Caco-2 cells was introduced and adapted. The adhesion was performed in an anaerobic glove box in standard 24-well plates at neutral pH for 30 min. The best method for separation of the adhered bacteria from Caco-2 cells appeared to be homogenization with an automatic pipette. The number of adhered bacteria was too small to be determined microscopically, so a new approach, i.e. detection of the apparent lag phase in liquid growth medium was tested. Under the selected assay conditions 1.04 bacterial cells from the late exponential phase adhered to one Caco-2 cell, which confirms the adhesion capability of P. xylanivorans Mz5(T). The adapted adhesion test using Caco-2 cells is suitable for estimation of adhesion capability of anaerobic bacteria.     

Adhesion and growth rate of Clostridium cellulolyticum ATCC 35319 on crystalline cellulose.

The rate of tritiated-thymidine incorporation into DNA was used to estimate Clostridium cellulolyticum H10 growth rates on Avicel cellulose, taking into consideration both the unattached cells and the cells adhered to the substrate. The generation time on cellobiose calculated from the data on cell density (4.5 h) agreed well with the generation time calculated by tritiated-thymidine incorporation (3.8 h). Growth on Avicel cellulose occurred when bacteria were adhered to their substrate; 80% of the biomass was detected on the cellulose. Taking into consideration attached and free bacteria, the generation time as determined by thymidine incorporation was about 8 h, whereas by bacterial-protein estimation it was about 13 h. In addition to the growth rate of the bacteria on the cellulose, the release of adhered cells constituted an important factor in the efficiency of the cellulolysis. The stage of growth influenced adhesion of C. cellulolyticum; maximum adhesion was found during the exponential phase. Under the conditions used, the end of growth was characterized by an acute release of biomass and cellulase activity from the cellulose. An exhaustion of the accessible cellulose could be responsible for this release.     

Growth models of the continuous bacterial leaching of iron pyrite by Thiobacillus ferrooxidans

The leaching of iron pyrite by Thiobacillus ferrooxidans was studied in a continuous stirred tank reactor at a variety of dilution rates (0.012-0.22 h(-1)), pyrite surface areas (18-194 m(2)/L), and inlet soluble substrate (Fe(2+)) concentrations (0-3000 ppm). The bacterial leaching rate was found to increase with increasing pyrite surface area, dilution rate, and inlet Fe(2+) concentration. The concentration of bacteria in solution was related to the concentration of bacteria attached to the pyrite surface by a Langmuir-type adsorption-desorption relation. Fitting the experimental data to this relation yielded a value for the area occupied per bacterium of 86 mum(2). This result is consistent with the concept of preferential bacterial attachment of certain sites on the solid. A bacterial growth model was developed that included both bacterial growth in solution and growth of bacteria attached to the pyrite surface. The specific growth rate of the attached bacteria was calculated from this model and was found to increase with increasing solid dilution rate and to decrease with increasing pyrite surface area and soluble substance concentration. An explanation of these results based on an active-inactive site mechanisms was also developed.     

Alginate Beads as Synthetic Inoculant Carriers for Slow Release of Bacteria That Affect Plant Growth

Uniform synthetic beads were developed as carriers for the bacterial inoculation of plants. The beads are made of sodium alginate and skim milk and contain a large reservoir of bacterial culture which releases the bacteria at a slow and constant rate. The beads are biodegradable and produce no environmental pollution. The strength of the beads, the rate of bacterial release, and the time of their survival in the soil can be controlled by several hardening treatments. The final product, lyophilized beads, is simple to use and is applied to the seeds concomitantly with sowing. The released bacteria are available for root colonization immediately at seed germination. Dry beads containing bacteria can be stored at ambient temperature over a long period without loss of bacterial content; storage requires a limited space, and the quality control of a number of bacteria in the bead is simple. The level of plant inoculation with beads was similar to that with previously used peat inoculants, but the former method yielded more consistent results, as the frequency of inoculated plants was much higher. The former method provides a different approach for inoculation of plants with beneficial rhizosphere bacteria.     

Bacterial growth on organic polymers

Mathematical models are developed describing unsteady-state bacterial growth on organic polymers that are hydrolyzed by extracellular enzymes secreted by the bacteria to yield low-molecular-weight oligomers that may be directly transported across bacterial cytoplasmic membranes and hence metabolized. Two different modes of extracellular enzyme action on the organic polymer are considered. In one case, the enzyme is exoacting yielding a transportable oligomer with each polymer bond hydrolyzed. In the other case, the enzyme is endoacting yielding a series of oligomers upon random cleavage of the polymer bonds with oligomers up to a certain chain length assumed to be transportable. These models are exploited to show under what circumstances the rate of hydrolysis by the extracellular enzyme to yield transportable oligomers influences the bacterial growth rate. It is shown that an initial lag period, an accentuated declining growth phase, and a low overall rate of bacterial growth will be some of the manifestations when the rate of hydrolysis has a strong influence.     

