In this issue

Flow Cytometry Phage Display Assay

Flow cytometry analysis of virus particles has so far been largely limited to enumeration and discrimination between different viruses in the same sample. Sokolenko and coworkers demonstrate that flow cytometry can also be used to assess protein display on the surface of a virus, using phage displaying green fluorescent protein as an example. While the expression of a fluorescent protein allowed an assessment of display based on fluorescence, side scatter was found to give a greater degree of discrimination, resulting in the observation of distinct phage sub-populations as a function of display conditions. Sub-population identification was facilitated by working with raw list-mode data using in-house scripts written in the R programming language, allowing a greater degree of processing control over smoothing and visualization of the data. Overall, the results suggest that viral protein display can be readily assessed with flow cytometry, even without the use of fluorescent dyes. 1

Illustration 1.

In this issue: page 1031

Efficient viability assessment in mixed bacterial communities

The large diversity of bacteria with regard to the cell wall structure and metabolic capabilities hamper the establishment of staining methods for viability assessment in mixed communities. The uptake of dyes and the interaction of dye with intracellular targets vary with the species of interest and the physiological state. Rüger and coworkers took this into consideration for the establishment of a new flow cytometric method for viability assessment of Staphylococcus aureus and Burkholderia cepacia in mixed culture using SYBR®Green I and propidium iodide. Extensive testing of staining protocols for each species at different growth phases in pure culture revealed conditions for the efficient determination of viability in mixed communities. Simultaneous use of Gram-specific fluorescence staining using wheat germ agglutinin enabled the discrimination of species. The presented method was successfully tested in mixed culture cultivations. Data showed viability alterations in comparison to pure cultures suggesting interspecies effects in mixed culture. 2

Illustration 2.

In this issue: page 1055

Seeds are getting ready for germination

Endoreduplication, during which nuclei undergo repeated rounds of DNA replication without mitosis, is very common in plants; however, its significance is unclear. It often occurs in specialized cells/tissues, such as trichomes, and endosperm, but not in all species. Rewers and Sliwinska analyzed endoreduplication in the cotyledons and embryo axis during development of seeds of five Fabaceae species differing in genome size, type of seedling establishment, and life cycle. In annual species endoreduplication intensity is higher in the cotyledons than in the embryo axis and can reach 128C; in a perennial species few or no endopolyploid cells occur in either seed region. In the cotyledons, which are storage organs in Fabaceae seeds, for most species the proportion of endoreduplicated cells increases during development and then decreases at seed physiological maturity, when the seed population reaches maximum germination. Therefore, changes in endoreduplication in the embryo, detected by flow cytometry, can be used as a marker for establishing the optimal seed harvest time. 3

Illustration 3.

In this issue, page 1067

Acoustophoresis: Acoustic toolbox for cell handling

The somatic cell count (SCC) in raw milk is a widely used indicator for milk quality and livestock health. Grenvall and coworkers demonstrate how acoustophoresis—acoustic forces acting on suspended particles—can be used to remove lipid particles from a raw milk sample while retaining cells, to facilitate direct optic or Coulter-based SCC measurements. The new approach, which simplifies the pretreatment protocols for the raw milk samples, yield SCC numbers comparable to those achieved with industry standard FACS measurements. Previous work in the group has shown that levels of other milk solids in the samples (e.g. casein and lactose) are unaffected by the acoustophoretic treatment. The research indicates that acoustophoresis provides a promising acoustic toolbox for sample handling to allow simple and fast cytometry and sample pretreatment that can be integrated into small scale systems. 4

Illustration 4.

In this issue, page 1076