BD ACCURI C6 PLUS

Overview

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.

Resources


 

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.

Resources

Sample Data

Bacterial viability on the BD Accuri C6 Plus

Bacterial viability on the BD Accuri C6 Plus

SYBR® Green and propidium iodide (PI) were used to discriminate live vs dead E. coli bacteria after treatment with varying concentrations of ethanol. Ethanol’s bactericidal effect on cell viability was dose-dependent. Cell counts were similar using direct volume measurement in BD Accuri C6 Plus software compared to a normalized internal reference bead control.

Resolution of four microbial populations using light scatter and an autofluorescence trigger
Resolution of four microbial populations using light scatter and an autofluorescence trigger
Row 1: Triggering on the forward scatter signal of lake water samples does not allow resolution of autofluorescent from non-fluorescent species and debris. Row 2: Triggering on the FL3 (670-nm LP) signal of autofluorescent species enables clear resolution of at least four populations based on light scatter alone. Table: Accurate event counts per mL of water sample can then be determined using BD Accuri C6 software.
Live/dead discrimination of E. coli using the BD Cell Viability Kit
Live/dead discrimination of E. coli using the BD™ Cell Viability Kit

E. coli cells were grown in LB broth overnight and treated with Conflikt® Detergent Disinfectant (1%) at room temperature for 5 minutes to induce cell death. The treated and untreated samples were stained with the BD Cell Viability Kit (Cat. No. 349483) and acquired on a BD Accuri C6 flow cytometer for 30 seconds on the Fast flow rate (66 µL/min) with SSC-H threshold = 10,000 to exclude debris. Results: A. Cells were initially gated on an FL2-A vs SSC-A plot as described in the product insert. B, C. Simultaneous staining with thiazole orange (TO) and propidium iodide (PI) allows distinction among live (TO+PI), dead (TO+PI+), and injured (TO+PIint) cell populations, revealing increased cell injury and death in the treated sample as expected. The TOPI+ population was excluded from the analysis as debris.

Conflikt is a registered trademark of Decon Labs, Inc.

Detection of GFP expression in bacteria
Detection of GFP expression in bacteria
Samples containing wild-type (left column) or GFP-transfected (middle column) Escherichia coli B, or a mixture of the two (right column), were acquired on a BD Accuri C6. In the mixture, transfected and non-transfected populations were resolved clearly.
Data courtesy of Tim F. Cooper, Dept. of Biology and Biological Chemistry, University of Houston, Houston, TX, USA.
 

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.

Resources

Sample Data

Algae analysis
Algae analysis
In three environmental water samples, autotrophic phytoplankton were identified based on detection of chlorophyll a and b, and characterized as blue-green algae (cyanobacteria) or red algae based on Phycoerythrin and Phycocyanin fluorescence. Discrimination of phytoplankton from background noise was achieved by triggering on FL3 and by setting an appropriate threshold value. Coastal and bay water contained a variety of algae with distinct autofluorescence signatures, while chlorinated pond water contained a single dominant population.

Data generated on the BD Accuri C6.

Water quality analysis using the Eawag water quality template
Water quality analysis using the Eawag water quality template

Drinking water samples were stained according to the Eawag protocol, acquired on a BD Accuri C6 using the Eawag water quality template, and analyzed using BD Accuri C6 software. A. When a sample is stained with SYBR® Green I, all bacteria appear within the template’s single, fixed gate, while noise and debris are excluded. B. When the sample is co-stained with SYBR® Green I and propidium iodide (PI), damaged bacteria are shifted out of the gate, leaving only viable bacteria within. C. Each water sample generates a unique flow cytometric fingerprint.
Data courtesy of Frederik Hammes, Eawag Department of Environmental Microbiology, Dübendorf, Switzerland.

Water samples analyzed on the BD Accuri C6
Water samples analyzed on the BD Accuri C6
A. Bacterial cells in six water samples from different origins, and in one pure E. coli culture (included as a control), were stained with SYBR® Green I and visualized on FL1-A vs FL3-A density plots on the BD Accuri C6. A standard gate (P1) was applied to each sample to exclude debris and background. Bacterial concentrations within each gate are reported in a white paper. B. A histogram overlay of gated SYBR® Green I fluorescence (P1) in all seven samples highlights each sample's distinct bacterial fingerprint.
 

Aquatic 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)
Aquatic Research - Rugged Inline Graphic
Rugged design allows continuous operation of the BD Accuri C6 aboard ship

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.

Resources

Sample Data

Cyanobacterial profiling: Lake Michigan

Cyanobacterial profiling: Lake Michigan
Data prepared in collaboration with Juli Dyble and Gary Fahnensteil, NOAA Great Lakes Environmental Research Laboratory, Ann Arbor, MI, USA.

Data generated on the BD Accuri C6.

Autofluorescence profiles for three phytoplankton species on the BD Accuri C6
Autofluorescence profiles for three phytoplankton species on the BD Accuri C6
All three species have high-intensity chlorophyll fluorescence (x-axis, FL3 670 LP optical filter), which varies from a median channel value of 3 x 105 (Chlamydomonas) to 5 x 106 (Cylindrotheca) on a scale with the maximum channel value of 16.7 x 106. The PE signals (y-axis, FL2 585 ±20 optical filter) vary from a median channel value of 800 (not above background, Chlamydomonas) to 4 x 105 (Rhodomonas). Data courtesy of Jason Adolf, PhD, University of Hawaii, and Juli Dyble Bressie, PhD, NOAA, Seattle, WA.
Phytoplankton counts in oyster nursery inflows and outflows
Phytoplankton counts in oyster nursery inflows and outflows
Chlorophyll autofluorescence (measured in FL3) and forward scatter (FSC) were used to characterize and enumerate phytoplankton in water inflows and outflows of an oyster nursery over the course of a day. The oscillating curve indicates tide levels. By comparing inflows and outflows over time, the researchers determined that the oysters fed heavily on larger phytoplankton (20–100 µm and 5–20 µm) at low tide in the early morning. Data courtesy of Gary Wikfors, PhD, NOAA, Milford, CT.

Data generated on the BD Accuri C6

 

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).

Resources

Sample Data

After fluorescence gating, light scatter can distinguish bacteria and yeast
After fluorescence gating, light scatter can distinguish bacteria and yeast
Cultures of E. coli (nutrient broth, Carolina Biologicals, 155068), L. acidophilus (tomato juice/yeast extract milk medium, Carolina Biologicals, 155110) and S. cerevisiae (yeast malt agar, Carolina Biologicals, 156250) were diluted in sterile water and stained with SYBR® Green I (1:10,000 dilution, Sigma S9430) at room temperature for 15 minutes. Samples were collected on the BD Accuri C6 with thresholds FSC-H = 10,000, SSC-H = 8,000. Debris was excluded from the analysis using the BD Accuri C6 Eawag Water Quality Template. Results: FSC vs SSC plots show relative light scatter of individual samples of (A) E. coli, (B) L. acidophilus, and (C) S. cerevisiae spiked into sterile water. Regions were drawn based on these individual samples. D. The samples were mixed and collected again.

Determination of bacterial strain by PNA FISH

Determination of bacterial strain by PNA FISH
Method: Determination of bacterial strain using S. aureus PNA FISH® reagents (AdvanDx, Inc.).

Data generated on the BD Accuri C6.



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.