BD Accuri C6

Overview

Many modern industries employ biological processes that require precise monitoring and control. Bacterial bioprocessing produces ingredients for food, drugs, cosmetics, and more; algae can be used to manufacture biofuels; yeast fermentation produces wine and beer. By offering rapid analysis of heterogeneous samples, flow cytometry is an ideal method for monitoring these processes.

The BD Accuri™ C6 flow cytometer offers particular advantages for industrial and agricultural applications. Compact and rugged, it can be transported into labs and processing facilities, and even into the field. Its open fluidics system allows it to connect easily to bioreactors and fermentation units for automatic sampling and continuous culture monitoring.

The following sections and resources illustrate the rich data you can generate by using the BD Accuri C6 for industrial applications in bioprocessing, biofuels, agriculture, fermentation, and other enterprises.


 

Bioprocessing

Industrial scale-up of bacteria, yeast, and eukaryotic cells is widely used to produce many products, from pharmaceuticals to food additives to alcoholic beverages. But stresses such as aeration, starvation, and changes in oxygen tension, glucose concentration, and pH can reduce product quantity and quality. Precise control and optimization of cell growth are crucial.

Flow cytometry offers a powerful and effective methodology for monitoring cellular status and growth in bioreactors by rapidly characterizing complex cultures at the single-cell level. Flow cytometry applications common in bioprocessing include cell counting, viability, cell cycle, apoptosis, transfection efficiency, cell line development, and media optimization.

Offering both performance and simplicity, the BD Accuri C6 allows bioprocess engineers to use light scatter signals to discriminate cells and fluorescence signals to measure cell viability, vitality, and other important cell characteristics. The BD Accuri C6 also interfaces smoothly with automated bioreactor sampling systems so that samples can be taken continuously and analyzed automatically. Bioprocess engineers can use the resulting real-time, online bioreactor data for pinpoint process control.

Resources

Sample Data

Using apoptotic cell counts to trigger passage of a CHO cell culture
Using apoptotic cell counts to trigger passage of a CHO cell culture
A 1-L bioreactor was batched with 900 mL of medium and inoculated with 100 mL of spinner flask culture of Chinese hamster ovary (CHO) cells at 106 cells/mL. The graph depicts the live and non-viable cell concentrations as a function of time. The dashed line indicates the passage trigger point. Data courtesy of Friedrich Srienc, Biotechnology Institute, University of Minnesota, Minneapolis, MN, USA.
Batch growth and GFP fluorescence of E. coli cells, autosampled every 15 minutes
Batch growth and GFP fluorescence of E. coli cells, autosampled every 15 minutes

E. coli BL21 pRSET-GFP cells were grown in lysogeny broth, inoculated into a bioreactor as described as follows, and acquired and analyzed on the MSP FlowCytoPrep and BD Accuri C6. Samples were collected for 1.5 minutes at the Medium flow rate (35 μL/min), with an acquisition threshold of FSC-H = 11,000 to exclude debris. GFP fluorescence was detected in FL1 (533/30) using the standard emission filter.

A. The cultured E. coli cells were inoculated into LB in a bioreactor, grown, stirred, and analyzed every 15 minutes. Cell concentrations increased to maximum within 8 hours, while the percentage of GFP+ events decreased. B. The cultured E. coli cells were transferred from LB to M9 minimal medium and grown overnight before similar inoculation into the bioreactor and analysis of samples every 15 minutes. Cell concentrations increased more slowly than in LB, taking nearly 30 hours to reach a maximum, while the percentage of GFP+ events increased, reached a plateau, and then decreased.

 

Biofuels

Some authorities argue that algal biofuel is the best renewable candidate to fully replace fossil petroleum. Research laboratories racing to find or develop strains of microalgae suitable for biofuel development need efficient analytical tools to monitor algal cultures—rapidly and in real time—for size, growth, viability, chlorophyll autofluorescence, and lipid content.

Flow cytometry offers a powerful and effective method for screening microalgal cultures, and the BD Accuri C6 is especially well suited to the task. Microalgae can vary greatly in size, can live as single cells or in colonies, and cultures grown in open ponds often contain debris and other contaminating organisms. These characteristics can make them difficult to process through a flow cytometer’s fluidics system, even when pond water is filtered before acquisition. But the direct-drive, peristaltic pump system of the BD Accuri C6 enables independent regulation of both the sheath and sample flow rates, and facilitates the removal of clogs that might form during sampling with built-in unclog and cleaning cycles. Using lipophilic dyes, the BD Accuri C6 can rapidly estimate neutral lipid storage in small samples of live algal cells, usually without needing to remove the alga from its growth medium.

