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Spore inoculum quality in filamentous bioprocesses is a critical parameter influencing pellet morphology and, consequently, process performance. It is essential to determine the concentration of viable spores before inoculation, to implement quality control and decrease batch-to-batch variability. The ability to assess the spore physiologic status with close-to-real time resolution would offer interesting perspectives enhanced process analytical technology (PAT) and quality by design (QbD) strategies. Up to now, the parameters contributing to spore inoculum quality are not clearly defined. The state-of-the-art method to investigate this variable is colony-forming unit (CFU) determination, which assesses the number of growing spores. This procedure is tedious, associated with significant inherent bias, and not applicable in real time. Here, a novel method is presented, based on the combination of viability staining (propidium iodide and fluorescein diacetate) and large-particle flow cytometry. It is compatible with the complex medium background often observed in filamentous bioprocesses and allows for a classification of the spores into different subpopulations. Next to viable spores with intact growth potential, dormant or inactive as well as physiologically compromised cells are accurately determined. Hence, a more holistic few on spore inoculum quality and early-phase biomass composition is provided, offering enhanced information content. In an industrially relevant model bioprocess, good correlation to CFU counts was found. Morphological parameters (e.g. spore swelling) that are not accessible via standard monitoring tools were followed over the initial process phase with close temporal resolution. [ABSTRACT FROM AUTHOR]

In stirred-tank photobioreactors agitation causes fragmentation of filamentous cyanobacteria. Here, we introduce a flow cytometric approach for high-throughput measurements of trichome dimensions, heterocysts and metabolic activity of Anabaena sp. cultures. The longest characterized trichome had 1135 μm chain length. This technology could potentially be used for monitoring and control purposes. • High-throughput method for morphological characterization of filamentous cyanobacteria • Physiological characterization via fluorescence staining methods • Potential on-line process analytical technology for photobioreactor cultivations [ABSTRACT FROM AUTHOR]

Aims The application of broad-spectrum intense light pulses is an innovative nonthermal technology for the decontamination of packaging materials, liquids or foodstuffs. The objective of this study was the fundamental investigation of the cellular impact of a pulsed light treatment on Listeria innocua and Escherichia coli. Methods and Results Flow cytometry in combination with different fluorescent stains, conventional plate count technique and a viability assay were applied to investigate the effects of a pulsed light treatment on the physiological properties of L. innocua and E. coli. The results showed that loss of cultivability occurred at considerably lower fluences than the shutdown of cellular functions such as the depolarization of cell membranes, the loss of metabolic, esterase and pump activities or the occurrence of membrane damage. Therefore, a considerable proportion of cells appeared to have entered the viable but nonculturable ( VBNC) state after the pulsed light treatment. A high percentage of L. innocua was able to maintain certain cellular vitality functions after storage overnight, whereas a further decrease in vitality was observed in case of E. coli. The loss of culturability was on the other hand directly accompanied by the formation of reactive oxygen species ( ROS) and DNA damages, which were assessed by the ROS-sensitive probe DCFH- DA and RAPD- PCR, respectively. Conclusions A significant discrepancy between conventional plate counts and different viability staining parameters was observed, which shows that a pulsed light treatment does not cause an immediate shutdown of vitality functions even when the number of colony-forming units already decreased for more than 6 log10 sample−1. Oxidative stress with concomitant damage to the DNA molecule showed to be directly responsible for the loss of cultivability due to pulsed light rather than a direct rupture of cell membranes or inactivation of intracellular enzymes. Significance and Impact of the Study The presented results suggest an UV light-induced photochemical rather than a photothermal or photophysical inactivation of bacterial cells by pulsed light under the applied experimental conditions. Flow cytometry in combination with different viability stains proved to be a suitable technique to gain deeper insight into the cellular response of bacteria to inactivation processes like a pulsed light treatment. [ABSTRACT FROM AUTHOR]

Abstract: Many research projects in cell biology now use flow cytometry for analysis or for isolation of specific cell types. In such studies, cell viability is obviously a crucial issue. However, many studies appear to rely upon light-scattering characteristics to identify and gate out non-viable cells, despite the fact that reliable identification of such cells can only be achieved through staining with impermeable fluorescent nuclear dyes such as propidium iodide or 7-amino actinomycin. In this paper we apply mathematical analysis to the theoretical problem of quantifying cell sub-populations labeled with two or more fluorescent markers, comparing situations in which dead cells have been identified with those in which cell viability has not been assessed. We demonstrate that in all cases in which dead cells are present within the population, percentages of live sub-populations in different subsets are mis-estimated. In cases where the pattern of marker expression differs greatly between live and dead cells, or where the proportion of dead cells is high, this mis-estimation will be aggravated; the subsets pattern will therefore be biased in a population selected only on the basis of light-scatter behavior. The importance of accurately detecting and gating out dead cells is illustrated by an experimental example accompanying the mathematical analysis. To conclude, identification of dead cells by means of viability stains should be an absolute routine in practical flow cytometry, so as to avoid mis-estimation in sorting or analysis. [Copyright &y& Elsevier]

Abstract: Pseudomonas fluorescens SBW25, a plant growth promoting bacterium, has been widely studied due to its potential as an inoculum for improving crop yields. Environmental inoculants are usually applied on seeds or directly to soil and to effectively promote plant growth they need to be viable and active. However, it is difficult to study the physiological status of specific microorganisms in complex environments, such as soil. In this study, our aim was to use molecular tools to specifically monitor the physiological status of P. fluorescens SBW25 in soil and in pure cultures incubated under different nutritional conditions. The cells were previously tagged with marker genes (encoding green fluorescent protein and bacterial luciferase) to specifically track the cells in environmental samples. The physiological status of the cells was determined using the viability stains 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) and propidium iodide (PI), which stain active and dead cells, respectively. Luciferase activity was used to monitor the metabolic activity of the population. Most of the cells died after incubation for nine days in nutrient rich medium. By contrast when incubated under starvation conditions, most of the population was not stained with CTC or PI (i.e. intact but inactive cells), indicating that most of the cells were presumably dormant. In soil, a large fraction of the SBW25 cell population became inactive and died, as determined by a decline in luciferase activity and CTC-stained cells, an increase in PI-stained cells, and an inability of the cells to be cultured on agar medium. However, approximately 60% of the population was unstained, presumably indicating that the cells entered a state of dormancy in soil similar to that observed under starvation conditions in pure cultures. These results demonstrate the applicability of this approach for monitoring the physiological status of specific cells under stress conditions, such as those experienced by environmental inoculants in soil. [Copyright &y& Elsevier]