Showing papers by "Douglas B. Kell published in 1994"

Influence of Viable Cells on the Resuscitation of Dormant Cells in Micrococcus luteus Cultures Held in an Extended Stationary Phase: the Population Effect

Abstract: A high proportion of Micrococcus luteus cells in cultures which had been starved for 3 to 6 months lost the ability to grow and form colonies on agar plates but could be resuscitated from their dormancy by incubation in an appropriate liquid medium (A. S. Kaprelyants and D. B. Kell, Appl. Environ. Microbiol. 59:3187-3196, 1993). In the present work, such cultures were studied by both flow cytometry and conventional microbiological methods and were found to contain various numbers of viable cells. Pretreatment of such cultures with penicillin G, and subsequent dilution, was used to vary this number. When the initial number of colony-forming cells per 30-ml flask was approximately nine (±five) or more, resuscitation of 10 to 40% of the cells, and thus culture growth, was observed. The lag period before the appearance of a population of cells showing significant accumulation of the fluorescent dye rhodamine 123 (i.e., of cells with measurable membrane energization) decreased from 70 to 27 h when the number of viable cells was increased from 30 to 105 per flask, while the lag period before an observable increase in the number of colony-forming cells occurred was almost constant (at some 20 h). Provided there were more than nine (±five) initially viable cells per flask, the number of initially viable cells did not affect the final percentage of resuscitable cells in the culture. The lag period could be ascribed in part to the time taken to restore the membrane permeability barrier of starved cells during resuscitation, as revealed by flow cytometric assessment of the uptake of the normally membrane-impermeant fluorescent DNA stain PO-PRO-3 {4-[3-methyl-2, 3-dihydro-(benzo-1, 3-oxazole)-2-methylidene]-1-(3′-trimethylammonium propyl)-pyridinium diiodide}. Although cell populations which contained fewer than nine ±five viable cells per flask failed to grow, 4 to 20% of the cells (of 1.2 X 106) were able to accumulate rhodamine 123 after 80 to 100 h of incubation, showing the ability of a significant number of the cells in the population at least to display “metabolic resuscitation.” Resuscitation and cell growth under such conditions were favored by the use of a 1:1 mixture of fresh lactate medium and supernatant from late-logarithmic-phase M. luteus cultures as the resuscitation medium. We conclude that the presence of a small fraction of viable cells at the onset of resuscitation facilitates the recovery of the majority of the remaining (dormant) cells. The cell density dependence of the kinetics, or population effect, suggests that this recovery is due to the excretion of some factor(s) which promoted the transition of cells from a state in which they are incapable of growth and division to one in which they are capable of colony formation.

Analytical or monitoring apparatus and method

Abstract: Cellular biological material (such as living tissue) is analysed or monitored by applying an AC electrical potential across the biological material so as to produce a nonlinear dielectric spectrum, and obtaining a detectable signal corresponding to the resulting spectrum. The potential is of a first frequency and the measured response at one or more second frequency substantially not overlapping with the first frequency.

Estimation of dormant Micrococcus luteus cells by penicillin lysis and by resuscitation in cell-free spent culture medium at high dilution

Abstract: Micrococcus luteus starved for 2–7 months in spent medium following growth to stationary phase in batch culture exhibited a culturability (as estimated by direct plating on nutrient agar plates) of < 0.001%. However, following a lag, some 70% of the cells could be lysed upon inoculation into and cultivation in fresh lactate minimal medium containing penicillin, showing the capability of a significant portion of the cells at least to enlarge (and thus potentially to resuscitate). When the viable cell count was estimated using the most probable number method, by incubation of high dilutions of starved cells in liquid growth media, the number of culturable or resuscitable cells was very low, and little different from the viable cell count as assessed by plating on solid media. However, the apparent viability of these populations evidenced with the most probable number method was 1000–100 000-fold greater when samples were diluted into liquid media containing supernatants taken from the stationary phase of batch cultures of the organism, suggesting that viable cells can produce a factor which stimulates the resuscitation of dormant cells. Both approaches show, under conditions in which the growth of a limited number of viable cells during resuscitation is excluded, that a significant portion of the apparently non-viable cell population in an extended stationary phase is dormant, and not dead.

Dielectric properties of human blood and erythrocytes at radio frequencies (0.2–10 MHz); dependence on cell volume fraction and medium composition

