Flow cytometry is used to measure and analyze multiple physical and chemical characteristics of cells as they flow single file, in a fluid stream, through a beam of light. The technology is employed extensively in life-sciences laboratories for counting and sorting cells based on their individual characteristics and for assessing cell viability. It can also be used for complex studies of immune function, apoptosis (programmed cell death), cancer, stem cells, and supporting drug discovery. 
While flow cytometry is routinely used for life-science and clinical applications, the size, complexity, cost and maintenance requirements of conventional flow cytometry systems have historically confined their use to core facilities and large laboratories with expert users. These factors can limit use of this powerful technology by companies engaged in cutting-edge biofuels R&D as they are unlikely to have access to a central core lab or a flow cytometry expert on staff.  

The recent development of benchtop flow cytometry instruments, combined with user-friendly software and turnkey assay kits, is now enabling use of this technology right at the lab bench by experts and novices. New microcapillary systems require smaller sample volumes, generate significantly less waste, have lower operating costs, enable high sample throughput, and are easier to set up and run than traditional flow cytometers.  

The Guava easyCyte 8HT benchtop flow cytometry system by EMD Millipore offers a high-speed, automated, cost-effective method for assessing lipid content and other characteristics of algae cultures including cell numbers and culture densities; relative chlorophyll content with identification of bright, dim, and negative subpopulations; and estimates of potential lipid production. 

Thousands of strains of algae exist, but there can be significant variations in lipid content between different strains. Identification of high-lipid-producing strains is a prerequisite for sustainable, cost-effective production of algae biofuel.   

Benchtop flow cytometry offers a high-speed, automated, cost-effective method for assessing lipid content in various algal strains. In addition to identifying which algal strains contain the highest lipid levels, the type of chlorophyll present in the strain must also be considered. Algae strains contain different combinations of chlorophyll molecules, designated A, B and C. Algal strains containing chlorophyll A produce lipids that are best suited for biofuel development. Some algal strains may have high lipid content but the lipid has not been generated via a process involving chlorophyll A. Therefore, the lipid may not be optimal for biofuel production.

Chlorophyll A-positive populations can be identified by flow cytometry by their fluorescence. Applying a gate on chlorophyll A-positive cells allows these cells to be evaluated directly for lipid content. Both chlorophyll A and lipid content can be evaluated in an algal sample in less than three minutes on the Guava easyCyte system. Samples in 96 well plates can be placed in the Guava system allowing for walk-away automation.

Benchtop flow cytometry is also used for cell counts and to monitor algal cultures for contamination. The forward scatter function of the flow cytometer can distinguish algal cells from bacteria based on size. The elimination of sheath fluid in benchtop systems, which allows for small instrument footprint, also provides additional benefits. 

Bard Holdings headquartered in Morrisville, Pa., has developed a unique patent-pending modular system to cultivate algae in a closed-loop, sustainable process. Charles Clerecuzio, chief operating officer, describes flow cytometry as being critical to their operations. One guava flow cytometry system sits in their support lab for R&D and production and is used to adjust feeling requirements and to evaluate biomass levels.

“On the R&D level, the system evaluates the strains themselves and tracks the conversion from reproducing to producing lipids and fats for energy storage when the algae is under stress,” Clerecuzio said. “We want to feed the cells of a lot of rich nutrients and grow up as much biomass as possible—build the factories and then you switch conditions to stress the cells out. There’s various ways to stress the cells so we also look at pre- and post-stressing and what methods are effective using the flow cytometer.”

Prior to adoption of flow cytometry, more labor intensive, time-consuming techniques such as hemocytometry were used. Clerecuzio noted that the guava system delivers more robust, reproducible results. Bard runs three shifts; with the ease-of-use and automation offered by the system, human error is minimized.

Author: Jim Mulry
Clinical Development Manager, EMD Millipore
(510) 427-4641
Jim_mulry@merckgroup.com