Use of flow cytometry in industrial microbiology for strain improvement programs

Abstract

A flow cytometry (FCM) system was chosen to analyze and sort microbiological samples, e.g., bacteria, bacterial spores, yeasts, and fungal spores, without major changes in the commercially available state. The system was further improved by addition of a stepping motor-driven scanning table that accepts standard petri dishes or microtiter plates. The electronics of the sorting system were changed to enable the sorter to deliver only one particle at a time, working in a "handshake" mode with the scanning table. Appropriate parameters, depending on the biological material and including all fluorescent stains that do not impair growth and productivity of cells were chosen to sort distinct bioparticles under aseptic conditions and to clone colonies or cultures out of them. A mutagenized sample of spores entering the germination cycle can be followed and thus provide a means to pick only viable growing cells despite the killing effect of the mutagen. One example of a typical strain improvement is illustrated. From a spore suspension of Rhizopus arrhizus, a subpopulation of morphologically different spores comprising about 5-10% of the whole population was cloned. From approximately 8,000 clones, 10 were isolated that produced approximately five-to six-fold the amount of fungal lipase activity, compared to the original strain or to reisolated clones from the mean population of clones.

Applications of Flow Cytometry to Clinical Microbiology

“…The promising tool described by Boye and Steen in 1983 became a "potent illuminating light" in the 1990s (38), as was stated in the book edited by David Lloyd, Flow Cytometry in Microbiology (186a), from which most microbiological cytometrists have learned their trade. In the last years of the 1990s, the applications of FCM in microbiology have significantly increased (9,28,103,148,291).…”

Flow sorting in aquatic ecology

“…In this way, strains of the ß-carotene producing chlorophyte Dunaliella salina that produced twice the amount of the carotenoid than the wild type, was successfully sorted using their specific fluorescence and scatter characteristics, and grown (Benamotz, 1991). Betz et al (1984) sorted and cultured subpopulations of the bacterium Rhizopus arrhizus that were different in their light scatter characteristics; some of these subpopulations showed an increased lipase production, and could subsequently be selected for further lipase production. An elegant way of selecting for a desired property of a specific cell type is the coupling of that property with the inherent fluorescent characteristics of the cells, which can be readily measured by the flow cytometer.…”

Assessment of bacterial viability status by flow cytometry and single cell sorting

“…Single cell sorting is another feature of flow cytometry that has not been exploited fully. While the technique has been used in industry for a long time for cloning (Betz et al 1984), there are very few applications in the literature using differential stains such as lipid stains, lectins, antibodies or nucleic acid probes (Bloodgood and Salomonsky 1987 ;De Stefano et al 1991 ;Francisco et al 1993 ;Nebe-von Caron et al 1994 ;Porter et al 1995a,b), or for the assessment of viability stains (Liu et al 1994 ;Nebevon Caron et al 1994). Comparison of cell growth with the viability stains tested has so far lacked conclusive correlation due to remaining discrepancies (Kaprelyants and Kell 1992 ;Durodie et al 1995 ;Lopez-Amoros et al 1995).…”