Abstract: The dielectric properties of human erythrocytes (red blood cells) suspended in whole blood and in isotonic media at various volume fractions (haematocrits) have been studied in the frequency range 0.2-10 MHz, in which the so-called beta-dispersion due to the Maxwell-Wagner effect is known to occur. The capacitance and conductance at 25 degrees C were measured by an instrument interfaced to a computer. The rectangular sample cavity (1 ml volume) contained four pure gold electrode pins, and the sample could be circulated by a roller pump. The frequency-dependence of the permittivity and conductivity were fitted by non-linear least squares regression. Corrections were applied for non-linearity in the dielectric increment at high haematocrit, and for electrode polarisation when diluting the blood in saline. Data were interpreted in terms of a simple equivalent resistor-capacitor circuit. From the measured haematological values the specific membrane capacitance (Cm) and the conductivities internal and external to the cells (sigma i' and sigma o' respectively) were estimated. The conductivities behaved in a predictable manner with a mean of 0.458 S.m-1 (s.d. +/- 0.044) for sigma i', whereas the value of Cm (and indeed the actual capacitance of the suspension) was dependent on the amount of plasma present. Hence, in stationary normal (anticoagulated) whole blood samples, Cm was as high as 2.98 mu F.cm-2 (s.d. +/- 0.40), in contrast to about 0.9 mu F.cm-2 in blood diluted more than two-fold (to less than 20% hct) in isotonic media. The high value remained when the diluent was plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid identification using pyrolysis mass spectrometry and artificial neural networks of Propionibacterium acnes isolated from dogs.

Abstract: Curie-point pyrolysis mass spectra were obtained from reference Propionibacterium strains and canine isolates. Artificial neural networks (ANNs) were trained by supervised learning (with the back-propagation algorithm) to recognize these strains from their pyrolysis mass spectra; all the strains isolated from dogs were identified as human wild type P. acnes. This is an important nosological discovery, and demonstrates that the combination of pyrolysis mass spectrometry and ANNs provides an objective, rapid and accurate identification technique. Bacteria isolated from different biopsy specimens from the same dog were found to be separate strains of P. acnes, demonstrating a within-animal variation in microflora. The classification of the canine isolates by Kohonen artificial neural networks (KANNs) was compared with the classical multivariate techniques of canonical variates analysis and hierarchical cluster analysis, and found to give similar results. This is the first demonstration, within microbiology, of KANNs as an unsupervised clustering technique which has the potential to group pyrolysis mass spectra both automatically and relatively objectively.

Rapid and Quantitative Analysis of the Pyrolysis Mass Spectra of Complex Binary and Tertiary Mixtures Using Multivariate Calibration and Artificial Neural Networks

Abstract: Binary mixtures of the protein lysozyme with glycogen, of DNA or RNA in glycogen, and the tertiary mixture of cells of the bacteria Bacillus subtilh, Escherichia coli, and Staphylococcusaurecls were subjected to pyrolysis mass spectrometry. To analyze the pyrolysis mass spectra so as to obtain quantitative information representative of the complex components of the mixtures, partial least-squares regression (PLS), principal components regression (PCR), and fully interconnected feedforward artificial neural networks (ANNs) were studied. In the latter case, the weights were modified using the standard back-propagation algorithm, and the nodes used a sigmoidal squashing function. It was found that each of the methods could be used to provide calibration models which gave excellent predictions for the concentrations of determinands in samples on which they had not been trained. Neural networks were found to provide the most accurate predictions. We also report that scaling the individual nodes on the input layer of ANNs significantly decreased the time taken for the ANNs to learn. Removing masses of low intensity, which perhaps mainly contributed noise to the pyrolysis mass spectra, had little effect on the accuracy of the ANN predictions though could dramatically speed up the learning process (by more than 100-fold) and slightly improved the accuracy of PLS calibrations.

Rapid screening for metabolite overproduction in fermentor broths, using pyrolysis mass spectrometry with multivariate calibration and artificial neural networks.

Abstract: Binary mixtures of model systems consisting of the antibiotic ampicillin with either Escherichia coli or Staphylococcus auresu were subjected to pyrolysis mass spectrometry (PyMS). To deconvolute the pyrolysis mass spectra, so as to obtain quantitative information on the concentration of ampicilin in the mixtures, partial least squares regression (PLS), principal components regression (PCR), and fully interconnected feedforward artificial neural networks (ANNs) were studied. In the latter case, the weights were modified using the standard backpropagation algorithm, and the nodes used a sigmoidal squsahing funciton. It was found that each of the methods could be used to provide calibration models which gave excellent predictions for the concentrations of ampicillin in samples on which they had not been trained. Furthermore, ANNs trained to predict the amount of ampicilin in E. coli were able to generalise so as to predict the concentration of ampicillin in a S. aureus background, illustrating the robustness of ANNs to rather substantial variations in the biological background. The PyMS of the complex mixture of ampicilin in bacteria could not be expressed simply in terms of additive combinations of the spectra describing the pure components of the mixtures and their relative concentrations. Intermolecular reactions took place in the pyrolysate, leading to a lack of superposition of the spectral components and to a dependence of the normalized mass spectrum on sample size. Samples from fermentations of a single organism in a complex production medium were also analyzed quantitatively for a drug of commercial interest. The drug could also be quantified in a variety of mutant-producing strains cultivated in the same medium. The combination of PyMS and ANNs constitutes a novel, rapid, and convenient method for exploitation in strain improvement screening programs. (c) 1994 John Wiley & Sons, Inc.