Josep Vives-Rego

Fate and effect of monoalkyl quaternary ammonium surfactants in the aquatic environment.

The effect of the alkyl chain of quaternary ammonium-based surfactants on their aquatic toxicity and aerobic biodegradability has been studied. Two families of monoalkylquats surfactants were selected: alkyl trimethyl ammonium and alkyl benzyl dimethyl ammonium halides. Acute toxicity tests on Daphnia magna and Photobacterium phosphoreum were carried out and EC50 values in the range of 0.1-1 mg/l were obtained for the two series of cationic surfactants. Although the substitution of a benzyl group for a methyl group increases the toxicity, an incremental difference in toxicity between homologs of different chain length were not observed. Biodegradability of the different homologs was determined not only in standard conditions but also in coastal water, both tests yielding similar results. An increase in the alkyl chain length or the substitution of a benzyl group for a methyl group reduces the biodegradation rate. The degradation of these compounds in coastal waters was associated with an increase in bacterioplankton density, suggesting that the degradation takes place because the compound is used as a growth substrate.

Assessment of E. coli and Salmonella Viability and Starvation by Confocal Laser Microscopy and Flow Cytometry Using Rhodamine 123, DiBAC4(3), Propidium Iodide, and CTC

Assessment of cell viability using methods which do not require cell culture is essential in the field of aquatic microbiology, since many bacteria known to be present in aquatic environments cannot be grown in culture. The study of bacterial biofilms, which previously needed an epifluorescent microscope, has recently been enhanced by the use of flow cytometry and confocal scanning laser microscopy (CSLM). A method based on the combination of several membrane potential related dyes, a membrane integrity dye and a redox probe was used to measure cell viability by flow cytometry and confocal laser microscopy. Rhodamine-propidium iodide (PI) double staining was used to discriminate viable from nonviable cells in CSLM observations. Membrane depolarization during E. coli and Salmonella starvation measured by DiBAC4(3) incorporation (flow cytometry and CSLM) was found to be in concordance with respiratory activity as detected by a tetrazolium salt (CTC) reduction.

Rapid flow cytometry - Nile red assessment of PHA cellular content and heterogeneity in cultures of Pseudomonas aeruginosa 47T2 (NCIB 40044) grown in waste frying oil

The accumulation of cytoplasmic polyhydroxyalkanoates (PHAs) and the heterogeneity of bacterial populations were analysed by flow cytometry and SYTO-13 and Nile red staining in rhamnolipid-producing Pseudomonas aeruginosa cultures grown in waste frying oil as carbon source. A combination of SYTO-13 and Nile red fluorescence with cytometric forward and side scatter values may allow increases in the final production of polyhydroxyalkanoates (PHA) by two basic mechanisms: (i) rapid assessment of polyhydroxyalkanoate content and (ii) definition of flow cytometric cell sorting protocols to select high polyhydroxyalkanoate (PHA)-producing strains. We report a rapid (less than 30 min) flow cytometric assessment of PHAs in Pseudomonas aeruginosa 47T2 following Nile red staining: (i) to estimate cellular PHAs content; (ii) to study heterogeneity of the batch cultures producing PHAs and (iii) to establish the basis for sorting sub-populations with a high capacity to accumulate PHAs.

Mathematical modelling methodologies in predictive food microbiology: A SWOT analysis

Predictive microbiology is the area of food microbiology that attempts to forecast the quantitative evolution of microbial populations over time. This is achieved to a great extent through models that include the mechanisms governing population dynamics. Traditionally, the models used in predictive microbiology are whole-system continuous models that describe population dynamics by means of equations applied to extensive or averaged variables of the whole system. Many existing models can be classified by specific criteria. We can distinguish between survival and growth models by seeing whether they tackle mortality or cell duplication. We can distinguish between empirical (phenomenological) models, which mathematically describe specific behaviour, and theoretical (mechanistic) models with a biological basis, which search for the underlying mechanisms driving already observed phenomena. We can also distinguish between primary, secondary and tertiary models, by examining their treatment of the effects of external factors and constraints on the microbial community. Recently, the use of spatially explicit Individual-based Models (IbMs) has spread through predictive microbiology, due to the current technological capacity of performing measurements on single individual cells and thanks to the consolidation of computational modelling. Spatially explicit IbMs are bottom-up approaches to microbial communities that build bridges between the description of micro-organisms at the cell level and macroscopic observations at the population level. They provide greater insight into the mesoscale phenomena that link unicellular and population levels. Every model is built in response to a particular question and with different aims. Even so, in this research we conducted a SWOT (Strength, Weaknesses, Opportunities and Threats) analysis of the different approaches (population continuous modelling and Individual-based Modelling), which we hope will be helpful for current and future researchers.

