BD Accuri News

 

January 2017


Spotlight

Water quality update

Water Quality Spotlight - Thumb

Maintaining the quality of our drinking water is critical. Yet conventional plate-based methods of water testing take too much time and miss viable but non-culturable (VBNC) microbes. Flow cytometry is increasingly being used to provide near-real-time (or even real-time) analysis of drinking water. We checked in with two sets of previous BD Accuri News interviewees whose recent publications highlight these advances.
Read the article »

Application Highlight

Microbiology research with the BD Accuri C6 Plus

In the diverse field of microbiology research, flow cytometry is a versatile and powerful technique for analyzing microorganisms, including bacteria and yeast. Light scatter data can reveal basic information about microbes' size, shape and surface features, while fluorescent stains can assess their cell viability, metabolic activity and concentration. The BD Accuri C6 Plus can provide data for a range of microbiological applications, such as measurement of gene expression, monitoring bacterial and yeast fermentations, recombinant protein production in bacteria, environmental research, food processing and monitoring drinking water.1

Perhaps the most common task in microbial analysis is to identify and count microbes. The unique fluidics system of the BD Accuri C6 Plus, driven by peristaltic pumps, allows it to determine sample volume and count cells rapidly, directly and automatically from the software, eliminating laborious plate counts.

SYBR® Green is a cell-permeable DNA dye often used as a total cell stain to tag bacteria in environmental samples such as drinking water. This approach, called fluorescence triggering, provides increased separation of SYBR® Green+ bacteria from background, compared to the use of light scatter to identify bacteria based on size. When the cell-impermeable DNA dye propidium iodide (PI) is added to the staining protocol, it can discriminate SYBR® Green+PI viable bacteria (with intact membranes) from SYBR® Green+PI+ damaged or dead bacteria (with disrupted membranes). This assay, which can be streamlined using a free downloadable software template, was standardized by Frederik Hammes and his colleagues at Eawag, whose work is profiled in this month’s Spotlight feature.

Microbio App Highlight - Fig 1 Thumb
Figure 1. Bacterial viability on the BD Accuri C6 Plus
Microbio App Highlight - Fig 1 Large
Figure 1. Bacterial viability on the BD Accuri C6 Plus

SYBR® Green and 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.

Figure 1 shows co-staining of an E. coli culture with SYBR® Green and PI. When treated with increasing concentrations of ethanol, damaged bacterial cells shift from the Live to the Dead cell gate on the FL1 vs FL3 plot. The line graph shows nearly identical results when the cells are counted directly by volumetric counting on the BD Accuri C6 Plus, compared with the use of counting beads.

Researchers analyzing aquatic samples on the BD Accuri C6 Plus can identify the broad categories of phytoplankton using their characteristic fluorescence profiles, reflecting their relative production of chlorophyll, phycocyanin and phycoerithrin (PE), detected in FL3, FL4 and FL2 respectively (Table 1).

Fluorophore Exciting Laser Major Emission Wavelength Detector (filter)
Chlorophyll 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)
Table 1. Detecting microbial autofluorescence on the BD Accuri C6 Plus
Naturally occurring fluorescent pigments in phytoplankton and the primary BD Accuri C6 Plus detectors where their fluorescence signals are detected.

These naturally produced fluorophores allow researchers to use flow cytometry to directly detect, discriminate, analyze by morphology and enumerate these organisms using fluorescence-detection methods on BD Accuri C6 Plus, without the addition of extraneous dyes or probes. You can see an example in last month’s interview with BD scientist Mirko Corselli.

Compact size, rugged design and portability make the BD Accuri C6 Plus ideal for environmental microbiological research in the field. Fixed optics and capillary sheath flow fluidics enable continuous operation, even during motion and vibration. BD Accuri™ flow cytometers have traveled to field sites across the globe, from the peaks of the Himalaya to the forests of China, from the Great Lakes to the Gulf of Finland and from the Arctic to the Antarctic.

Download the BD Accuri C6 Plus brochure »

Visit the BD Accuri Microbiology page »

Browse kits and templates »


 

Tips & Tricks

Use BB515 to detect dim antigens

BB515 Tips and Tricks - Fig 2 Thumb
Figure 2. Use BB515 instead of FITC to resolve dim antigens
BB515 Tips and Tricks - Fig 2 Large
Figure 2. Use BB515 instead of FITC to resolve dim antigens

In a typical Treg panel, cells were stained with CD3 PerCP-Cy™5.5, CD4 PE, CD127 APC, and either CD25 FITC (upper plots) or CD25 BB515 (lower plots). (With CD25 expressed at variable levels, CD25brightCD127low/neg cells have been found to express FoxP3, the classic intracellular Treg marker.) Results: In the left plots, BB515 resolved two different subpopulations. In the right plots, BB515 distinguished a CD25-expressing population that FITC did not resolve.

The popular fluorochrome FITC is not always bright enough to detect dim antigens. A brighter alternative is BD Horizon Brilliant™ Blue 515 (BB515). Its excitation peak exactly matches the 488-nm laser in BD Accuri flow cytometers, and it emits a narrower band of fluorescence that is detected in the FL1 channel.

Figure 2 shows the results of two different regulatory T-cell (Treg) panels in which the dim antigen CD25 was paired with either FITC (upper plots) or BB515 (lower plots). In the dot plots on the left, the mean fluorescence intensity (MFI) of the cells stained with BB515 was shifted right, indicating brighter staining. In the histograms on the right, BB515 was able to resolve a CD25mid population that FITC did not detect.


 

Publication Picks

This section highlights interesting recent research articles using BD Accuri flow cytometers.

New melanoma therapy

Alderman C, Sehlaoui A, Xiao Z, Yang Y. MicroRNA-15a inhibits the growth and invasiveness of malignant melanoma and directly targets on CDCA4 gene. Tumor Biol. 2016;3713941-13950. PubMed

Gut microbiota

Liu X, Zeng B, Zhang J, et al. Role of the gut microbiome in modulating arthritis progression in mice. Sci Rep. 2016;6:30594. PubMed

Telomerase RNA analysis

Xu J, Wang Y, Yang L, Gao Y, Li B, Jin Y. A cytometric assay for ultrasensitive and robust detection of human telomerase RNA based on toehold strand displacement. Biosens Bioelectron. 2017;87:1071-1076. PubMed

CRISPR-Cas genome editing

Benakanakere M, Finoti LS, Tanaka U, Grant GR, Scarel-Caminaga RM, Kinane DF. Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing. Sci Rep. 2016;6:31180. PubMed


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

1All reagents and kits are compatible with both the BD Accuri™ C6 and the new BD Accuri C6 Plus flow cytometer systems. Data was generated on the BD Accuri C6 Plus. Information about BD reagent kits, BD Accuri™ C6 and BD Accuri™ C6 Plus software templates, and BD CSampler™ and BD CSampler Plus automation options is available at www.bdbiosciences.com/accuri.

Cy™ is a trademark of GE Healthcare. Cy™ dyes are subject to proprietary rights of GE Healthcare and Carnegie Mellon University, and are made and sold under license from GE Healthcare only for research and in vitro diagnostic use. Any other use requires a commercial sublicense from GE Healthcare, 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA.

SYBR® is a registered trademark of Life Technologies Corporation.