Resources

Sample Data

Light-scatter profiles for microalgae of varying size
Light-scatter profiles for microalgae of varying size
A. FSC-A histograms for 11 species of microalgae, ranging in size from 2 to 15 µm, are overlaid across the entire dynamic range of the BD Accuri™ C6 (left) and “zoomed” for easier comparison (right).
B. FSC-A vs SSC-A plots of the smallest, largest, and an intermediate species (A, F, and H) using logarithmic axis scaling. The maximum channel values of the axes are identical for each plot (FSC-A=1.25 x 106, SSC-A=2.0 x 106). Growth conditions for each strain were a 12-h light:12-h dark photoperiod, 100 µE/m2/s, and 20°C. Cultures were sampled and analyzed using Medium fluidics speed (core size 16 µm) at the transition from logarithmic to stationary growth phase. Ten thousand events were measured for each sample and gated to exclude debris.
Data courtesy of James Barker and Rose Ann Cattolico, University of Washington.
Neutral lipid content in Isochrysis sp., analyzed with Nile Red
Neutral lipid content in Isochrysis sp., analyzed with Nile Red
Isochrysis cells, either (A) unstained or (B) stained with 1 µg/mL of Nile Red, were analyzed on a BD Accuri C6 to detect neutral lipid content. Nile Red is intensely fluorescent in organic solvents and hydrophobic environments. Data is plotted on 2D density plots of chlorophyll fluorescence (x-axis: FL3; ex: 488 nm, em: 670 nm LP) vs Nile Red fluorescence (y-axis: FL2; ex: 488 nm, em: 585 ±20 nm). Staining resulted in a 200-fold increase in FL2 signal, while the increase in FL3 fluorescence (chlorophyll a autofluorescence) was minimal.
C. Data from unstained (black) and stained (red) cells are plotted on a single-parameter histogram (Nile Red; FL2). Debris was excluded from the analysis by gating.
Data courtesy of Gordon Wolfe, California State University (Chico).
 

Plant Biology

Of the approximately 500,000 species of angiosperms (flowering plants) in the world, only about 2% have been described even minimally at the molecular level. Because anthropogenic change is driving many species extinct each year, a global molecular and genomic census of angiosperms is needed.

Flow cytometry can provide a rapid and efficient way of quantifying DNA content using dyes such as propidium iodide (PI). However, plant nuclear DNA content (ploidy) varies over extreme ranges, and nuclear DNA measurements of plants are often hampered by excessive cellular and subcellular debris and autofluorescence from other prevalent cellular components, such as chloroplasts.

Several factors make the BD Accuri C6 ideal for determining the nuclear DNA contents of plants. It is readily transportable into the field to analyze freshly isolated samples, before degradation can occur during transport or storage. Its sophisticated digital signal processing system has a seven-decade dynamic range that encompasses almost the entire known span of plant C-values (haploid nuclear DNA content). Its user-controllable, nonpressurized fluidics system can handle particles of 100 µm or more in almost any kind of sample tube. Finally, its blue laser optimally excites PI. A high correlation between PI-DNA emission in the FL2 and FL3 channels places the nuclear events in a narrow diagonal region from which it is easy to exclude fragments and debris.

Resources

Sample Data

Distinguishing DNA content and ploidy in A. thaliana root tissues
Distinguishing DNA content and ploidy in A. thaliana root tissues
Arabidopsis thaliana plants were germinated from seeds and grown under sterile conditions. Root tissues were chopped with a fresh razor blade, stained with PI, and acquired on the BD Accuri C6. A. Nuclear contents form a linear cluster on the biparametric contour plot of FL2-A vs FL3-A fluorescence emission, corresponding to the endoreduplicative series of 2C (diploid), 4C, 8C, 16C, and 32C cells. The cluster outside the rectangular region of interest (R1) contains cellular debris. B. Enlargement of the R1 region shows nucleic clusters in polygonal region P1, comprising only 1.8% of the detected events. C. A uniparametric histogram of FL2-A fluorescence, gating on region P1 of panel B, shows a near-perfect linear correlation with clear peaks. %CVs for the first four peaks are about 3%, and day-to-day reproducibility of the 2C peak is about 7%. Abbreviations, 2C, 4C, etc, indicate the ploidy of each peak. Data courtesy of David W. Galbraith, School of Plant Sciences and Bio5 Institute for Collaborative Bioresearch, University of Arizona, Tucson, AZ, USA.
 

Food, Beverage, and Veterinary

In industrial processes, microbes can represent contaminants to be controlled or beneficial agents to be encouraged. In the first two interviews below, researchers describe how they use the BD Accuri C6 to detect foodborne pathogens and monitor fermentations. In the third, a team describes how they use flow cytometry to optimize cryopreservation of semen in their efforts to preserve the endangered Asian elephant.

Resources