Enumeration, viability and heterogeneity in Staphylococcus aureus cultures by flow cytometry

Abstract Several fluorochromes (rhodamine 123, bis-oxonol, propidium iodide, SYTO-13 and calcein) were tested by flow cytometry for their ability to determine cell density, viability and heterogeneity in Staphylococcus aureus cultures exposed to heating (60–70–80°C for 2 min), formaldehyde 2% for 20 min and gramicidin-S at 2–5–10 μg/ml for 20 min. Results were validated by viable plate count and counts performed with a particle analyser. Flow cytometry gave quicker results and more accurate information about intermediate states and heterogeneity of S. aureus cultures than viable plate counts. Rhodamine 123 and oxonol were found to be efficient dyes for the assessment of bacterial viability. SYTO-13 was an excellent marker for total counts and calcein can be used to assess metabolic activity.

Second-order functions are the simplest correlations between flow cytometric light scatter and bacterial diameter

Second-order mathematical relationships between bacterial cell diameter determined by electric particle analyser and flow cytometric forward light scatter in axenic cultures are obtained and discussed. Since it is technically impossible today to obtain both measurements for each individual cell, standard regression techniques cannot be applied. To overcome this limitation, we assume that these two parameters are related by a monotone increasing function that enables their mathematical relationships to be studied. Our conclusion is that forward light scatter data cannot be linearly transformed into bacterial size values by an accurate and universal function. However, second-order relationships seem to be the simplest satisfactory relationships between cell diameter and forward light scatter in eubacteria.

Analysis and IbM simulation of the stages in bacterial lag phase: Basis for an updated definition

The lag phase is the initial phase of a culture that precedes exponential growth and occurs when the conditions of the culture medium differ from the pre-inoculation conditions. It is usually defined by means of cell density because the number of individuals remains approximately constant or slowly increases, and it is quantified with the lag parameter lambda. The lag phase has been studied through mathematical modelling and by means of specific experiments. In recent years, Individual-based Modelling (IbM) has provided helpful insights into lag phase studies. In this paper, the definition of lag phase is thoroughly examined. Evolution of the total biomass and the total number of bacteria during lag phase is tackled separately. The lag phase lasts until the culture reaches a maximum growth rate both in biomass and cell density. Once in the exponential phase, both rates are constant over time and equal to each other. Both evolutions are split into an initial phase and a transition phase, according to their growth rates. A population-level mathematical model is presented to describe the transitional phase in cell density. INDividual DIScrete SIMulation (INDISIM) is used to check the outcomes of this analysis. Simulations allow the separate study of the evolution of cell density and total biomass in a batch culture, they provide a depiction of different observed cases in lag evolution at the individual-cell level, and are used to test the population-level model. The results show that the geometrical lag parameter lambda is not appropriate as a universal definition for the lag phase. Moreover, the lag phase cannot be characterized by a single parameter. For the studied cases, the lag phases of both the total biomass and the population are required to fully characterize the evolution of bacterial cultures. The results presented prove once more that the lag phase is a complex process that requires a more complete definition. This will be possible only after the phenomena governing the population dynamics at an individual level of description, and occurring during the lag and exponential growth phases, are well understood.

Statistical analysis and biological interpretation of the flow cytometric heterogeneity observed in bacterial axenic cultures

Histogram comparison and meaningful statistics in flow cytometry is probably the most widely encountered mathematical problem in flow cytometry. Ideally, a test for determining the statistical equality or difference of flow cytometric distributions will identify the significant differences or similarities of the obtained histograms. This situation is of particular interest when flow cytometry is used to study the heterogeneity of axenic bacterial populations. We have statistically measured the heterogeneity of successive cytometric measures, the modifications produced after 20 transfers from the same culture, and the differences between 20 subcultures of identical origin. The heterogeneity of the bacterial populations and the similarity of the obtained 360 histograms were analysed by standard statistical methods. We have studied bacterial axenic cultures in order to detect, quantify and interpret their cytometric heterogeneity, and to assess intrinsic differences and differences produced by laboratory manipulations. We concluded that the standard axenic cultures have a considerable intrinsic cellular and molecular heterogeneity. We suggest that the heterogeneity we have detected basically has two origins: cell size diversity and cell cycle variations.

Involvement of RNA and DNA in the staining of Escherichia coli by SYTO 13

Aims: To assess the extent to which DNA and RNA bacterial content contributes to fluorescent response of SYTO 13.

Flow cytometric narrow-angle light scatter and cell size during starvation of Escherichia coli in artificial sea water

Flow cytometry was used to study starvation of Escherichia coli in artificial sea water. Flow cytometric narrow‐angle light scatter was compared and assessed in relation to the cell sizes obtained by scanning electron microscopy at low temperature, and by image analysis. A correlation between narrow‐angle light scatter and cell size was not observed, although an acceptable correlation (γ= ‐0.845) between narrow‐angle light scatter and the starvation period was observed. On the other hand, the distribution of narrow‐angle light scatter at any given moment of culture is asymmetric and may be associated with the cell size distribution at the specific moment of starvation.