IGEM:IMPERIAL/2007/Projects/Cell by date/Implementation/Flow Cytometry

Summary
The flow cytometric method produces direct total cell counts with high speed (10 – 15 minutes), high accuracy (less than 5 % instrument error), and high sensitivity (lower detection limit of about 200 cells/mL). It does not permit differentiation of bacterial cells on the basis of taxonomy, metabolic activity, culturability or viability, and estimations of biomass or biovolume from light scattering data are complex and subjective and difficult to do. The method requires a flow cytometer operator that can distinguish stained microbial cells from background noise signals on the basis of relevant control samples and experience. The method assumes proper maintenance, standardization and calibration of the flow cytometer used. The method consists of staining an aqueous sample of suspended bacteria with a DNA-targeting fluorochrome and enumeration of the stained cells by counting with a flow cytometer. Fixatives for storage of samples are optional (see Standard Methods 9216 B, Greenberg et al. (1993)) but long-term storage before analysis is not recommended.

Equipment
1. Flow cytometer, equipped with a 488 nm blue laser with a minimum power of 15 mW1.

2. Filters, a 520 nm bandpass filter for green fluorescence detection, a 630 nm bandpass or longpass filter for red fluorescence detection.

3. Counting modus, volumetric counting hardware are inclusive to some instruments and preferable, though this can be compensated for by the use of commercially available bead count standards.

4. Flow cytometry measuring tubes, comes standard for different brands of instruments.

Reagents
1. Fluorochrome, SYBR® Green I4, diluted 1:100 from original stock solution in 0.2 μm filtered DMSO, and stored at -20 °C until use.

2. Dilution liquid, preferably from the same solution in which the bacteria are suspended, filtered with a 0.1 μm sterile syringe filter prior to use.

3. EDTA stock solution, 500 mM EDTA suspended in nanopure water and sterile filtered (0.2 μm) prior to use.

4. Lysis buffer stock solution, comprising 10 % v/v Triton X-100, 5 % v/v Tween 20, 10 mM Tris-HCl, and 1 mM EDTA. - Partec GmbH or equivalent - Partec GmbH or equivalent - CountBright (Invitrogen) or equivalent - Or equivalent DNA binding stains e.g. SYBR Green II or SYTO9

Protocol
1. Take 1 mL of the sample in a sterile Eppendorf tube and add 10 μL of the fluorochrome and 10 μL of EDTA stock solution, mix thoroughly by vortexing and incubate in the dark at room temperature for at least 10 minutes. If the bacteria concentrations are expected (or found) to exceed 1 x 107 cells/mL, it is advised to increase the concentration of fluorochrome accordingly (10 μL of respectively fluorochrome and EDTA for every 1 x 107 cells per mL).

2. If the flow cytometer is not equipped with volumetric counting hardware, add just before measurement a commercially available bead standard of known concentration as prescribed by the manufacturer.

3. Run the sample through a flow cytometer using the instrumental settings appropriate for the filter set-up and trigger on the green fluorescence channel.

4. If the bacterial concentration in the sample exceeds 2 x 105 cells/mL, dilute the sample to a concentration between 0.1 – 2 x 105 cells/mL after staining and just before measurement with the dilution liquid.

5. If the bacteria are present in clusters (can be controlled with fluorescence microscopy or evidenced through FCM light scatter data), dispersion of clustered cells can be achieved with the addition of 1 % v/v of lysis buffer prior to staining, coupled with vortexing or gentle sonication.

Calculations (Under construction)
Optimal separation of stained bacteria from background noise is achieved on a two dimensional dotplot of green fluorescence (520 nm) and red fluorescence (630 nm). Background noise and stained bacteria are separated from each other by means of electronic gating (see section 3 of this report), using the flow cytometry software. Depending on the flow cytometer used, the software usually performs the counting and calculations automatically. Any sample dilution should be accounted for by the operator.