How to Count Cells
An Overview of Cell Counting Methods
#cell counting #acridine orange #Hoechst #AO/HO staining #high content imaging #high content analysis #HCAdownload pdf
The microscope is then focused on an area of the counting chamber and the cells are counted using a tally counter. This is typically done using the 1 mm2, 100 nl area of the counting chamber and a 4x or 10x objective, but the precise area and objective used will depend on the size of your cells and their density in suspension. This process is generally repeated using four different 1 mm2 areas and the results are averaged. If determining cell viability, separate counts should be made for live and dead cells, with the dead cells appearing blue due to the permeability of their damaged membranes to trypan blue.
- Fig 1 Schematic representation of a hemocytometer grid.
Automated cell counters were designed to be a faster, easier, automated alternative to manual counting. They use the same principles of operation as hemocytometers; 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). Automated cell counters may either operate as standalone devices or require a connection to a computer. In addition to a cell count, most counters also provide statistical information on cell size.
Benefits & Drawbacks: For general purpose cell counting and cell viability applications, automated cell counters are an affordable and high-throughput solution. The cost of operation is low, they are easy to use, and they greatly reduce the amount of human effort required to count cells. They are both precise and reliable, but may have difficulty obtaining accurate measurements of cells that are highly irregularly shaped, are extremely small, or are in cell suspensions that are extremely dilute or contain a large variety of cells that need to be distinguished. For most cell types and most applications, automated cell counters provide excellent counting performance at a relatively low cost.
Benefits & Drawbacks: Because of their relative speed compared to manual counting and their ability to accurately count cells of differing size, Coulter counters are frequently used for complete blood counts in hospitals, where red blood cells and white blood cells need to be quickly and accurately distinguished. However, Coulter counters are not capable of distinguishing live cells from dead cells, nor do they accurately count cells which form clusters or clumps. They also require more maintenance.
Use of a flow cytometer is quite simple (load and run), and therefore the overall ease of use depends more heavily on the experimental set-up.
Benefits & Drawbacks: Flow cytometers are extremely powerful cellular analysis tools but are also extremely expensive, with costs ranging from $40,000 to over $100,000. Because of this, they are rarely used for general cell counting applications.
To estimate cell density using a spectrophotometer, place the cell suspension in a cuvette and measure the absorbance. If you are looking to get a relative density measurement, you may simply compare to another sample. Otherwise, you must compare to cell suspensions of known density in order to estimate an absolute cell density.
Plating is another method of counting cells, although only in colony-forming cells such as bacteria. To count cells with plating, the cells are heavily diluted and streaked onto a plate. After given sufficient time for colony growth, the number of colonies are counted. Based on the dilution and the known volume of suspension that was streaked onto the plate, the density of the original suspension can be determined.
Plating is only a useful method for microbes, and due to the time required for colony formation it is also the slowest method.
Using the LUNA Reusable Slide for accurate cell counting with automated cell counters
Application of a non-hazardous vital dye for cell counting with automated cell counters
Quick Start Guide2018-04-16
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