A FLOW CYTOMETRIC APPROACH FOR CHARACTERIZATION AND DIFFERENTIATION OF BACTERIA DURING MICROBIAL PROCESSES

The analysis of growing or resting bacterial populations by flow cytom etry offers several advantages over traditional methods for determinin g mean-value parameters. This method has been applied here to measure both the distribution of single-cell fluorescence intensity and the li ght-scatter behaviour of the methylotrophical strains of Methylobacter ium rhodesianum MB126 and Methylocystis GB25 as well as Pseudomonas fl uorescens and a strain isolated from the soil. The four different bact erial populations were analysed concerning the DNA and the poly-3-hydr oxybutyrate (PHB) content. A new cell-preservation method is presented . Optimized staining methods for each strain were developed in detail, in two cases DNA had to be dehybridized before staining with a mixtur e of mithramycin/ethidium bromide. Nile red is used for detecting PHB. Both stains were excited by an argon-ion laser at 488 nm; fluorescenc e emission for mithramycin/ethidium bromide was measured from 520 nm a nd for Nile red from 600 nm onwards. It is shown that changes in the D NA content and in the forward-lightscattering behaviour of the bacteri al strains chosen were measurable. These changes could be related to d ifferent cultivation conditions and correlated, in the case of strains that accumulate PHB, with alterations of that biopolymer content. In addition it was found that these methods provide a contribution to the differentiation of mixed bacterial populations.