Cell Counting & Hemacytometers

When Robert Hooke first discovered the cell in the 17th century, researchers were just becoming aware of the microscopic units that built up all life. Since that discovery, scientists have taken great pains to refine how we see the human body through a microscopic lens. Such efforts have culminated in a deep appreciation for the cell’s role in keeping our bodies healthy and our world together.

With investigations into cell biology come additional efforts to count cells. For centuries, researchers have known that disturbances in blood strongly indicate that a person is sick. After cells were first discovered, subsequent research allowed researchers to count all kinds of cells in the blood. Inventions such as the hemocytometer made manual cell counting possible, but further technological advances have since automated this process. With such advances, cell counting has become that much easier and more accurate. 

At Excedr, we will help you push your cell counting efforts forward by giving you a primer on the subject and showcasing the cell counters we can lease.

What Is Cell Counting?

Cell counting represents any method researchers use to count the number of cells in a sample. Researchers can count cells through several approaches, both manually and automated (insert link to automated liquid handlers here). Scientists also classify cell counting under cytometry, the ability to measure cells by their size and characteristics. 

In most cases, scientists measure cell viability, which determines the number of viable cells and dead cells in cell culture. However, researchers can also measure many other traits in cells, including cell size, cell type, activity level, DNA content, and the proteins they produce inside and at the membrane. 

Measuring these traits allows researchers to distinguish different kinds of cells as they conduct total cell counts. Irrespective of the approach, having an accurate cell count represents the primary goal of cell counting techniques.

Uses of Cell Counting

Having an accurate count of viable cells is essential for many experimental pipelines. In fact, many protocols require researchers to accurately count the number of cells for downstream experiments:

• Cell transfections: Cell transfections are a process where foreign nucleic acids are introduced into cell suspensions. These experiments allow researchers to study a gene’s function and the proteins they encode. The number of live cells affects how effectively the transfection occurs.
• Quantitative PCR (qPCR): qPCR measures gene counts within a sample preparation. Although qPCR quantifies gene abundance, normalizing gene counts to cell concentration would help account for differences in gene abundance stemming from technical variation.
• Cytotoxicity experiments: Cytotoxicity refers to the extent to which exposure to a chemical damages a cell. Counting the concentration of cells by viability helps measure these impacts by distinguishing between live and dead cells.

Irrespective of the experimental workflow, cell counting always involves three steps: sampling and processing, cell counting, and calculations. 

Sampling & Processing

Distinguishing cells by their characteristics depends on having a way to differentiate them when counting cells. Staining a cell sample represents one such approach. Cell staining relies on dyes that interact with cellular components to color them. Scientists have developed several dyes to characterize cells, most of which distinguish a cell membrane’s integrity:

• Trypan blue: Trypan blue staining is the most common approach for determining the total number of cells and the number of live and dead cells. Trypan blue only enters cells that have broken membranes, a strong indicator of a dead cell.
• Propidium iodide (PI): PI is a molecular dye that binds to DNA inside cells. Once bound, the dye emits a red color when excited. Like trypan blue, the dye only stains cells that have damaged cell membranes.
• Acridine orange: Acridine orange is a fluorescent dye that binds to DNA in cells. It is commonly used to determine total cell counts, emitting a green color when excited. 

Irrespective of the dye employed, preparing samples for cell counting is a sequential process. From the total volume of the sample, cell lines must first be pelleted with a centrifuge. Then, the cell pellet must be isolated and washed. After obtaining a purified cell fraction, staining can occur. Scientists then use a pipette to transfer the sample volume into a cell counter for counting.

Amid that process, scientists must consider several factors to ensure the success of cell counting efforts:

• Minimizing sample artifacts: Sample artifacts refer to any component of the sample that either does not belong to the sample or are not the cells of interest. Artifacts arise when reagents and equipment are not properly sterilized. In such situations, researchers should use ethanol to clean slides and equipment to prevent debris from accumulating. Dyes can also stain biomolecules other than those from the target cell. For instance, trypan blue staining only occurs with serum-free fractions because trypan blue also stains serum proteins. 
• Operator error: Counting cells can be a highly subjective process. How one determines whether a cell is viable or not depends on the user conducting the experiments. Even though the counters will have the most experience, disagreements in cell counts can arise between research groups. Users may also conduct these counts at different magnifications or have other differences in protocols that affect their calculations. In these cases, standardized cell counting workflows are essential for minimizing operator error.
• Are the cells adhering to a surface? Adherent cells must first be detached before cell counts can be performed. Detaching these cells requires a careful protocol that does not kill the cells in the process. Adherent cells can be detached with physical detachment or with enzymes. However, the approach selected must not affect the cell’s characteristics or cause the cells to die. 
• Dilution factor: Larger dilution factors increase the coefficient of variation when performing cell counts. Researchers must ensure that the dilution factor is not too high when selecting a hemocytometer or cell counter when counting the total number of cells. 

Obtaining Cell Counts with a Hemocytometer

Hemocytometers (also spelled hemacytometers) are inexpensive tools used to manually count cells with light microscopy. All hemocytometers look like microscopy slides, but they have large squares and smaller squares that make manual cell counting easier. 

