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Cell Counting Techniques

Cell culture is a critical step in cell biology research. To ensure healthy cell growth and facilitate downstream experiments, researchers must determine cell growth curves and understand the growth state of cells in culture. Therefore, cell concentration, viability, and concentration need to be measured before downstream experiments.

Counting Chambers / Hemocytometers

The hemocytometer is a widely used cell counting chamber that is commonly employed for manual cell counting. The objective is adjusted at different magnifications under the microscope. The cell state is manually observed by staining to determine whether the cells are dead or alive and counted. The number of cells in a certain volume of homogeneous cell suspension is detected. Afterward, the number of cells per ml is converted to obtain the cell concentration of the cell suspension.

Automated Cell Counter

Automated cell counters were designed to be a faster, easier, automated alternative to manual counting. They use the same principles of operation as hemocytometers and use similar disposable counting chambers; they perform multiple counts of cells within a known area and average out the results. They also can discern live cells from dead cells using dye exclusion methods (such as trypan blue).

Coulter Counters

  • Cells are relatively poor conductors. When cells are suspended in an electrolyte, a small-bore tube is inserted into the cell suspension so that its tube is filled with a dilute solution. Electrodes located on both sides of the small-bore tube generate a steady current.
  • Coulter counters are not optical instruments but rather measure the electrical resistance across one or more microchannels. When a cell passes through a small pore, a pulse signal is instantaneously induced by a voltage change. The larger the cell volume, the larger the pulse caused and the higher the amplitude of the generated pulse.

Image Method

Obtain high-definition pictures of the sample through the camera lens. Based on the morphology and color of the living and dead cells, the software intelligently analyzes the results of the pictures. The instruments currently on the market using this method are broadly divided into two categories, one is the bright field cell counter which requires only simple Taipan blue staining, and the other is the fluorescent field cell counter which requires the appropriate fluorescent dye.

  • Trypan blue staining. Trypan Blue is a cellular transmembrane dye. The membrane structure of living cells is intact, and Trypan blue staining cannot pass through them; the membrane permeability of dead cells is changed, and Trypan blue staining can cross the membrane of dead cells and enter the cells, causing the cells to deepen in color.
  • AO/PI fluorescent staining. AO/PI is a nuclear dye. AO is acridine orange, a small molecule dye that can cross all cell membranes to enter the cell and label nucleated cells with green fluorescence. PI is propidium iodide, a large molecule dye that can only enter the cell across the membrane after the permeability of the dead cell is changed, causing the nucleus of the dead cell to be labeled with red fluorescence.

Creative Bioarray Relevant Recommendations

  • Creative Bioarray provides Trypan blue staining, commonly used for the determination of cell viability. And we provide many fluorescent dyes that are highly specific to a variety of organelles and can be used to monitor cell health and cell death. Our cell counting kit also performs well in practice. We are always committed to providing customers with high-quality products and services.

Flow Cytometers

  • Flow cytometry is used to cause individual cells to refract, diffract and scatter the light beam as it passes through the detection area irradiated by the laser. The scattered light detector receives and generates pulses to convert the light signal into an electrical signal. The pulse size is proportional to the size of the illuminated cells, and the number of pulses represents the number of illuminated cells.
  • Flow cytometry requires the use of more expensive specific fluorescent reagents and corresponding excitation light and filters, which are more expensive to use. Therefore, flow cytometry is not used for cell counting, but for cell population analysis.


Techniques Hemocytometers / Automated Cell Counter Coulter Counters Image Method Flow Cytometers
Trypan blue staining AO/PI fluorescent staining
Application Conventional cells Single suspension cells Conventional cells PBMC, primary cells, immune cells Currently rarely used for cell counting
Advantages & Disadvantages The most primitive technical method, time-consuming and labor-intensive. It needs to clean the line periodically, with no visualization of cell morphology. It cannot exclude the interference of nucleated cells. It uses specific staining principles to compensate for the disadvantage of Trypan blue stains. It is commonly used for cell type detection.
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