Counting Cells by Manual and Automated Methods

A prerequisite of many cell-based assays is knowing the total number of cells in a sample or the cell density per well. Thus, the ability to count cells accurately is an important first step in many workflows, so minimizing variability in one’s data due to counting inaccuracies should be a priority. Cell counting can be done manually using a hemocytometer or an automated cell counter. This article reviews the main cell counting methods in practice today and offers considerations for choosing a method or type of cell counting instrument.

Manual counting with hemocytometers

The traditional method of cell counting involves adding a cell sample to a hemocytometer, and then counting cells by eye using a brightfield microscope. A hemocytometer is a glass slide with a specially designed and calibrated grid that has been etched into the glass, so that you can count cells visible within a measurable area. Viability dyes can be added to the cell sample to distinguish live versus dead cells, allowing a viability estimate of the overall sample.

But the manual counting method is laborious and less reliable than the newer, more efficient method of automated cell counting (see below). As automated cell counters have become more affordable, reasons for relying on manual counting have diminished. “There aren't too many scenarios for researchers to continue using a manual-based method, but one reason would be for students to learn the technique and the mathematics behind cell counting when they are learning the basics of cell culture in the lab,” says Andrew Jones, Market Development Manager at DeNovix.

Infrequent needs for counting, coupled with patience, can certainly warrant hemocytometer use. “Manual counting is an adequate approach to estimating cell counts,” says Mahesh Dodla, Global Product Manager for Monitoring Tools at MilliporeSigma. “Hemocytometers can be used when researchers are not constrained by time and do not require the more precise accuracy of an automated cell counter.” Cell sample type might also be a factor to consider. “If the cells are highly clustered and difficult to isolate into single cells, manual cell counting may work better compared to using an automated cell counter,” adds Neon Jung, CEO at Logos Biosystems.

Automated counting by electrical resistance

Most automated cell counters operate using either impedance (also referred to as resistance in this context) or imaging to count individual cells. Impedance-based counters measure the impedance across a small channel. When a cell with greater electrical resistance than the surrounding medium blocks the channel, the impedance measurement increases and the cell is detected.

Impedance-based cell counters, used in hematology for decades, work well for measuring the number and sizes of single round cells (i.e., blood cells). However, most impedance-based counters don’t measure cell viability, which could be a deal-breaker for labs that rely on it. “Most of the cells used in laboratories have complex morphologies, exist in a clustered form, and require viability measurement in order to monitor cell health,” says Jung. Despite this, impedance-based counters can rival their image-based counterparts for accuracy. “Impedance-based cell counters are more accurate and have a lower coefficient of variability,” explains Dodla. For a portable model with a small lab footprint, MilliporeSigma’s hand-held Scepter™ 3.0 is an impedance-based counter that can be used anywhere, including within the cell culture hood.

Automated counting with imaging

Image-based automated counters use microscopy and image analysis algorithms to count cells, and the algorithms are capable of distinguishing between live and dead cells. Automating this process removes the subjectivity brought to cell counting and live/dead identifications by individual researchers, leading to less variable results overall. “[Image-based] counters will also usually perform calculations such as cell density, viability and dilution volume, avoiding potential errors for downstream processing and costly failures in downstream applications,” says Jones.

Image-based counters can also provide information on cell morphology and the expression of fluorescently tagged molecules of interest. “Cells can be stained with a variety of fluorescent markers to assess cell viability, apoptosis and transfection efficiency,” says Bikram Chakraborty, Strategic Marketing Manager at Agilent. “Furthermore, image analysis algorithms can be devised for a wide range of quantitative microscopy applications where cells can be counted based on the expression of a desired phenotype using a fluorescent probe.”

Image-based counters also provide images that are helpful in monitoring cell health. “This serves as a great quality control before moving to downstream processes,” says Jones. “Maintaining a record of cell images is often an important factor in troubleshooting with technology suppliers for downstream applications.”

Some researchers may be concerned about the additional consumables cost and biohazard waste generated by an automated system, but consumable-free alternatives are emerging. “The DeNovix CellDrop Automated Cell Counter using DirectPipette™ technology was developed to eliminate plastic waste and consumable costs from this process,” notes Jones.

On the horizon

Automated cell counters with new features continue to evolve. “Some new counters can be placed in the cell culture incubator all the time, which allows cell culture videos and cell counting right in the incubator,” says Dodla. Label-free cell counting and analysis techniques may become more available in the next couple of years, notes Chakraborty. “[I also anticipate] new software algorithms for label-free cell counting, which can help researchers study cell images without the need for perturbing stains, while saving money on reagent costs,” they say.

Some labs using cell counters also need to count nuclei. “There’s been a push to improve the throughput of the new generation of automated cell counters to serve labs moving into applications such as single-cell sequencing workflows,” says Jones. “Many of these single-cell sequencing labs require nuclei counts rather than just cell counts, so cell counters have started to provide customized applications to quickly and accurately give these labs the answers they require for counting isolated nuclei.” Though disparate in methods, all of the cell counting tools available today are designed to make it easier for you to count cells accurately and reliably.