For its ability to The cell counting apparatus also contains a slit where samples are introduced in microliter volumes. Then once the samples are added, researchers place a coverslip on top to prevent samples from evaporating. 

Hemacytometers were first developed to count red blood cells. Since then, researchers also count unicellular organisms and other cells dispersed from multicellular organisms with a hemocytometer. Over the next century, scientists developed multiple kinds of hemocytometers with varying counting grid arrangements. 

The different layouts produce varying counting areas that allow researchers to count different types of cells at varying abundances.

• Malassez: The Malassez was the first kind of hemocytometer produced. It comprises an irregular grid of 200x250 um rectangles subdivided into 40x50 um rectangles. 
• Improved Neubauer: The improved Neubauer is the most common hemocytometer grid. It contains a large square subdivided into nine sets of 1x1 mm smaller squares. The corner squares within this arrangement are further subdivided into 16 small squares bearing 200x200 um dimensions. The center square also contains those small squares, but each is also subdivided into 16 smaller squares with 50x50 um dimensions. 
• Bürker-Türk/Thoma: This hemocytometer is like the improved Neubauer. However, it also contains additional grid lines to subdivide the large squares into 16 group squares with 200x200 um dimensions.
• Fuchs-Rosenthal: This hemocytometer contains a simple 4x4 large square grid comprising 1x1 mm squares. Each of the 1x1 squares is further subdivided into 16 um 250x250 um squares. However, unlike the hemocytometers mentioned above, the Fuchs-Rosenthal has a depth of 0.2 mm instead of 0.1 mm. 
• Jessen: This hemocytometer is similar to the Fuch-Rosenthal, but has a 5x5 large square grid and a depth of 0.4 mm.
• Speirs-Levy: Like the Fuch-Rosenthal, the Speirs-Levy bears a depth of 0.2 mm. However, the hemocytometer adopts a 5x2 rectangular grid subdivided into 1x1 mm squares. 

The Need for an Automated Cell Counter

Hemocytometers are an affordable tool for counting cells within a sample. However, the variation in sample counts increases with more dilute samples, leaving a lower limit of detection of 25000 cells/mL of sample. This stems from the 10000-fold dilutions that take place when pipetting the sample volumes into the hemacytometer. 

Counting multiple sample aliquots can increase accuracy. Nonetheless, obtaining multiple aliquots can be challenging should little samples be available. That’s why researchers have also turned to cell counters for their cell counting efforts.

What Is an Automated Cell Counter?

Cell counters are the hardware that detects characteristics inherent in specific cell types. The first automated cell counters harnessed the Coulter Principle to count cells. Under the Coulter Principle, cells are insulators that reduce electric current within a saline solution. 

The first cell counter had an aperture where an electric current was passed through. There, the cells would pass through the aperture and cause transient reductions in electric current. Since then, newer cell counters have automated identifying cells through algorithms.

Excedr Leases an Array of Automated Cell Counters

Excedr boasts several automated cell counters from diverse vendors that streamline your cell counting efforts. Each of these counters boasts unique features that will help you with any of your experimental needs:

• Beckman Coulter: The company hosts an array of cell counters that provide fast and accurate cell counting results. They host a Vi-Cell BLU Cell Viability Analyzer, which measures cell viability with trypan blue. The automated cell counter can count viable cells in a 96-well plate format and has a 24-position carousel to process samples at a high-throughput rate. 
• Denovix: Denovix produces the CellDrop Automated Cell Counter. It boasts two permanent optical sapphire surfaces that form a chamber of a precisely defined height. This setup allows smaller microliter volumes to be pipetted into the chamber. Using optical sapphire surfaces also makes it easier for slides to be cleaned with ethanol. The system also reduces plastic usage from using and disposing of individual hemocytometers. 
• Logos Biosystems: Logos boasts the Luna-FX7 automated cell counter. Luna-FX7 is equipped with an 8- and 3-chamber slide layout for counting multiple samples concurrently. The Luna-FX7 also contains a CountWire System software for users to access and manage data remotely. The software also supports counting samples over time to make growth curves for cell cultures of interest.
• ThermoFisher Scientific: ThermoFisher Scientific’s  Invitrogen Countess 3 Automated Cell Counter uses algorithms to automate identifying live and dead cells in hemacytometer counts. The machine allows researchers to identify cells with trypan blue and only requires 10 uL of sample to conduct. It also has a broader cell concentration range from 10000-10000000 cells. Finally, the machine has a counting area of more than 3 times the size of the standard hemacytometer.

Speak with Us Today

Cell counting is one of the earliest techniques researchers use to diagnose a person’s health through blood. In recent years, researchers have also expanded its uses to count other kinds of cells, both human and microbial. 

While hemacytometers have historically enabled manual cell counting efforts, an automated cell counting process will allow more experiments to be done quickly and reproducibly. With many experiments requiring accurate cell counts as controls, companies are following suit with their lines of cell counters.

If you want reproducible counts for your experiments, speak with us today. We can provide you with cost-effective hemacytometer and automated cell counter leases for high-throughput cell counting. Because we don’t carry an inventory, you can decide which system works best for your research needs. Take advantage of our brand-agnostic leasing program and push your cell counting efforts forward today!

Are you interested in leasing a hemacytometer or a cell counter? Let us know!