BD ACCURI C6 PLUS
In the diverse field of microbiology research, flow cytometry is a powerful technique for analyzing microorganisms including bacteria and yeast, and offers many advantages over more conventional techniques. Light scatter data can reveal basic information about microbes' size, shape, and surface features, while fluorescent stains can assess microbes' cell viability, metabolic activity, and concentration. In some cases, this information might be enough to identify specific microorganisms in a heterogeneous sample. In others, additional techniques such as fluorescence in situ hybridization (FISH) can be added.
These characteristics allow flow cytometry to be used in diverse microbiological applications, such as measurement of gene expression, monitoring bacterial and yeast fermentations, recombinant protein production in bacteria, and food processing. On BD Accuri™ flow cytometers, triggers and thresholds help to accurately identify small particles such as microbes. Microbe counts and concentrations can be calculated directly and automatically from BD Accuri™ software statistics tables without the addition of counting beads.
The following sections and resources illustrate the rich data you can generate by using BD Accuri systems for microbiology applications. See also the separate tab for Industrial Applications that use microbes for bioprocessing, food and beverage processing, and biofuels.
- White Paper: Life Science Application of Personal Flow Cytometry
- Nature.com Webinar Replay: The Power of Flow Cytometry in Microbiology Applications
- BD Accuri™ C6 Plus Brochure
- Webinar Replay: Flow Cytometry as a Tool for Microbial Analysis
- Bulletin: Threshold and Analysis of Small Particles on the BD Accuri™ Flow Cytometer
Microbe Counts and Viability
Perhaps the most common task in microbial analysis is to identify and count microbes. On BD Accuri flow cytometers, triggers and thresholds help you accurately identify small particles such as microbes and distinguish them from instrument noise, background signals, and debris. Microbe counts and concentrations can be calculated rapidly, directly, and automatically from the software, eliminating laborious plate counts. If microbes are transfected with green, yellow, red, or other fluorescent proteins, they can be detected and clearly differentiated from non-transfected populations.
The BD™ Cell Viability Kit provides a simple, two-color method to monitor microbial cell viability on BD Accuri flow cytometers. Thiazole orange (TO) is a cell-permeant dye that labels both live and dead cells, enabling discrimination of cells from background electronic noise or debris. Propidium iodide (PI) is impermeable to healthy cells with intact membranes, but permeates cells with compromised membranes such as dead cells. When used in combination in the kit, these dyes provide a rapid, simple method to distinguish live, dead, and injured bacteria, yeast, or eukaryotic cells.
- Technical Bulletin: A Novel, Transportable Flow Cytometer Facilitates Algal Quantification in Cultures and Environmental Samples
- Product Information Sheet: BD Cell Viability Kit
Water Quality Analysis
Rapid and accurate quantitation of bacteria in drinking water is essential to monitor, control, and optimize treatment processes and to illuminate the biology of low-nutrient water systems. Historically, laboratories have relied on heterotrophic plate counts (HPCs) to monitor water quality, but this method is unreliable and time intensive. Flow cytometry can rapidly quantitate bacteria and discriminate them from debris.
Researchers at Eawag Aquatic Research in Zurich, Switzerland, have developed a standard flow cytometric staining protocol for BD Accuri flow cytometers to discriminate bacteria from debris in drinking water samples. Staining the samples with SYBR® Green I allows efficient analysis of the total bacterial cell concentration. Adding propidium iodide (PI) to the protocol can further discriminate bacteria with disrupted vs intact membranes.
BD Accuri systems are ideal for monitoring drinking water because of their transportability, open fluidics systems, and ability to determine sample volume and calculate cell concentrations directly. A free software template for the Eawag protocol simplifies setup and analysis.
- Product Information Sheet: BD Accuri™ Eawag Water Quality Template
- White Paper: Assessing Water Quality with the BD Accuri™ Flow Cytometer
- Nature.com Webinar Replay: Using Flow Cytometry in Drinking Water Microbiology
- Customer Interview: Frederik Hammes on Water Quality Research
Environmental research on marine and freshwater ecosystems often focuses on their microbiomes. The productivity of phytoplankton and cyanobacteria species responsible for harmful algal blooms is of critical concern. Fortunately for researchers, most aquatic microorganisms contain natural chlorophylls, phycobilins, and other intrinsic fluorescent pigments (see table) that can readily be detected by flow cytometry.
|Fluorophore||Exciting Laser||Major Emission Wavelength||C6 Detector (filter)|
|Chloropyll a,b||488||640 nm||FL3 (670 LP)|
|Phycoerythrin||488||575 nm||FL2 (585 ±20)|
|C-phycocyanin||640||650 nm||FL4 (675 ±12.5)|
|R-phycocyanin||640||646 nm||FL4 (675 ±12.5)|
|Allophycocyanin||640||660 nm||FL4 (675 ±12.5)|
Handling particles as small as 0.5 µm, BD Accuri flow cytometers can help speed sample analysis for biologists studying marine and freshwater ecosystems. Fixed optics and capillary sheath flow fluidics enable continuous operation even during motion and vibration. BD Accuri systems have traveled to aquatic field sites across the seas, from the Great Lakes to the Netherlands, from the Gulf of Finland to the Gulf of Mexico, from the Arctic to the Antarctic.
As with other applications, a major advantage of using flow cytometry in aquatic research is multiparametric analysis at the single-cell level, avoiding the need to average data across multiple subpopulations. This allows analysis of conditions relating to microbial population growth rates, species succession, infection, competition for resources, and other aspects of aquatic ecology.
- White Paper: Multiparametric Analysis of Aquatic Organisms Using Flow Cytometry
- Article: Flow Cytometry in Aquatic Research
- Webinar Replay: Multi-Parametric Analysis of Aquatic Organisms Using Flow Cytometry
- Customer Interview: Leila Tirichine on the Tara Oceans Expedition
- Customer Interview: Peeter Laas on Flow Cytometry on the High Seas
- Customer Interview: Julie LaRoche on Monitoring Marine Microbes
Microbial Species Identification
By combining fluorescence and light scatter, flow cytometry can distinguish microbes from noise and debris. In some cases, depending on the species involved, this information might be enough to distinguish bacteria from yeast or even identify specific microorganisms in a heterogeneous sample. In other cases, additional techniques such as fluorescence in situ hybridization (FISH) can be added and even run on a flow cytometer (Flow-FISH).
SYBR® is a registered trademark of Life Technologies Corporation.
All reagents and kits are compatible with both the BD Accuri C6 Plus and BD Accuri C6 flow cytometer systems. Platforms referred to as "BD Accuri" represent both the BD Accuri C6 Plus and BD Accuri C6. Data was generated on either the BD Accuri C6 Plus or the BD Accuri C6 as indicated in figure legends.