Flow cytometric measurements. Flow

Flow cytometric measurements.
Flow cytometry was used to determine the final cell concentration and average biovolume after growth.
A 10-
l aliquot of SYBR green stain (Molecular Probes, Basel, Switzerland), diluted 100 times in
dimethyl sulfoxide (Fluka Chemie AG, Buchs, Switzerland), was added to 1 ml of a bacterial suspension and incubated for 15 min at room temperature in the dark before analysis.
For outer membrane permeabilization, EDTA (pH 8) was added (5 mM final concentration) to the sample together with the stain (1).
If asample contained more than 10cells ml1, a dilution prior to the staining procedure was done.
All samples were measured on a CyFlow Space flow cytometer (Partec, Mu¨nster, Germany) equipped with a 200-mW solid-state laser emitting a fixed wavelength of 488 nm and equipped with volumetric counting hardware.
The trigger was set on the green fluorescence (520-nm) channel, and signals for total cell counting were collected on the combined 520-nm/630-nm(red fluorescence) dot plot.
For cellular biovolume estimations, additional signals were collected on the combined 520-nm/side scatter (SSC) dot plot.
An experimentally derived correlation factor was then used to determine the cellular
biovolume (15).
The quantification limit of the instrument was below 1,000 cells ml1 with an average standard deviation of less than 5% (14).