Steric-Hyperlayer Sedimentation Field Flow Fractionation and Flow Cytometry Analysis Applied to the Study of Saccharomyces cerevisiae

Abstract

Sedimentation field flow fractionation separation associated with flow cytometry has been used for the characterization of several commercial Saccharomyces cerevisiae yeasts used for wine production. A new type of channel 80 microm thick and new operating conditions, such as sample introduction when field and flow are established and a channel inlet connected to the accumulation wall, were used. Good repeatability (5% RSD) and reduced analysis time (2-10 min) were obtained. The avoidance of the stop-flow relaxation process in conjunction with the use of a channel of reduced thickness has demonstrated that an effective "steric-hyperlayer" mode driving to a major focusing effect of the species in the channel thickness is involved in the elution of the yeast cells. Flow cytometry analyses were performed, and the forward scattering and side scattering yeast characteristics correlation maps were obtained. Field flow fractionation and flow cytometry information obtained indicated that the fractogram profiles of the yeast cell depended not only on the size, but also on the shape and density.

Rice starch granule characterization by flow cytometry scattering techniques hyphenated with sedimentation field–flow fractionation

“…In recent papers, our group has shown that substantial information can be deduced using association of flow cytometry (FC) with sedimentation field-flow fractionation (SdFFF) in the case of cell sorting [1,2]. It is therefore suggested that flow cytometry can be extended to other micron-sized species like, in this report, starch particles.…”

“…Such strategies already developed in the qualitative mode for cell separations [2,36] can be optimized quantitatively for organic particles, for example SS signal calibration attempts linked to particle composition or surface properties. In that regard, the versatility of fraction collections of predicted properties (size, polydispersity) opens new possibilities.…”

“…The SdFFF system used in this work consists of a separation channel connected to a UV detector and to a classical HPLC pump, with a sample chromatographic-like injection device [1,2]. The FFF channel was made of two polystyrene plates, one described as the depletion wall and the other called the accumulation wall, separated by a Mylar spacer in which the channel was manually carved.…”

“…Qualitatively, it can be stated from experimental data that particles of the same density and shape are eluted according to their volume (or size) while particles of identical shape and volume are eluted according to their density. Hyphenation of information provided by FFF in terms of retention with that provided by FC provides a rather sophisticated description of the sample population in terms of size, density, surface and composition characteristics, with the help of complex multi dimensional data analysis procedures or strategies [1,2]. Moreover, the separation power of SdFFF permits purification of fractions characterized by their multi dimensional FC pattern (population fingerprint) using FS in combination with side scattering (SS) and/or fluorescence.…”

“…Size characterization was performed by flow cytometry on collected fractions all along the elution time. Fractions were collected and designated as follows: (1) the total peak fraction: TP, from 1 min 35 s to 5 min 30 s; (2) peak fractions F1-F4: F1, 1 min 45 s/2 min 40 s; F2, 2 min 50 s/3 min 25 s; F3, 3 min 30 s/4 min 20 s; and F4, 4 min 25 s/5 min 30 s.Elution procedures already described for cell sorting were systematically applied in order to limit or avoid channel poisoning[1,2].…”