Growth Rates of Sulfolobus acidocaldarius in Nature

Turnover times for water passing through several Sulfolobus acidocaldarius-containing springs were determined by measuring the dilution rates of small amounts of sodium chloride that were added to the springs. Chloride was diluted out exponentially, while concentrations of the bacteria remained constant. Additionally, temperature, pH, and chemical composition of the springs also remained constant during the time that the chloride was being diluted. The springs are thus steady-state systems, and since the rates of bacterial growth must be at least equal to the chloride dilution rates, minimal doubling times for the bacterial populations can be calculated. Half-times for chloride dilution, equivalent to bacterial doubling times, were on the order of 10 to 20 h for springs ranging in volume from about 20 to 2,000 liters, but approximately 30 days for two larger springs of about 1 million liters. Formaldehyde-fixed cells of a serologically distinguishable strain of S. acidocaldarius were also added as markers to four of the smaller springs, and the dilution rates of these bacteria were compared with the chloride dilution rates. The rates agreed reasonably well, thus verifying the growth rates obtained from the chloride dilution rates. In three springs, exponential growth was studied by draining the springs and allowing them to refill with bacteria-free water. Exponential doubling times were on the order of a few hours, much more rapid than steady-state doubling times. The methods used in this work may have wider utility in aquatic environments.     

Toxic Effects on Bacterial Metabolism of the Redox Dye 5-Cyano-2,3-Ditolyl Tetrazolium Chloride

The monotetrazolium redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) has been used as a vital stain of actively respiring bacteria for several years. In this study, inhibitory effects on bacterial metabolism of this redox dye have been examined in a brackish water environment (Kiel Fjord, Germany) and a freshwater environment (Elbe River, Germany). As the results from time series experiments (1 to 10 h) show, bacterial growth and respiration of the investigated natural communities were clearly reduced by CTC supply. Compared with untreated controls (100%), CTC-treated samples showed distinctly lower heterotrophic bacterial plate counts (0 to 24 and 11.8 to 23.7%, respectively), bacterial production (0.9 to 14.1 and 1.1 to 9.6%, respectively), bacterial respiration (4.1 to 9.4 and 6.8 to 43.8% for several concentrations of (sup14)C-labeled glucose), and [(sup14)C]glucose incorporation (0.2 to 4.2%). Additionally, toxicity of CTC was demonstrated by luminescence in a Microtox bioassay. CTC concentrations of 0.1 and 5.0 (mu)M required only 15 min for decreases of approximately 50 and 100%, respectively. The suppression of CTC on several bacterial metabolic processes suggests that determination by the CTC technique underestimates the actual number of active cells distinctly. This conclusion is confirmed by the comparison of generation times calculated on the basis of thymidine uptake data and active bacterial counts determined by the CTC assay and microautoradiography. While unrealistic short generation times (0.5 to 5 h) resulted from the CTC assay, the generation times calculated according to microautoradiography ranged within values (7 to 21 h) reported elsewhere for comparable aquatic environments. The inhibitory effect of CTC demonstrated in our experiments is an aspect with regard to the application of this tetrazolium dye for the estimation of active bacteria in natural aquatic environments which hitherto has not been considered.     

Kinetics of adherence of Actinomyces viscosus to saliva-coated silica and hydroxyapatite beads

Adherence of 14C-labelled strains of Actinomyces viscosus to uncoated and saliva-coated silica and hydroxyapatite beads had both loose and firm components, probably reflecting different subpopulations of bacteria within a single culture. Adherence was characterized by the proportion of bacteria available for each type of adherence and a constant (Kb) for each combination of bacterial strain and bead surface. Loose adherence, which was greater with silica than with hydroxyapatite beads, always involved many more bacteria than firm adherence. Firm adherence was greater with A. viscosus WVU627 than A. viscosus TF11. The association rate constants (Ka) for loose and firm adherence were similar, indicating simultaneous processes, but the dissociation rate constant (Kd) was lower for loose adherence than for firm adherence. Removal of loosely adhering bacteria by washing may only reflect their distance from the bead surface. Silica beads were convenient for studying bacterial adherence and formed an acceptable coating of salivary glycoprotein.     

How generation intervals shape the relationship between growth rates and reproductive numbers

Mathematical models of transmission have become invaluable management tools in planning for the control of emerging infectious diseases. A key variable in such models is the reproductive number R. For new emerging infectious diseases, the value of the reproductive number can only be inferred indirectly from the observed exponential epidemic growth rate r. Such inference is ambiguous as several different equations exist that relate the reproductive number to the growth rate, and it is unclear which of these equations might apply to a new infection. Here, we show that these different equations differ only with respect to their assumed shape of the generation interval distribution. Therefore, the shape of the generation interval distribution determines which equation is appropriate for inferring the reproductive number from the observed growth rate. We show that by assuming all generation intervals to be equal to the mean, we obtain an upper bound to the range of possible values that the reproductive number may attain for a given growth rate. Furthermore, we show that by taking the generation interval distribution equal to the observed distribution, it is possible to obtain an empirical estimate of the reproductive number.