BD Accuri News

Spotlight

Identifying the Microbes in Your Gut

Walton & Tejero - Thumbnail

Gemma Walton, PhD, and Sandra Tejero, PhD, are researchers in the Food and Nutritional Sciences Department at the University of Reading, UK. Their research interests include gut microbiology, prebiotics and probiotics, and the role of gut bacteria in health and disease. They told us how they adapted the Flow-FISH methodology to identify gut microbes using the BD Accuri™ C6 flow cytometer.

Read the full interview »

Noteworthy

Personal Flow Cytometry in the Core Facility

The BD Accuri C6 is increasingly proving its value in flow cytometry core facilities at major universities and medical centers as well as in individual research labs. In a BD Biosciences white paper, core lab managers discussed how a personal flow cytometer helps them fulfill their missions.

Download the white paper, The Personal Flow Cytometer in the Flow Cytometry Core Facility »

Read extended interviews with core facility managers Tim Bushnell and Ian Dimmick »

Application Highlight

Flow Cytometry in Aquatic Research

Flow cytometry is now an accepted methodology in marine and freshwater research, but this was not always so. "The introduction of flow cytometry to marine science was not always met with great enthusiasm, and there were many who said it would never catch on," recalled Sandra E. Shumway at a recent webinar sponsored by BD Biosciences and Elsevier. Dr. Shumway, research professor of marine sciences at the University of Connecticut, pioneered the use of flow cytometry for marine research in the early 1980s. "I was fortunate to have the support of Jeff Becton [of Becton Dickinson]. By loaning us BD flow cytometers and troubleshooting them, Jeff was as key as any scientist in making flow cytometry a reality for marine science."

Accuri News - August - Figure 1 -

Figure 1. Rugged design allows continuous
operation of the BD Accuri C6 aboard ship

A BD Biosciences white paper explored the benefits of using flow cytometry for aquatic research, with data examples drawn from the BD Accuri C6, which offers particular advantages for marine science. Handling particles as small as 0.5 µm, the BD Accuri C6 flow cytometer 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 (Figure 1). The BD Accuri C6 has 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.

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 (Table 1) that can readily be detected by flow cytometry.

Table 1. Naturally occurring fluorescent pigments in phytoplankton and their detection on the BD Accuri C6.

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)
Accuri News - August - Figure 2 - Thumb
Figure 2. Autofluorescence profiles for three phytoplankton species on the BD Accuri C6
 
Accuri News - August - Figure 2 - Large
Figure 2. 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.

The native chlorophylls, phycoerythrins (PEs), and/or phycocyanins in many phytoplankton can aid in species identification and enumeration. Because chlorophyll a or b is the dominant fluorophore in most classes of autofluorescent phytoplankton, the BD Accuri C6 FL3 detector will pick up the strongest signals. If PE is present, FL2 will also detect a strong signal. Figure 2 shows examples of three cultured phytoplankton species analyzed on the BD Accuri C6. All three species are high in primary chlorophyll fluorescence (x-axis), but can be distinguished by their secondary PE fluorescence (y-axis).

Gary Wikfors of the National Oceanic and Atmospheric Administration (NOAA) used the BD Accuri C6 to assess oyster feeding activity by analyzing the phytoplankton content of inflowing and outflowing water of an oyster nursery over the course of a day's tides. As Figure 3 shows, his team found substantial variability over time, with the oysters feasting heavily on the larger phytoplankton at low tide in the early morning.

Accuri News - August - Figure 3 - Thumb
Figure 3. Phytoplankton counts in oyster nursery inflows and outflows
 
Accuri News - August Figure 3 - Large
Figure 3. 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.

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.

Dr. Wikfors praised the instrument's ruggedness and transportability. "We used to pick up our [80-pound] BD FACScan™ cytometer and drag it all over the place," he said at the webinar. "A major benefit for onsite work is being able to pack the BD Accuri C6 in a protective pelican case. The optics are bolted down and remain in alignment when you move the instrument, which has enabled us to do more field work."

Learn more »

Download the white paper, Multiparametric analysis of aquatic organisms using flow cytometry »

Replay the webinar, Multiparametric analysis of aquatic organisms using flow cytometry »

Read how the BD Accuri C6 weathered a severe storm off the Estonian coast »


 

Tips & Tricks

Determining Cell Counts and Concentrations

Because the BD Accuri C6 flow cytometer can count cells and measure sample volume directly, it can simplify cell analysis by calculating cell concentrations (per unit sample volume) automatically. Accurate cell concentrations are essential in many research and clinical applications, including enumerating leucocytes, B cells, T cells, and platelets in human blood, measuring microorganism concentrations in purified water, and determining the viability of cultured cell lines. Direct counts on the BD Accuri C6 correlate highly with counting beads, and are more precise than hemocytometer counts.

A BD Biosciences technical bulletin contains recommendations, tips, techniques, and troubleshooting suggestions to help maximize the accuracy of cell counts and concentrations on the BD Accuri C6. The recommendations are summarized in Table 1.

Download the technical bulletin, A Guide to Absolute Counting Using the BD Accuri™ C6 Flow Cytometer »

Table 1. Summary of recommendations for absolute cell counting on the BD Accuri C6

Area Recommendations
Preventative maintenance Follow recommended preventive maintenance routines.
Sample concentration 1,000–5 x 106 cells/mL
Cell suspension Assess and minimize cell clumping.
Sample medium Calibrate fluidics when necessary to account for liquid viscosity.
Sample type Cell lines
Primary cells
Beads
Bacteria*
Sample volume 12 x 75-mm tube: 300 μL–2 mL
Users should verify other tube/plate types, calibrate fluidics when necessary.
Fluidics speed Standard settings: Medium or Fast only
Custom settings: Minimum settings are listed below. Appropriate flow rate and core size combinations are experiment-specific and should be validated by the user.
- Flow rate: ≥15 μL/min
- Core size: ≥16 μm
Using the BD CSampler™ Use the agitate function if necessary to maintain a homogeneous suspension.
Avoid V-bottom, flat-bottom, and deep-well plates.
Sample volume:
- 96-well round- or U-bottom plates: 40%–50% well capacity (150 μL–200 μL)
- 12 x 75-mm tubes: 300 μL–2 mL
Troubleshooting See the Troubleshooting section of the technical bulletin.

*For special considerations when counting bacteria and other small particles, see the BD Biosciences technical bulletin, Threshold and Analysis of Small Particles on the BD Accuri™ C6 Flow Cytometer.

In addition, two BD Biosciences white papers present sample data and guidance for specific cell counting applications. The first covers viable cell concentrations in cultured cell lines; immune cell concentrations in human peripheral blood; and platelet counts in whole, unlysed human blood. The second paper reports on a more detailed study validating human platelet concentrations measured by direct volume on the BD Accuri C6, without using a hematology analyzer.

Download the white paper, Determining Cell Concentration by Direct Volume on the BD Accuri™ C6 »

Download the white paper, Platelet Counting with the BD Accuri™ C6 Flow Cytometer »

Events

Meeting – August 22–27, 2013 – Milan, Italy
International Congress of Immunology »

Meeting – September 27–29, 2013 – Detroit, MI
Great Lakes International Imaging and Flow Cytometry Association Meeting »

BD Webinar Replay
Design of Multicolor Flow Cytometry Panels Incorporating BD Horizon™ Brilliant Violet™ Dyes »


For Research Use Only. Not for use in diagnostic or therapeutic procedures.

Class 1 Laser Product.